Biochemistry and Medicinal Chemistry of the Dengue Virus Protease

Review pubs.acs.org/CR

Biochemistry and Medicinal Chemistry of the Dengue Virus Protease Christoph Nitsche,† Steven Holloway,‡ Tanja Schirmeister,‡ and Christian D. Klein*,† †

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany ‡ Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, Staudingerweg 5, D-55128 Mainz, Germany infections exists, and the only (unspecific) preventive activities are protective measures against the transmitting mosquitos. This is of particular, current concern because the disease spreads considerably, probably due to climate change and socioeconomic factors such as global travel and urbanization in tropical and subtropical countries. Today approximately 40% of the world’s population, more than 2.5 billion people, live in areas and countries of infection risk.1 In recent decades, the number of occurring infections, CONTENTS severe cases, and countries reporting autochthonous infections for the first time has dramatically increased. The World Health 1. Introduction 11348 Organization estimates 50−100 million infections per year. 2. Structural Biology of Dengue Protease 11350 2.1. Fundamental Properties and Structural RelaApproximately 2 million clinical cases are reported annually, tionship to Other Proteases 11350 including ca. 500 000 severe cases of DHF and DSS (25%), of 2.2. X-ray Crystallography of Dengue Protease 11351 which 2.5% die. Another recent study suggests a much higher 2.3. NMR Spectroscopy of Dengue Protease 11353 number of annual infections using statistical and cartographic 3. Protease Constructs, Substrates, and Assays 11355 analyses of infection reports worldwide.2 From these exhaustive 3.1. Protease Constructs Used for in Vitro evaluations, a total number of infections for the assessment year Applications 11355 2010 of 390 (credible interval 284−528) million infections was 3.2. Mutagenesis Studies 11358 calculated, of which 96 (67−136) million are clinically manifest 3.3. Substrate Specificity 11359 with characteristic symptoms. 3.4. Substrates for Enzymatic Assays 11362 The dengue virus (DENV) belongs to the family of flaviviridae, 3.5. Assay Conditions 11363 genus flavivirus, which also contains the West Nile virus (WNV), 3.6. Protease Constructs, Substrates, and Assays: tick-borne-encephalitis virus (TBEV), yellow fever virus (YFV), Concluding Remarks 11364 and several other pathogenic viruses.3 Another close relative that 4. Inhibitors of Dengue Virus Protease 11364 also belongs to the flaviviridae family is the hepatitis C virus 4.1. General Remarks 11364 (HCV). The dengue vectors are the mosquitos Aedes aegypti 4.2. Peptidic and Peptidomimetic Inhibitors 11364 (Stegomyia aegypti) and Aedes albopictus (Stegomyia albopicta). 4.3. Nonpeptidic Inhibitors 11370 The presence of these vectors, which are traditionally associated 4.4. Inhibitors: Concluding Remarks 11377 with tropical and subtropical climates, is closely connected to Author Information 11377 dengue outbreaks. Therefore, the recent appearance of Aedes Corresponding Author 11377 albopictus on the northern side of the Alps has caused concern in Author Contributions 11377 middle-European countries.4,5 Notes 11377 Four serotypes of dengue virus, which can be further divided Biographies 11377 into a number of genotypes, have been described in the literature. Acknowledgments 11378 The emergence of a presumptive fifth serotype was described Abbreviations 11378 recently.6 The genetic difference between the dengue serotypes References 11379 is so pronounced that they are sometimes considered as separate species and show a distinct pathological behavior.7,8 The severity of dengue infections, reflected by the incidence of DHF and DSS, 1. INTRODUCTION is often considered to be most pronounced for serotype 2, Dengue virus is the pathogenic principle behind several medical followed by serotypes 1 and 3, with serotype 4 being conditions. The most common disease caused by this virus is comparatively rarely associated with severe disease.9 In the past dengue fever, characterized by high fever and flu-like symptoms, decades, increasing intercontinental travel led to the global which is highly unpleasant but usually self-limiting. A small fraction of dengue-infected patients develops more serious Special Issue: 2014 Drug Discovery and Development for Neglected conditions, in particular dengue hemorrhagic fever (DHF) and Diseases dengue shock syndrome (DSS). These are associated with hemorrhages and volume depletion and can be fatal. Currently, Received: April 28, 2014 no specific preventive or therapeutic measure for dengue Published: September 30, 2014 © 2014 American Chemical Society

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significant effect after the disease has reached the life-threatening, later stages. Replication of dengue virus requires the formation of a virusspecific biomolecular machinery at the rough endoplasmatic reticulum that is known as the “replication complex” and consists of vesicular membrane structures. In these structures, the the viral polyprotein is anchored to the ER membrane by numerous transmembrane domains (Figure 1). The polyprotein is processed by several proteases, including host proteases such as furin and signalase, which act from the ER lumen (signalase) or at a later stage of virus maturation in the trans-golgi-network (furin). At the cytoplasmatic side of the membrane-bound polyprotein, the viral protease performs several cleavages. The activity of the viral protease depends crucially on the physicochemical microenvironment in which virus replication occurs. As indicated in Figure 1, the protease domain, being localized at the N-terminal region of NS3, interacts with domains of the NS2B protein that contribute to the substrate recognition region of the protease. Furthermore, the interaction of NS3 with the membrane- and protein-microenvironment within the replication complex appears to be involved in proper folding and, consequently, proteolytic activity of NS3. Under in vitro conditions, this is reflected by the inactivity of isolated NS3 in the absence of a certain NS2B subdomain, and by its unusual buffer requirements: the pH of in vitro assay buffers for dengue protease is usually in the basic range, and cosmotropic additives such as glycerol or ethylene glycol are present in significant concentrations, often along with ionic or nonionic detergents. These conditions can be expected to have an influence on inhibitor recognition and may have a detrimental effect on the in vitro/in cellulo/in vivo correlation of assay results. This highlights the importance of cellular assays for the characterization of protease inhibitors. Further details on the genome organization, replication, processing, and maturation of dengue virus can be obtained from several recent review articles and book chapters.16−18 Vaccines against dengue have been pursued for decades. In the past, these efforts did not result in a clinically useful product and future will show whether the currently studied candidate vaccines will provide an effective protection. Vaccine development for dengue virus is, in contrast to other viruses of the flaviviridae family such as yellow fever virus or tick-borne encephalitis virus, hampered by several difficulties.19 Animal testing of dengue vaccines is difficult and has only limited significance. A fundamental problem of dengue vaccination is the risk of antigen-dependent enhancement, as described above, and a dengue vaccine must therefore have a lasting, tetravalent effect against all four dengue serotypes. The emergence of a novel dengue serotype, which may transfer from a sylvatic life cycle to humans, will put vaccinated persons at a particular risk of developing complicated dengue-related diseases such as DHF or DSS, due to antigen-dependent enhancement. Very recently, Vasilakis reported on the discovery of a presumptive new dengue serotype in Asia, but the full characterization of the virus isolate is still outstanding.6 The emergence of a novel serotype would put the efforts toward a tetravalent dengue vaccine at significant risk. It is therefore highly attractive to pursue approaches for the treatment and prevention of dengue infections that carry less risk than vaccination. Pharmacological interventions against dengue virus replication can be aimed at several targets. These can be classified into viral proteins and host factors that are essential for replication. Among the viral proteins, the most attractive target is the viral protease.

distribution of all dengue serotypes. The immune response towards an infection with one serotype leads to a significantly increased risk for aggravated disease and complications such as DHF when infection with another serotype occurs at a later time.10 This phenomenon is described as “antigen-dependent enhancement” and is discussed as a major obstacle in vaccine development. The dengue virus contains a single-stranded RNA genome, which is replicated by the translation mechanisms of the host in conjunction with the viral RNA-dependent RNA polymerase. In other RNA viruses, particularly those that do not require an arthropod vector for transmission, HCV being the prime example, the low fidelity of the RNA polymerase leads to an extremely high mutation rate and a large genetic diversity of the virus. While this effect was initially also expected for dengue virus, recent work has shown that the genetic stability of the dengue virus is higher than expected.11,12 In the context of drug development, this means that viral proteins as potential drug targets will probably have a lower tendency to experience resistance-causing mutations than in the case for HCV. The RNA genome of dengue virus consists of about 11 kilobases, which encode for a polyprotein that contains nonstructural (NS) and structural proteins (see Figure 1). The

Figure 1. Polyprotein of dengue virus at the membrane of the rough endoplasmatic reticulum. Cleavage sites of the viral protease and of the host proteases are indicated by arrows. Host proteases that act on this polyprotein include, in particular, furin and signalase. The NS3 protein (red), including the protease functionality, is localized in the vicinity of the NS2B protein (green), which contributes to the formation of the substrate binding region of NS3 protease. Data from refs 16−18.

capsid (C) and envelope (E) proteins and a membrane proteinprecursor (prM) are located at the N-terminus of the polyprotein, followed by the nonstructural proteins NS1−NS5. The functions of the nonstructural proteins are partially known. Most significantly, NS3 contains a protease domain, commonly denoted as NS3pro, and a helicase domain. NS5 contains an RNA methyltransferase and a RNA polymerase domain. Other nonstructural proteins are supposed to be part of the replication complex. The primary host cells of dengue virus in humans are dendritic cells and other cells of the immune system.8,13,14 In the first few days following infection, there is significant viraemia, accompanied by high fever. 15 Notably, most life-threatening complications such as DHF evolve at a later stage, when virus replication has declined. Pharmacological interventions that aim at the replication process of dengue viruses will therefore have to be applied in the early stages of disease or as a prophylactic measure during dengue outbreaks, and will probably not have a 11349

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consequence of ligand or substrate binding, with the ligand/ substrate causing an induced fit of the cofactor. However, the “open/closed” terminology is somewhat unfortunate because the conformational changes do not resemble a closure. The protease of dengue virus is devoid of cysteine residues and therefore does not contain disulfide bonds. There are also no indications of essential posttranslational modifications. These properties make a heterologous expression of the protein in bacteria relatively straightforward. A large number of expression systems, as outlined in the section on biochemical assays (section 3.1), have been described. A comparison of the tertiary structures of dengue protease, trypsin, and thrombin is shown in Figure 3. The trypsin-like fold,

The protease of HCV, a closely related virus, is the target of numerous highly effective anti-HCV drugs, which recently entered the market or are currently in advanced clinical development,20,21 and may thus serve as a paradigmatic example for the discovery of dengue therapeutics and their therapeutical value. Very recently, a strong correlation between the proteolytic cellular activity of dengue virus protease and activation of the host transcription factor NF-κB was confirmed in an animal model.22 Activation of NF-κB is linked to apoptosis of endothelia cell and therefore responsible for severe cases of dengue hemorrhagic fever, underlining the attractiveness of DENV protease as drug target. In the present work, we will review the protease of dengue virus from several directions, with a particular focus on those aspects related to medicinal chemistry.

2. STRUCTURAL BIOLOGY OF DENGUE PROTEASE 2.1. Fundamental Properties and Structural Relationship to Other Proteases

The proteases of dengue and other flaviviruses are serine proteases with a trypsin-like fold. The tertiary structure of dengue protease is shown in Figure 2 (PDB code 3U1I).23 As in all

Figure 3. Structural alignment of the dengue NS3 protease domain (red, 3U1I chain B), human thrombin (yellow, 1AHT chain H), and bovine trypsin (gray, 3UQO chain A). The side chain atoms of the catalytical triads are shown in stick representation. This and all following structural alignments were generated by matching homologous residues of the complete X-ray structures. Chimera145 was used for matching and generation of the graphic. Figure 2. Tertiary structure of dengue protease from serotype 3, PDB code 3U1I. The unit cell of this X-ray structure contains two nonidentical assemblies of the NS3 protease domain and a partial sequence of NS2B. Shown here are chains A (NS2B fragment, blue) and B (NS3 protease domain, red). The termini of the protease and cofactor chains are indicated by “C” and “N”, respectively. A disordered part of the NS3 protease fragment between residues 10 and 16 is indicated by a dotted line. This graphic was generated using Chimera.145

common to all three proteases, can be recognized, but significant differences between the viral and the eukaryotic proteases, particularly in the peripheral regions, are obvious. Figures 4 and 5 show the structural relationship of dengue protease to the proteases from West Nile virus and hepatitis C virus, respectively. The similarity to WNV protease is significant on both the structural and the sequence levels (45% identity, 60% similarity), whereas the HCV protease, albeit being folded similarly, has a much lower degree of sequence similarity to DENV protease (18% identity, 35% similarity). The specificity pockets of dengue protease are indicated in Figure 6. This figure is based on the X-ray structure 3U1I, which contains a tetrapeptide aldehyde covalently bound to the catalytic serine. It is therefore possible to deduce the specificity pockets from the location of the four peptide residues. However, only the S1−S3 pockets are relatively well-defined, and the S4 “pocket” is, at best, a superficial, hydrophobic region with significant solvent exposure. This part of the tetrapeptide ligand and the protein interacts closely with a crystallographic neighbor in the 3U1I structure and may therefore not resemble the binding mode of the P4 residue under dilute conditions. However, the 3U1I structure is extremely valuable for a detailed understanding of the ligand binding behavior and will therefore be discussed in

further molecular graphics in this Review, the NS3 protease domain is shown in red and the NS2B cofactor fragment is shown in blue. This structure of dengue protease is the first in which the NS2B cofactor is folded in a way that allows interaction with the ligand, and contributes to the formation of the S2 and S3 pockets. As further outlined below, the NS2B cofactor in previous X-ray structures of dengue protease is folded differently, so that the catalytical competence and biological significance of the folds observed earlier is questionable. Therefore, the 3U1I structure serves as a reference structure for all further molecular graphics in this Review. In the literature, the observed folds of the NS2B cofactor are sometimes denoted as “closed” or “open”, with the closed state being the one in which NS2B contributes to the S2 and S3 pockets. Formation of the “closed” state is often interpreted as a 11350

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Figure 6. Specificity pockets. Dengue protease in complex with the tetrapeptide aldehyde Bz-nKRR-H covalently bound to the catalytic serine (3U1I).23 The residues near the substrate-mimicking peptide inhibitor are covered with a solvent-accessible surface, which is colored according to the underlying atom type (CPK colors, C = gray, N = blue, O = red). The residues of the catalytic triad are covered by a yellow surface and denoted by single-letter code. The S1, S2, and S3 pockets, indicated by numbers, are clearly defined by the respective residues of the inhibitor. The S4 pocket is less well-defined, because the P4 norleucine residue points outward and interacts mostly with residues from a crystallographic protein neighbor. This is even more pronounced for the N-terminal benzoyl cap, which is not shown in this graphic. The putative position of the S1′ pocket, which is not occupied by the ligand, is also indicated. This graphic was generated using Chimera.145

Figure 4. Comparison of the dengue and West Nile virus proteases (3U1I and 2FP7). The dengue NS3 protease domain is shown in red and the dengue NS2B cofactor in blue, while the analogous WNV proteins have a lighter color. This graphic was generated using Chimera.145

The first three-dimensional structure of dengue protease was solved by X-ray crystallography and reported by Erbel and coworkers in 2006,24 along with a structure of the West Nile virus protease. Several essential features of this structure, with the PDB code 2FOM, are listed in Table 1 along with all other X-ray structures of dengue protease reported to date. In the 2FOM structure, the trypsin-like fold of the protease domain is clearly evident (Figure 7). The N-terminal part of NS2B cofactor is halfway wrapped around the protease domain, and only a very small section is folded in a helical conformation. A part of the cofactor domain appears to be disordered, because electron density is not defined for residues 77−84. A complete dengue NS3 protein, including the protease and helicase domains, was successfully crystallized and analyzed by Luo et al., resulting in the structures 2VBC, 2WHX, and 2WZQ.25,26 These structures show a clear structural separation between the two domains, which are structurally clearly distinct from each other (cf. Figure 8). Luo and co-workers speculate that basic surface residues of the protease domain are implicated in the binding of the helicase domain to its nucleic acid substrate. They also made the surprising observation that even minor changes in the linker region, such as introduction of an additional glycine residue, have a detrimental effect on helicase activity. The two structures of the complete NS3 protein indicate that the influence of the helicase domain on the protease function is probably negligible; this underscores the validity of in vitro assays of the isolated protease domain. Chandramouli et al. presented two X-ray structures of the NS3 protease domain in complex with a mutated NS2B fragment, in which the cofactor shows a significantly altered fold (PDB codes 3L6P, 3LKW).27 The significance of these results for drug design remains unclear.

Figure 5. Comparison of the dengue and HCV proteases (3U1I and 4KTC). Note the similar overall fold, but significant discrepancies, particularly in the cofactor (indicated by red arrows) and in the Nterminal part of the protease domains. In the dengue protease, the Nterminal part is partially disordered and localized at the “upper” part of the figure (upper black arrow), whereas in HCV protease, the Nterminal part forms an α-helical region (lower black arrow) of 10 residues that is completely absent in DENV or WNV protease. This graphic was generated using Chimera.145

more detail below. In contrast to other proteases such as trypsin, the binding “pockets” of dengue protease are relatively shallow and superficial and may more aptly be described as “binding regions” or “channels”. 2.2. X-ray Crystallography of Dengue Protease

With dengue protease being a highly attractive target for medicinal chemistry, there is considerable interest in the elucidation of X-ray crystallographic structures that may help guide the rational development of inhibitors. However, albeit being a relatively small protein that can be obtained in good yield from heterologous expression in E. coli cells, the crystallization of dengue protease does not appear to be straightforward. Up to now, only two structures with bound inhibitors were reported. 11351

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Table 1. Three-Dimensional Structures of Dengue Protease Solved by X-ray Crystallography, Sorted by Date of Publicationa PDB code

construct and mutations

serotype

resolution [Å]

ligand

remarks

pH

ref

2FOM

NS2B(H)-gly-NS3pro

2

1.5

none

NS2B cofactor present and mostly defined, but does not contribute to active site or substrate recognition pockets

8.5

24

2VBC

NS3 helicase and protease domains, shortened NS2B fragment

4

3.15

none

full-length NS3 with protease and helicase domains; closely related to 2WHX and 2WQZ; NS2B does not contribute to substrate recognition site

6.5

25

2WHX

NS3 helicase and protease domains, shortened NS2B fragment

4

2.2

none

identical construct to 2VBC, alternative conformation indicates significant flexibility between the protease and helicase domains; NS2B only partially resolved, without contribution to substrate binding site

7.4

26

2WZQ

NS3 helicase and protease domains, shortened NS2B fragment, mutant NS2B(H)-gly-NS3pro with N-terminal deletion in NS3pro NS2B(H)-gly-NS3pro, S135A mutant NS2B(H)-gly-NS3pro

4

2.8

none

insertation mutant of 2VBC with additional glycine in position 174 between protease and helicase domains; fold practically identical to 2VBC

1

2.2

none

NS2B does not contribute to substrate recognition site; NS2B forms peripheral loop

7.5

27

1

2.0

none

active site mutant of 3L6P: S135A; structure highly similar to 3L6P

7.5

27

3

2.3

Bz-Nle-LysArg-Arg-H; Arg-Arg

NS2B is resolved and contributes to substrate recognition site; two slightly different monomers in unit cell: tetrapeptide aldehyde covalently bound to S135 of one monomer; Arg-Arg near active site of other monomer

6.8

23

3 2

1.8 1.46−2.7

aprotinin none

similar to 3U1I, but with aprotinin ligand; NS2B only partially resolved 4M9T crystallized in the presence of the thiol-reactive probe DTNB, but no electron density found for the probe

6.5 5.5, 8.5

23 32

3L6P

3LKW 3U1I

3U1J 4M9K, 4M9M, 4M9I, 4M9F, 4M9T 1BEF, 1DF9, 2QID a

NS2B(H)-gly-NS3pro NS2B(H)-gly-NS3pro wild-type and A125C mutant at various pH values fraudulent structures; obsolete

26

these PDB entries are no longer valid, and the corresponding publications were withdrawn

All constructs were expressed in E. coli cells. Note that some of the early structures of dengue protease were later shown to be fraudulent.

Figure 8. Structure of the complete NS3 protein, with protease domain (left, red) and helicase domain (right, orange). PDB code 2WHX. An Nterminal fragment of the NS2B cofactor (blue) was included in the expression construct and the crystallization experiment and is partially resolved. The orientation of this figure differs from the other figures to make the interdomain linker clearly visible. This graphic was generated using Chimera.145

protease and aprotinin.23,28 The 3U1I structure (cf. Figures 2 and 6) is of special significance because it allows, for the first time, the study of the enzyme in its “active” state, with the NS2B cofactor in a position where it participates in substrate recognition. Biochemical data had previously indicated a crucial importance of NS2B for the binding of peptide substrates, but this was not reflected by earlier X-ray structures, in which the cofactor was positioned distant from the active site, or was not resolved. In 3U1I, the cofactor is folded in a significantly different way and nearly surrounds the whole protease domain. A detailed analysis of the binding mode of the tetrapeptide inhibitor reveals only relatively few highly energetic or specific interactions (Figure 9). The most important molecular

Figure 7. 2FOM, the first reported X-ray structure of dengue protease, in comparison with the more recently described 3U1I structure. The 2FOM structure, shown in lighter colors, is unliganded, whereas the 3U1I structure bears a covalently bound tetrapeptide aldehyde in the active site. The presence of the ligand appears to induce a rearrangement of the cofactor, indicated by black arrows, which then contributes to the binding site. This graphic was generated using Chimera.145

Two highly interesting structures were reported by Noble and co-workers in 2012: 3U1I, containing a covalently bound tetrapeptide aldehyde (6); and 3U1J, a complex of dengue 11352

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Figure 10. Crystallographic contacts in 3U1I. Shown in green is a crystallographic neighbor from an adjacent unit cell, and in yellow an Arg-Arg dipeptide that is located close to the active site of the neighbor. The N-terminal norleucine and benzoyl moieties of the tetrapeptide aldehyde inhibitor, shown with gray carbons, interact with protein and ligand atoms from the neighboring unit cell, and not with the “proper” protease domain. This graphic was generated using Chimera.145

Figure 9. Binding mode of the tetrapeptide aldehyde inhibitor to dengue protease (PDB code 3U1I). NS3pro in red, NS2B in blue. The inhibitor is shown in ball/stick representation with gray carbon atoms. The interacting residues of the target are shown in light brown and with oneletter codes. The inhibitor is covalently bound, via a hemiacetal function, to the catalytic serine. This graphic was generated using Chimera.145

relatively weak, particularly when the covalent-reversible binding mode is considered. In contrast, the noncovalent inhibitor aprotinin, cocrystallized with dengue protease in the 3U1J structure, binds to the target with a Ki in the lower nanomolar range (cf. section 4.2 for a detailed discussion).31 In the cocrystal structure of aprotinin and dengue protease, the NS2B cofactor does not participate in ligand recognition and its C-terminal domain is unresolved. Cys14 and Lys15 of aprotinin bind in the S1 and S2 pockets of dengue protease, respectively. The alignment of the aprotinin backbone in this region closely resembles the backbone of the tetrapeptide aldehyde ligand in 3U1I. Cys14 forms a disulfide bridge to Cys38, and this disulfide bridge of aprotinin neatly fits into the S2 specificity pocket in a way similar to that of the arginine residue in P2 of the tetrapeptide aldehyde. Hydrophobic contacts and hydrogen bonds to the protein backbone complete the interaction profile of dengue protease and aprotinin. It is remarkable, particularly with respect to the design of dengue protease inhibitors, that aprotinin achieves significant affinity toward the target with only minor contributions coming from charge−charge interactions. This indicates that the discovery of high-affinity, small molecular inhibitors with acceptable physicochemical properties should be feasible. One factor that may contribute to the high affinity of aprotinin, even in the absence of interaction with the NS2B cofactor, may be that its conformational flexibility is highly restricted. As with HCV protease inhibitors, one key to success in dengue protease inhibitor discovery may be the design of compounds with increased rigidity. Several additional, unliganded structures of the serotype 2 wild-type protease and cysteine mutants were described in 2013.32 These structures closely resemble the earlier 2FOM structure.

recognition events appear to be electrostatic interactions between the P1 side chain and Asp129, and polar interactions between the S2 side chain and Asn152, and perhaps also Asp75 of the catalytic triad. A few hydrogen bonds are formed between the inhibitor and protein backbone atoms, for example, between the P3 lysine and Met84. The B-factors of the ligand indicate a relatively large degree of thermal motion for the N-terminal residues, whereas the P1 arginine is tightly bound in the S1 pocket. The unit cell of the 3U1I structure contains two protease/ cofactor assemblies that are not identical. It is a most remarkable observation that the second assembly does not contain a tetrapeptide aldehyde ligand covalently bound in the active site, but additional electron density near the active site indicates the presence of a smaller ligand. This additional electron density was modeled by the authors with an Arg-Arg dipeptide, occupying the S1 and S2 pockets. Even the noncovalent binding of this small ligand is sufficient to stabilize “closed” conformation of the NS2B cofactor in the second monomer. An analysis of crystallographic contacts between adjacent unit cells of the 3U1I structure reveals that both ligands are involved in contacts between neighboring cells (cf. Figure 10): The P4 norleucine and the benzoyl cap at the N-terminus interact solely with partners from another unit cell, either from protein residues or from the putative Arg-Arg ligand. It appears likely that the position of the P4 norleucine residue and the benzoyl cap in 3U1I do not resemble the binding mode under “dilute” conditions, because these hydrophobic residues are not expected to be solvent-exposed. A certain extent of conformational reorganization in the target and the ligand may lead to a hydrophobic collapse of the structure that is observed in the crystallized state. The putative Arg-Arg ligand, which is not expected to have a high affinity toward the target and has relatively high thermal flexibility (B-factors), may be “held in place” by its crystallographic neighbors. The tetrapeptide aldehyde in the 3U1I structure was reported to bind to dengue protease with a Ki of 5.8 μM.29,30 This appears

2.3. NMR Spectroscopy of Dengue Protease

The published X-ray crystal structures discussed in the section above, in the presence or absence of a cocrystallized inhibitor, differ particularly in the localization of the C-terminal NS2B cofactor domain (often referred to as NS2Bc). In the catalytically active form, this essential cofactor domain is wrapped around the active site of NS3 as a short β-hairpin. This part of the protease11353

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sparsely populated. However, with increasing pH and salt concentrations, a more pronounced conformational exchange can be observed, indicating a crucial relevance of electrostatic interactions for the association of NS2Bc to the active site of NS3pro. Noteworthy, in contrast to the linked construct, which is stable at room temperature over longer incubation times, for the unlinked protease precipitation occurred during the experiments, indicating total dissociation of the complete cofactor (with N-terminal and C-terminal domains) from the protease. This effect was even more pronounced at high ionic strengths. Isolated NS3pro was previously reported to be practically insoluble, and therefore dissociation leads to precipitation of at least the NS3 domain, limiting the value of this construct for enzymatic assays and related purposes. Assignments for spectra of the dengue protease with aprotinin were published by Bi and co-workers.39 However, detailed binding studies and information regarding the conformational state of the protease complex were not presented in this work. Chen and co-workers performed a detailed study on the binding properties of aprotinin toward the unlinked construct of dengue protease using pseudo contact shifts.40 In this study, the “closed” conformation was also confirmed with (highly affine) bound aprotinin. Therefore, the lack of electron density for the Cterminal domain of NS2B observed in the crystal structure (3U1J) seems to be a crystallization artifact. The NMR data suggest an orientation of NS2Bc quite similar to the data presented in the crystal structure 3U1I with cocrystallized tetrapeptide inhibitor. Noteworthy, the NS2B-NS3-aprotinin complex was found to be less prone to sample degradation, indicating a stabilization of the protease cofactor interaction as well as an inhibition of dissociation by aprotinin. Li and coworkers additionally analyzed the binding of aprotinin to a related unlinked construct, underlining the previously discussed results of aprotinin binding to the active site of the closed protease conformation.41 In summary, the NMR-based studies give further indication for a high flexibility of the cofactor domain NS2B. However, it is obvious from various NMR data published in recent years that for both the West Nile and the dengue virus proteases the predominant conformation found in solution is the “closed” state with an NS2Bc β-hairpin wrapped around the catalytically active center of NS3pro, regardless of whether the cofactor and protease domains are covalently linked to each other or whether an inhibitor is present or not. Therefore, the “closed” state is the only relevant template for rational drug discovery. Consequently, the sole available model reflecting this template in a sufficient way is the 3U1I crystal structure solved by Noble and co-workers. However, the results provided by NMR techniques are not only useful for clarifying the structural basis for further applications. As ligand binding is influencing the obtained NMR spectra, this technique can also be considered for studying binding modes of certain ligands as an alternative approach to elaborate cocrystallizations. For the related West Nile virus protease, a recent study pinpointed the binding sites of several peptidic ligands with NMR-based methods.115 Future works should also consider this possibility for research on dengue protease, especially in drug discovery campaigns.

cofactor complex is crucial for substrate recognition and sufficient catalytic efficiency. In the crystal structure 2FOM (DENV-2) without any cocrystallized ligand, a disordered, flexible NS2Bc domain was observed (often referred as “open”), whereas for the cocrystal structure of DENV-3 protease with a tetrapeptidic ligand, the expected localization wrapped around the protease domain was observed (often referred as “closed”). The structure 3U1J (DENV-3) cocrystallized with aprotinin (BPTI) lacked electron density for the C-terminal domain of NS2B, indicating an “open” state with a flexible cofactor. These ambiguous results from elaborate crystallization studies were not able to reveal the relevant conformational state of the protease− cofactor complex. One may speculate about crystallization artifacts (such as crystal contacts, see above) or the artificial protease construct with a permanent, non-natural covalent linkage between the two domains as possible reasons for these results. Therefore, NMR studies were used to identify the predominant conformation in solution for various protease constructs. The first suitable NMR spectra and signal assignments of dengue protease were reported in works studying the commonly employed covalently linked truncated construct NS2B(H)-glyNS3pro.33−36 De la Cruz and co-workers investigated paramagnetic tags on different sites of the protease as well as combinatorial isotope labeling to build a model of the “closed” conformation using a “closed” state structure of the closely related WNV protease as a template. The “closed” state was supposed to be obtained upon ligand binding after a conformational change. The obtained spectra were more suitable for resonance assignments with much more uniform cross-peak intensities as compared to the absence of a ligand. However, the results observed for the latter case were later associated with protein degradation,37 indicating the “closed” state as the most relevant conformation for dengue protease regardless of ligand binding. This is in correlation with the observations made for the WNV protease, where the “closed” conformation was found to be the predominant state in solution in presence or absence of a ligand.34 As the artificial covalently linked protease construct might not properly reflect the biologically relevant model, alternative unlinked constructs were evaluated using NMR techniques. Kim and co-workers investigated an unlinked protease complex, which was catalytically active and suitable for NMR experiments.38 For this construct, disperse cross-peaks (HSQC) were found for the isolated protease without any ligand. Experiments based on paramagnetic relaxation enhancements indicate that the C-terminal cofactor forms a β-hairpin with the protease domain, representing the “closed” state; however, structural details were missing. Chemical shift perturbations were only observed in the presence of a peptidic substrate-based ligand (6), making this construct suitable for protein−ligand interaction studies. Evaluations of the relatively tight-binding ligand 6 toward the linked protease construct partially confirmed the previous observations made by de la Cruz and co-workers in that chemical shift perturbations were observed upon ligand binding. A quite similar, unlinked protease construct with internal cleavage site between NS3pro and NS2B(H) was used to study the protease in detail.37 Using lanthanide tags at various sites of the protease, detailed structural information could be obtained using pseudo contact shifts. The authors confirmed that for both the linked and the unlinked constructs, the “closed” conformation is the most applicable state in solution, appearing also in the absence of a ligand. The “open” conformation is 11354

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3. PROTEASE CONSTRUCTS, SUBSTRATES, AND ASSAYS

peptides with two basic residues in P1 and P2 and additional residues in P3. Noteworthy, chromogenic and fluorogenic substrates with a single arginine residue in P1 are cleaved by the NS3 protease without the cofactor domain,49,50 indicating the hydrophilic core sequence of NS2B as crucial for recognition in the S2 and S3 pockets, which was later confirmed by the cocrystal structure of NS2B-NS3 in complex with a tetrapeptidyl inhibitor (cf. Figure 9).23 Site-specific mutagenesis of the truncated cofactor of serotype 2 identified the residues W62 (highly conserved in all four serotypes), L75, and I79 as critical for NS3 activation.51 The role of the truncated NS2B in substrate binding and interaction with NS3 as well as the catalytic efficiency of protease mutants and similarity analysis for the four serotypes were analyzed in later studies (cf. section 3.2).52−54 Using truncated GST-tagged cofactor domains in combination with glutathione affinity chromatography, the noncovalent binding affinity between the cofactor and NS3pro was studied.50,55 Only in the presence of GST-NS2B(H) a fully catalytically active protease with the usual substrate specificity could be obtained. Using this construct, the binding affinity between NS2B(H) and NS3 could be quantified with a halfsaturation concentration of 177 nM.50 On the basis of the truncated construct, Leung et al. developed a nonapeptide (GGGGSGGGG) linked NS2B(H)-NS3 protease NS2B(H)gly-NS3pro (originally denoted as CF40.gly.NS3pro) for DENV-2 that can be expressed in E. coli culture and is less prone to autocatalytical cleavage, digestion, and precipitation.56,57 The linked construct has significant enzymatic activity and is relatively stable. It therefore became the preferred dengue protease construct and was used in numerous in vitro assay applications such as substrate specificity studies and inhibitor screenings. Biochemical characterization of the construct revealed maximal protease activity at pH 9 in the presence of 20% glycerol.56 Li et al. expressed and analyzed NS2B(H)-gly-NS3pro constructs based on genetic material of all four dengue virus serotypes for the first time.58 A related construct using only 18 residues of NS2B connected via a glycine linker to the full-length NS3 domain was successfully used to solve the crystal structure of the full-length NS3 protein, including the protease and helicase domains, of serotype 4.25,26 The truncated construct introduced by Leung et al. with at least 40 cofactor residues NS2B(H)-gly-NS3pro was also the subject of various structural biology studies, including solution (NMR) analysis (DENV-2) and X-ray crystallography (3L6P, DENV-1; 2FOM, DENV-2; 3U1I, DENV-3). These experiments demonstrated that inhibitor binding to the active site induces a “closed” conformation, which is most likely responsible for substrate binding and cleavage. In the “closed” conformation, the C-terminal part of the NS2B cofactor forms a β-hairpin structure and is wrapped around the NS3 protease domain.23,35 For the crystal structures of serotypes 1 (PDB 3L6P)27 and 2 (PDB code 2FOM)24 without inhibitor or substrate, the “open” conformation was observed, whereas the cocrystal structure of serotype 3 (PDB 3U1I)23 with a substratebased inhibitor is in the “closed” state (section 2.2). NMR experiments suggest a similar conformational change for the linked construct after addition of a nonpeptidic inhibitor.35,36 However, a recent publication indicates that the “open” state observed in solution experiments for the unliganded NS2B(H)gly-NS3pro can be traced back to the presence of degraded protein.37 Therefore, it seems that the closed conformation is the dominant species in solution, regardless of whether a ligand is present or not (section 2.3).

3.1. Protease Constructs Used for in Vitro Applications

Several protease constructs have been described that differ in details such as the type of linker construct between cofactor and protease, length of the cofactor domain, or the type of affinity tag (see Table 2). Some of the differences have a significant impact on ligand and substrate recognition. In this section, we describe the types of in vitro constructs for the expression of a catalytically competent dengue protease, which can be used to characterize potential enzyme inhibitors, including high-throughput screening. We subsumed the different constructs into subclasses and refer to the N-terminal protease domain as NS3pro. The truncated cofactor with a hydrophilic core domain of varied length is denoted as NS2B(H) and the full-length cofactor with additional hydrophobic domains as NS2B(FL). A permanent covalent linkage between the NS2B and NS3 domains based on linkers rich in glycine is denoted as -gly-. Most constructs use His6 affinity tag for purification; therefore, this part of the construct is not specially indicated. If the reader is further interested in the exact appearance of a particular protease construct, including vector design, transformation, expression, and purification procedures, she or he is kindly referred to the particular literature. The viral protease function, which is located N-terminally on the NS3 protein, consists of approximately 185 amino acids. It requires close interaction with a cofactor domain from the neighboring NS2B protein to obtain catalytic activity and selectivity. Initial studies on labeled cell lysates, expressed by recombinant vaccinia viruses, indicated that proteolytic activity (against cleavage sites of nonstructural polyprotein fragments of DENV-4) required simultaneous presence of the NS3 and NS2B domains.42,43 Further studies showed that a hydrophilic sequence of about 40 amino acids from the central region of NS2B is sufficient for activation of NS3 protease in vitro.44 This sequence is commonly denoted as “hydrophilic core sequence” or “cofactor” (CF40, NS2BH, NS2B(H)), in contrast to the fulllength NS2B (NS2B(FL)). An in vitro processing system of the full-length, catalytically active DENV-2 protease obtained by expression with recombinant vaccinia viruses in BHK-21 cells was shown to require microsomal membranes as an anchor for hydrophobic domains of the NS2B cofactor to establish proteolytic activity.45 A full-length NS2B(FL)-NS3 construct including hydrophobic cofactor domains and NS3 helicase domain without reported proteolytic activity and low yield after refolding was obtained from E. coli culture by Champreda and coworkers.46 The same group investigated active dengue NS2B(FL)-NS3pro with full-length NS2B after expression as inclusion bodies from E. coli followed by stepwise dialysis.47,48 The hydrophobic domains of the cofactor, however, are not essential for the proteolytic function. In correlation to previously reported results, the hydrophilic core sequence of NS2B activates the NS3 protease in the absence of microsomal membranes.45 Consequently, Yusof and co-workers reported the expression and isolation of a truncated DENV-2 protease construct from E. coli culture, including the hydrophilic core of NS2B(H) linked via 13 amino acids to the NS3 protease domain, in denaturated form. This could be refolded to obtain the active enzyme after autocatalytic cleavage at the NS2B/NS3 site.49 This protease construct showed proteolytic activity toward the natural substrate (NS4B-NS5 precursor) and synthetic fluorogenic 11355

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Table 2. Dengue Protease NS2B−NS3 Constructs and Substrates Used in Biochemical Assaysa

Review

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The different constructs are subsumed into subclasses, and the N-terminal protease domain is denoted as NS3pro. The truncated cofactor with a hydrophilic core domain of varied length is denoted as NS2B(H) and the full-length cofactor with additional hydrophobic domains as NS2B(FL). A permanent covalent linkage between the NS2B and NS3 domains based on linkers rich in glycine is denoted as -gly-.

a

Table 2. continued

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inhibition by aprotinin.60 Future studies will show whether these novel constructs, which may have more resemblance to the in vivo conditions under which the dengue protease acts within the membrane-anchored replication complex, can replace the wellestablished truncated and covalently connected protease constructs for in vitro studies.

The obvious conformational ambiguity in crystal structures might be an artifact from the covalently linked protease construct, which therefore does not reflect the biologically relevant state of the protein. Thus, NS2B and NS3pro domains, encoded on different plasmids, were coexpressed in E. coli culture to obtain an unlinked protease-cofactor complex for NMR solution experiments.38 Blending of NS2B and NS3pro, obtained by separate expression and purification, did not result in a soluble product due to the insolubility of isolated NS3pro. However, the presence of a 50 amino acids segment of NS2B was able to stabilize NS3pro and yielded a soluble complex, which could be purified with the cofactor domain ligated to His6-tagged NS3pro. NMR studies with the unlinked construct confirmed that the “closed” conformation is the predominant form in solution, independent of ligand presence (cf. section 2.3). Furthermore, this construct can be assayed using physiological conditions with an optimal activity at pH 7.5 without addition of glycerol. These findings were confirmed for a related DENV-2 construct without permanent covalent linkage, where the core domains from NS2B and NS3pro were expressed either by cell-free synthesis or in E. coli culture and subsequent autocatalytical cleavage, without the requirement of refolding.37 The two domains were connected using the natural NS2B/NS3 cleavage site EVKKQR. This dengue protease construct was reported to be slightly more active in substrate processing than the related linked NS2B(H)gly-NS3pro construct. In addition, in vitro protease constructs with the full-length NS2B domain have been reported. These constructs usually require the presence of special detergents, such as phospholipids forming micelles, which provide a membrane-like microenvironment stabilizing the NS2B structure by interaction with the hydrophobic domains of the full-length cofactor.59−61 Chokuspmanee and co-workers reported a fusion protein consisting of full-length NS2B (NS2B(FL)) and NS3pro with the membraneintegrating mstX (mistic) protein from Bacillus subtilis. The mstX-NS2B(FL)-NS3pro fusion protein can be purified in the presence of foscholine-14 detergent. The strong tendency of the resulting protein to autoproteolytic cleavage, which made isolation of the full-length protein difficult, prompted the authors to perform a mutagenesis study in search for autoproteolytical cleavage sites.60 Recently, a construct of full-length NS2B(FL)NS3pro that can be solubly expressed from E. coli culture using detergent micelles without any additional fusion protein was reported.59 For this construct, an in vitro assay with a fluorogenic substrate was developed, using assay conditions (buffer: 10 mM Tris-HCl, pH 8, 20% glycerol, 1 mM CHAPS) that are very similar to the conditions commonly employed for the NS2B(H)gly-NS3pro constructs in many other structural and functional studies (cf. section 3.5). Lyso-myristoylphosphatidylcholine (LMPC) was found to be the most suitable detergent, yielding an active protease despite of the absence of the assay detergent CHAPS. Foscholine-14 and foscholine-15 micelles also increased protease activity, however, only in the presence of CHAPS in assay buffer. A comparative inhibition analysis for the broadspectrum serine protease inhibitor aprotinin (BPTI) of the NS2B(FL)-NS3pro construct with the conventional truncated cofactor NS2B(H) showed a significant lower inhibition potential of aprotinin against the full-length NS2B construct. The NS2B(FL)-NS3pro construct remained active in the presence of 5 μM aprotinin, whereas no activity at 0.5 μM aprotinin was found for the truncated construct.59 In analogy, the autoproteolytic digestion of the full-length construct obtained via mstX-fusion construct in micelles was equally resistant toward

3.2. Mutagenesis Studies

Valle and Falgout examined the influence of mutagenesis of the dengue virus type 2 NS3 protease on its self-cleavage by generating 46 single-amino-acid substitutions in a cloned NS2BNS3 cDNA fragment of dengue virus type 2, which were assayed in vivo. The NS3 region chosen for the mutational analysis contained the catalytic Ser135 residue, five putative substrate binding residues, and several highly conserved residues among flaviviral and other serine proteases. Of these mutations, 12 (Thr134Asp, Ser135Ala, Ser135Cys, Gly148Ala, Leu149Ala, Leu149Arg, Tyr150Ala, Tyr150Val, Tyr150His, Gly151Ala, Gly153Ala, Gly153Val) yielded a complete or almost complete inhibition, and nine (Asp129Lys, Asp129Arg, Asp129Leu, Phe130Ala, Phe130Leu, Gly133Ala, Gly136Ala, Asn152Ala, Asn152Gln) caused a significant reduction of the protease activity; furthermore, 14 mutations (Val126Ala, Asp129Glu, Asp129Ser, Asp129Ala, Phe130Tyr, Thr134Ala, Ile139Leu, Ile139Ala, Ile140Ala, Gly144Pro, Leu149Ile, Tyr150Phe, Val154Ala, Val155Ala) decreased the cleavage of the enzyme moderately, and 11 mutations (Ser131Pro, Ser131Cys, Ile140Leu, Asp141Glu, Asp141Ala, Lys142Ala, Lys142Asn, Lys143Ala, Lys143Asn, Gly144Ala, Arg184Ala) led to similar protease activities as compared to the wild type.62 In 2004, the group of Katzenmeier examined the influence of six different residues within the NS2B cofactor on the catalytic activity of the dengue virus type 2 NS2B-NS3 protease by generating six single alanine mutants Trp62Ala, Ser71Ala, Leu75Ala, Ile77Ala, Thr78Ala, Ile79Ala and a Leu75Ala/ Ile79Ala double mutant. The replacement of the residues Leu75, Ile77, and Ile79 by alanine resulted in a decreased efficiency of autoproteolysis as compared to the wild type, whereas for the Trp62Ala and Leu75Ala/Ile79Ala mutants no self-cleavage could be detected at all, indicating that the conserved residues Trp62, Leu75, and Ile79 of the NS2B cofactor play an essential and critical role for the activation of the dengue virus NS3 protease.51 A further study was published in 2010 by the same group with the difference that alanine replaced individual residues located in the S1 and S2 binding pockets of the dengue virus NS3 protease. From the mutants created (Leu115Ala, Asp129Ala, Gly133Ala, Thr134Ala, Tyr150Ala, Gly151Ala, Asn152Ala, Ser163Ala, and Ile165Ala), only the Leu115Ala mutant showed a catalytic activity comparable to that of the wild-type enzyme, whereas the activity of the other mutants either decreased (e.g., Gly133Ala) or disappeared (Tyr150Ala, Gly151Ala). The results of this study confirmed predictions for the active site conformation based on earlier observations.52 The mechanism of the NS2B-mediated activation of the dengue virus NS3 protease and substrate binding was further analyzed by Zuo et al. by using several methods of molecular dynamics simulation, mutagenesis, and bioassays. Three different single and double mutants, Asp50Ala/Glu92Ala, Gln35Gly, and Gln27Gly/Arg54Gly, were screened to obtain important parameters of enzyme kinetics (kcat, Km, kcat/Km) as compared to those of the wild-type enzyme (WT), resulting in lower catalytic efficiency for the mutants as compared to the wild type, 11358

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Table 3. Important Natural Cleavage Sites of the Dengue Virus Protease for Essential Polypeptide Processinga serotype

capsid C

NS2A/NS2B

NS2B/NS3

NS3/NS4A

NS4B/NS5

DENV-1 DENV-2 DENV-3 DENV-4

RRKR↓SVTM RRRR↓XAGXb KRKK↓TSLC GRKR↓STIT

WGRK↓SWPL SKKR↓SWPL LKRR↓SWPL ASRR↓SWPL

KKQR↓SGVL KKQR↓AGVL QTQR↓SGVL KTQR↓SGAL

AGRR↓SVSG AGRK↓SLTL AGRK↓SIAL SGRK↓SITL

GGRR↓GTGA XTRR↓GTGNb TGKR↓GTGS TPRR↓GTGT

a

Sequences of cleavage sites reported regarding a literature selection.63,71−73,75 For different isolated viral strains with shifted genotypes, several slightly different sequences for the cleavage sites have been reported. bFor this cleavage site, different amino acids for the residue X are reported in the literature selection.

Table 4. Substrates Investigated in Early Studies by Yusof and Coworkers with Comparison of Kinetic Parameters for NS2B(H)NS3pro and NS3pro in the Absence of the Cofactor Domain49 kcat (s−1)

Km (μM)

a

kcat/Km (s−1 M−1)

substrate

NS3pro

NS2B(H)-NS3pro

NS3pro

NS2B(H)-NS3pro

NS3pro

NS2B(H)-NS3pro

Boc-GRR-AMC Boc-QRR-AMC Cbz-RR-AMC pGlu-RTKR-AMCa

82.3 128 50 85

180 93 102 134

4.3 × 10−6 1.8 × 10−6 1.3 × 10−6 2.7 × 10−6

0.031 0.01 0.009 0.013

0.052 0.014 0.026 0.03

172 107 88 97

pGlu = L-pyroglutamic acid.

with an arginine in P1 and a glutamine residue in P2, highly conserved in all four serotypes. For this important cleavage site, studies with synthetic peptides showed lower catalytic activity in vitro as compared to the other cleavage sites, which might result from the suboptimal P2 residue.48,56,58 Additional cleavage sites were identified in the other domains of the viral polyprotein, which often possess two or at least one basic residue(s) in P1 and P2. Examples are the internal cleavage sites within the NS3, NS2A, and NS4A domains as well as the prM/M cleavage region, with arginine (P1) and lysine (P2) residues of the latter being conserved in all four serotypes.63,72,74,75 However, although catalytic activity of the dengue protease is reported against this site, the prM/M cleavage is usually processed by the host protease furin just prior to the release of the virions from the host cell.69,74−76 Notably, all P1′ residues of the cleavage sites shown in Table 3 are small and neutral (Ser, Gly, Thr, Ala). Substrate specificity was studied both with synthetic peptides, preliminarily investigated as substrates for assay purposes, and with peptidic inhibitors. The latter often contained electrophiles that covalently target the dengue virus protease (cf. section 4.2). Both approaches confirmed the distinct preference for dibasic residues in P1 and P2 as assumed from the natural cleavage sites (Table 3). Herein, we review the substrate-related approaches. For a detailed discussion of substrate-based inhibitors, the reader is kindly referred to section 4.2. In addition to the dibasic P1/P2 essentiality, the preferences for other positions like small uncharged residues for the prime region can be deduced from the polyprotein cleavage sites, as mentioned above. However, more effort was needed to analyze the preferences for other positions between P4 and P4′. In early studies, Yusof and co-workers described substrates bearing two arginine residues in P1 and P2 as well as a glutamine or glycine in P3 with sufficient activity for the autocatalytically cleaved truncated NS2B(H)-NS3pro (DENV-2) construct.49 The authors compared the kinetics of four substrates at this construct with the NS3pro in the absence of the cofactor domain (Table 4). Noteworthy, they observed minor differences in Km values but an almost total loss of catalytic activity (kcat) for NS3pro as compared to the NS2B(H)-NS3pro construct, indicating that NS2B is not only relevant for substrate recognition but also for adequate catalytic efficiency. Regarding the kcat values of the

however, with no statistically significant difference of the values between the mutants. Therefore, the distinct influence of the particularly analyzed residues toward enzymatic activity is hard to deduce.53 Yildiz et al. identified regions of the dengue virus NS2B-NS3 protease, which are sensitive to allosteric inhibition with the help of cysteine mutagenesis and cysteine reactive probes. Eight mutant enzymes in which cysteine replaced certain individual residues were created and evaluated according to their kinetic parameters in contrast to those of the wild-type enzyme: Glu19Cys, Leu31Cys, Trp83Cys, Glu86Cys, Asn105Cys, Thr111Cys, Leu115Cys, and Ala125Cys. Most of these cysteine substitutions did not affect the catalytic activity of the protease, whereas the replacement of Trp83, Thr111, and Leu115 led to a total loss of activity. On the one hand, the Trp83 residue is completely buried, suggesting that the formation of a cavity and the destabilization of the folded form of NS2B-NS3pro may be responsible for this; on the other hand, the solvent-exposed Thr111 and Leu115 residues were not expected for their loss of function. The incubation of each cysteine mutant enzyme with cysteine reactive probes indicated that the allosterically sensitive site is located at the Ala125 residue, between two loops (120s and 150s) of the NS2B-NS3pro.32 3.3. Substrate Specificity

The most important natural cleavage sites for the processing of the viral polyprotein by dengue protease are shown in Table 3. A few cleavage sites have been shown to be processed by the host proteases such as signalase or furin, as observed for related flaviviruses.43,63−68 The viral protease cleavages are performed at the cytoplasmic side of the endoplasmatic reticulum membrane, whereas the host proteases process the polyprotein inside the endoplasmatic reticulum, except furin cleavage, which is most likely performed within the trans-Golgi network. The fully active NS3 protease connected to the relevant residues of NS2B has a strong preference for substrates with dibasic sequences in P1 and P2. For all four dengue serotypes, the primarily identified polyprotein cleavage sites, in the capsid protein (C) and between the nonstructural proteins 2A/2B, 3/ 4A, and 4B/5, contain basic residues in P1 and P2.63,69−73 The cleavage site between NS2B and NS3 deviates from this finding 11359

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substrates shown in Table 4, the GRR sequence containing a glycine residue in P3 showed the highest catalytic efficiency. For the glutamine (P3) analogue, significantly lower efficiency values were observed. Although this study was important to reveal the essential role of the cofactor domain for sufficient catalytic efficiency using synthetic peptide substrates, no significant consequences regarding structure−activity relationships can be drawn due to an insufficient number of analyzed peptides. Leung et al. discovered substrates for a similar nonlinked construct and the covalently linked protease system NS2B(H)gly-NS3pro (DENV-2) based on the natural cleavage sites. They found significantly higher catalytic activity for the latter (linked) construct, which is not prone to autocatalytical cleavage.56 Synthetic peptide inhibitors published in the same work (see section 4.2 for a detailed discussion) showed promising affinities with the nonprime sequence AGRR (P4−P1). The investigated substrates contained the P6−P1 residues of four cleavage sites (NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5) with mainly basic residues in P1 and P2 (Table 3). The sequences AcTTSTRR-pNA and Ac-EVKKQR-pNA, corresponding to the Cterminal part of the NS4B/NS5 and NS2B/NS3 cleavage sites, were processed most rapidly, giving the following ranking for kcat: 4B/5 > 2B/3 ≫ 2A/2B ≈ 3/4A. A different order was obtained for Km: 2B/3 ≫ 4B/5 ≫ 2A/B ≈ 3/4A. The relatively high Km values for the NS2B/NS3 cleavage site-based substrates might be related to the glutamine in P2 instead of a more privileged basic arginine or lysine residue. Noteworthy, these rankings were consistently obtained for both tested protease constructs NS2B(H)-NS3pro and NS2B(H)-gly-NS3pro. However, for the final catalytic efficiency (kcat/Km), the latter construct gave much higher values. These early studies focused on the protease construct validation as well as the identification of single, suitable sequences for assay purposes and were followed by reports on the systematic variation of the recognition sequence, with particular emphasis on the P1−P4 positions. A thorough analysis of synthetic substrates and their specificity considering all four serotypes was reported by Li and co-workers, using a covalently linked protease construct.58 More than 130 000 tetrapeptides (Ac-XXXX-ACMC) representing all possible amino acid combinations (with the exception of cysteine and methionine, but including norleucine (n)) for the four nonprime residues P1−P4 were analyzed, clearly confirming the preference for dibasic residues in P1 and P2 with higher preference for an arginine or lysine residue in P1. Although for P2 a clear preference for arginine was shown, a weaker priority for lysine was observed for this position. For an arginine in P1 the following preference ranking was achieved: R > T > Q/N/K for P2, K > R > N for P3, and n > L > K for P4, resulting in nKRR as the most preferred nonprime sequence for all four serotypes. This work clearly indicated that the P3 position contributes to substrate binding, because a change from the preferred lysine to the suboptimal threonine in P3 leads to a 3−10-fold decrease of Km (depending on the serotype). Steady-state kinetic parameters (DENV-2) for the identified Bz-nKRR-ACMC substrate and threonine-substituted analogues are given in Table 5. Regarding these kinetic results, the P2 amino acid is important for the substrate ground-state binding, which is shown by the increased Km value of the substrate Bz-nKTRACMC as compared to Bz-nKRR-ACMC. A suboptimal P3 substitution, such as in Bz-nTRR-ACMC, not only led to increased Km, but also to a significantly decreased kcat value. A threonine substitution in P4 (Bz-TKRR-ACMC) resulted in similar Km, but caused a significant decrease of kcat, indicating a

Table 5. Steady-State Kinetic Parameters of Tetrapeptidic Substrates Investigated by Li and Coworkers (DENV-2)58 substrate

Km (μM)

kcat (s−1)

kcat/Km (s−1 M−1)

Bz-nKRR-ACMC Bz-nKTR-ACMC Bz-nTRR-ACMC Bz-TKRR-ACMC Bz-TTRR-ACMC

12 34 46 11 76

1.4 1.4 0.76 0.20 0.17

112 100 40 300 16 700 18 300 2 200

minor importance of the P4 residue for ground-state binding, however, a crucial relevance for obtaining high turnover rates. An exchange of both P3 and P4 amino acids (Bz-TTRR-ACMC) consequently affected both Km and kcat and finally resulted in a crucial loss of catalytic efficiency. The enzymological properties (Km, kcat) within the spectrum of dengue serotypes do not differ significantly (Table 2), arguing for a highly conserved active site and other recognition elements of the protease in the four serotypes. However, for serotype 4 the highest catalytic efficiencies were observed for the optimal substrate Bz-nKRRACMC (380 000 s−1 M−1) as well as for suboptimal analogues shown in Table 5. On the basis of the optimized nonprime sequence, an analogous screening of more than 130 000 variants for the prime region (nKRRXXXX) was performed: P1′ and P3′ are preferentially small and polar residues, in particular serine.58 P2′ and P4′ appear to have even less relevance for substrate recognition. These results indicate a minor importance of the prime position residues between P4′ and P1′ for molecular recognition. No significant difference in serotype specificity was found for the P1′−P4′ positions. Another combinatorial approach to reveal the substrate specificity of dengue protease was executed using a slightly different autocatalytically cleaved protease construct (NS2B(H)NS3pro) without permanent covalent linkage.77 Starting from P4−P4′ octapeptides or substrates with more than eight residues derived from the natural cleavage sites (Table 3), the authors attempted to identify a minimum optimal substrate size for assay applications. The decapeptide sequence FAAGRKSLTL derived from the NS3/NS4A cleavage site (P6−P4′) was used as an initial point to derive different peptide frames by systematic truncations in the nonprime and prime regions. Studies for this cleavage site showed that, in correlation to the previously discussed results, four nonprime residues seem to be optimal for catalytic activity. A higher number of nonprime residues resulted in slightly higher catalytic rates, albeit accompanied by lower binding affinities (higher Km values). For the prime region the results suggest that the residues P1′−P3′ are most important, because the best catalytic efficiencies were obtained for substrates bearing these residues. The removal of prime residues within the FAAGRKXXXX and AGRKXXXX peptide frames resulted in decreased kcat values. However, identical Km values, especially in the case of the AGRK-based frame, were observed, indicating less importance of the prime residues for ground-state binding. The introduction of an arginine residue at P3 in the sequence AGRKSLTL, giving the peptide ARRKSLTL with a 4-fold higher kcat/Km ratio, is supportive for the importance of a basic residue in P3 as previously observed by Li and co-workers.58 The analysis of substrate preferences within the nonprime region proposed that a sequence ranging from P1 to P4 should be sufficient for a majority of binding interactions with the protease, because the substrates of the AGRK (P1−P4) frame showed 3-fold lower Km values than those of the FAAGRK (P1−P6) frame. For the capsid 11360

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Table 6. Dipeptidic Fluorogenic Substrates of the Sequence Bz-XR-AMC with Kinetic Parameters Evaluated at the NS2B(H)-glyNS3pro Construct of Serotype 275

prime site of the internal capsid cleavage site (KKQRSAGM) showed the highest turnover rate (kcat of approximately 0.1 s−1) within all investigated peptides. A ranking of the native polyprotein cleavage site substrates according to their catalytic efficiencies gave the following order: C ≫ 2B/3 > 2A/2B > 3A/ 4A > 4B/5. In summary, Niyomrattanakit and co-workers77 suggest heptapeptides as the preferred size for synthetic dengue protease substrates. From systematic variations of the peptide length and the sequences around the scissile bond for revealing the substrate specificity of dengue protease, they demonstrated: (i) that the preferred residues range from P4 to P3′, (ii) that the presence of basic side chains at P3 and P4 is beneficial for substrate recognition in the nonprime region and, (iii) that serine at P1′ is the preferred substrate residue in the prime region. However, as compared to the work of Li et al.58 discussed before, these conclusions are drawn from a relatively small number of analyzed peptides that are limited on the natural cleavage sites for the nonprime residues. An additional later study of various prime region sequences was performed by Prusis and co-workers for all four

cleavage sequence (RRRRSAGM, DENV-2, Table 3), an optimal kcat/Km coefficient was found with the sequence RRRRSAG. Each additional prime residue to the nonprime sequence RRRR (P4−P1) resulted in lower Km (higher binding affinity) and higher kcat values until the addition of the P4′ methionine, which led to a slightly improved Km but a much lower kcat. Niyomrattanakit et al. also studied the effect of various residues that are not present in natural cleavage sites in the prime position.77 The data suggest that nonprime residues influence the Km values, with the sequence RRRR (P1−P4) identified as the most active one, whereas the prime region has a greater impact on the rate constants (kcat). The most efficiently cleaved substrate sequence in the analyzed series was RRRRSAG (kcat/Km = 11 087 M−1 s−1). In contrast, Li and co-workers58 (see above) found higher catalytic efficiency for substrates based on the nonprime residues nKRR for the same serotype (Table 2). An NS3-NS5 cleavage site hybrid (AGRKGTGN), combining the nonprime residues of the NS3/NS4A site with the prime region of the NS4B/NS5 site, was identified as the substrate with the weakest kcat/Km ratio of approximately 20 M−1 s−1, whereas a hybrid substrate combining the nonprime site of NS2B/NS3 with the 11361

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serotypes.78,79 For the nonprime sequence RRRR, 56 peptides with modified prime sequences were analyzed (RRRRXXXX), resulting in the finding that residues P1′ and P2′ are influencing kcat values, whereas all four prime residues affect Km, supporting the previously discussed results. The cleavage preference of P1′/P2′ for DENV-2 and related flaviviral proteases was further studied by Shiryaev et al.71 They analyzed peptides of the general sequence GLKRXXAK (P4− P4′) and observed a flat structure−activity relationship without any significant preferences in P1′/P2′ for this particular sequence. From a screening of 30 substrates, Gouvea and co-workers discovered the hexapeptide AKRRSQ (P4−P2′) with a catalytic efficiency in the same range as for the quite similar prime region substrate nKRR discovered by Li and co-workers for the identical NS2B(H)-gly-NS3pro construct of DENV-2.75 These two works independently identified a consensus sequence KRR for P3−P1 and a nonpolar aliphatic residue in P4 (norleucine or alanine). Benzoyl-capped dipeptides with an arginine residue in P1 and various arginine mimetics in P2 were analyzed toward their enzymatic cleavability (Table 6).75 Three substitutions are noteworthy, also in the context of inhibitor design, because these side chains yielded higher kcat/Km coefficients than the native arginine residue. These are the 4-aminomethylphenylalanine (Amf), 4-aminocyclohexylalanine (Aca), and trans-4-aminomethylcyclohexylalanine (Ama) derivatives, with the highest kcat/Km ratio observed for the latter. All three noncanonical amino acids comprise an unsubstituted “free” amine functionality, obviously acting as a suitable replacement for a basic canonical side chain. Interestingly, the para-guanidinylphenylalanine derivative did not show relevant cleavability in this study. However, this side chain was identified as a suitable arginine mimetic for P1 in the peptide-based inhibitor Bz-nKRX-H in a related work using a similar protease construct (cf. section 4.2).30 Although the discussed studies showed slightly different results in certain details, a clear preference for basic residues, especially arginines, for the nonprime substrate region was clearly identified. The prime residues are less important for molecular recognition; however, catalytic efficiency can be improved if small and uncharged amino acids are present at the prime positions.

HPLC assay of dengue protease was described as well, using Nterminal 2-aminobenzoic acid as a fluorescence donor and Cterminal 3-nitrotyrosin as an internal quencher function.81 HPLC-based assays are relatively time- and materialconsuming methods and consequently not suitable for highthroughput screening campaigns that are frequently performed in the early stages of drug discovery. However, HPLC-based systems must be considered as orthogonal methods for the characterization of initial screening hits to exclude false positive candidates, which can appear due to inner filter effects from fluorogenic substrates.81,82 Because most substrates employed for homogeneous assays comprise chromogenic or fluorogenic groups, they can be used additionally for an HPLC-based assay system in a very suitable way to validate the screening results. In homogeneous assays of dengue protease, two types of substrates are used for screening and assay purposes: (i) Peptides that bear a non-natural chromogenic or fluorogenic moiety in P1′. Upon enzymatic cleavage, these substrates yield a product with pronounced absorption in the UV/vis range or fluorescent properties. (ii) Peptides with non-natural residues at the N- and C-termini that constitute an internally quenched system due to fluorescence (Förster) resonance energy transfer (FRET). Cleavage restores the fluorescence of the fluorophore at one of the termini. In the first group of substrates, the fluorophore or chromophore is located in P1′ near the cleavage site. This is not compatible with the preference for small/noncharged amino acids in P1′, and the catalytic efficiency with which these substrates are cleaved can be inferior to the natural substrates. In contrast, FRET substrates provide a considerably longer substrate sequence (e.g., P4−P4′), resulting in high-affinity substrate recognition and efficient cleavage. Chromogenic substrates have been described with the paranitroanilide (pNA) or thiobenzyl ester (SBzl) moieties in P1′. The latter is used in the presence of 4,4′-dithiodipyridine in assay buffer to allow the formation of the chromophore SBzlthiopyridine after enzymatic thioester cleavage.58 For the substrate Bz-nKRR-SBzl, the highest catalytic activity of all investigated dengue virus substrates was observed with a kcat/Km coefficient of 50 200 000 M−1 s−1 for DENV-4 protease. This is predominantly due to the faster cleavage of the thioester bond as compared to an amide bond, resulting in an exceptionally high rate constant (300 s−1).58 If the amide bond between the peptidic substrate and the strongly yellow colored para-nitroaniline moiety is cleaved, the chromogenic reaction can be followed using spectrophotometry.49,56,83−85 The pNA-based substrate with the best reported catalytic efficiency was investigated by Leung and co-workers (Ac-RTSKKR-pNA), representing the NS2A/NS2B cleavage site of serotype 2 with a kcat/Km of 903 and 191 M−1 s−1 for the NS2B(H)-gly-NS3pro and the NS2B(H)NS3pro constructs, respectively.56 As compared to the related UV/vis-active substrate (kinetically analyzed using HPLC) Dansyl-RTSKKRSWPLNE (kcat/Km = 6.3 M−1 s−1), which represents additional prime residues of the same cleavage site, the catalytic efficiency of the nonprime site pNA substrate is quite convincing.48 The coumarin derivatives AMC and ACMC are widely used as P1′ moieties in fluorogenic substrates. A detectable fluorescence signal is induced by cleavage from the P1 position of the nonprime site substrate.29−31,36−38,41,49−54,58,71,74,75,86−108,133 Benzoyl capped substrates with a C-terminal fluorescent coumarin-derived moiety were intensively analyzed by Li and co-workers with the highest catalytic efficiency for Bz-nKRR-

3.4. Substrates for Enzymatic Assays

On the basis of the available, comprehensive insights on substrate specificity of dengue protease, synthetic substrates with chromoor fluorogenic properties amenable for screening purposes were designed and investigated. These substrates comprise additional functionalities for signal detection by fluorescence or absorbance in the UV/vis range in assay systems that involve continuous monitoring or end point analysis. The latter may involve chromatographic separation of the products. Substrates for HPLC-based assays do not strictly require any chromo/ fluorogenic functions.80 However, quantitation of the products becomes difficult when chromophoric, aromatic groups (Phe, Trp, His, or Tyr) are absent in the substrate and cleavage products, as for most of the dengue protease cleavage sites shown in Table 3. Also, signal separation becomes challenging if highly heterogeneous mixtures are analyzed, as in the screening of crude natural products such as plant extracts. Therefore, fluorogenic moieties are often included in the substrates, for example, an Nterminal 5-(dimethylamino)naphthalene-1-sulfonyl (Dansyl) substituent to address this caveat. The Dansyl group can be used in UV/vis-absorption and fluorescence-based HPLCapplications in parallel.47,48 A FRET-based substrate for an 11362

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ACMC (kcat/Km = 51 700−380 000 M−1 s−1, depending on the serotype) for all four serotypes.58 To our knowledge, the ACMC group was not used in other dengue protease assays, but the use of several closely related Bz-nKRR-AMC substrates is frequently reported, however, with a lower catalytic efficiency (Table 2). The TTNTRR-AMC substrate, derived from the NS4B/NS5 cleavage site, showed very high catalytic efficiency using an alternative coexpressed DENV-2 protease construct without covalent linkage (kcat/Km = 147 910 M−1 s−1) and is therefore one of the substrates with highest reported efficiency for dengue serotype 2 protease (Table 2).38 Although this substrate sequence shows convincing catalytic activity, closely related compounds with a serine instead of an asparagine in P4 as well as partially additional nonprime residues reveal significantly lower efficiency performances, for example, Ac-TTSTRR-pNA (kcat/ Km = 883 M−1 s−1 for NS2B(H)-gly-NS3pro and 275 M−1 s−1 for NS2B(H)-NS3pro)56 and Dansyl-TTSTRRGTGNIG (kcat/Km = 10.9 M−1 s−1).48 The high efficiency of the substrate reported by Kim and co-workers might be a consequence of the unlinked coexpressed protease construct, which may support a higher cleavage rate.38 However, Leung and co-workers previously observed higher cleavage rates for the glycine linked construct NS2B(H)-gly-NS3pro, as compared to the related NS2B(H)NS3pro.56 With respect to Table 2, the two most frequently used substrates for assay purposes are the fluorescent substrates BznKRR-AMC, representing the “best” identified sequence from the comprehensive peptide screening of Li and co-workers,58 and Boc-GRR-AMC, which is much less efficiently cleaved but enjoys significant popularity due to its broad commercial availability. For the latter, catalytic efficiencies (kcat/Km) between 21.4 and 2905 M−1 s−1 (DENV-2) have been reported for various protease constructs and assay conditions, indicating that the substrate character is not the only relevant parameter for an effective in vitro assay system. Therefore, the most reliable way for comparison was given by Li and co-workers,58 who analyzed both substrates using the same conditions for all four dengue virus serotypes. The kcat/Km values for the Bz-nKRR-AMC substrate were 75−1000-fold higher than those for the BocGRR-AMC substrate, indicating a lower catalytic efficiency for the latter one. FRET substrates have also been used widely to characterize dengue protease. The following donor/acceptor pairs were described: 2-Abz/(3-NO3)-Y,77−79,81,109−114,116,134 Edans/ Dabcyl,53,80,117 5-TAMRA/QXL570,85 2-Abz/EDDnp,75 and methoxycoumarin/dinitrophenyl.58 Donor and acceptor groups can be placed arbitrarily on the N- or C-termini of the substrate. Both cases of a C-terminal donor combined with an N-terminal quencher and a C-terminal quencher combined with an Nterminal donor can be found in dengue protease-related literature. FRET substrates with high catalytic efficiency are 2Abz-RRRRSAG-(3-NO3)-Y and 2-Abz-AKRRSQEDDnp.75,77−79 However, the highest activity was observed for the Dabcyl-KQRRGRIE-Edans substrate with an exceptionally high rate constant kcat of 42.5 s−1 as well as high binding affinity (Km = 7.17 μM).53 FRET-based substrates were of particular importance for the enzymological evaluation of the prime positions.58,75,77,78 FRET substrates were also used to study the cleavage preference and solvation properties of various potential substrates connected to solid supporters.116 For dengue protease, a high selectivity for basic residues in P1 and P2 (also Q or Y were found to be tolerated) was found, in correlation with the results from other works discussed above.116

As in all fluorescence-based homogeneous assays, inner filter effects of the screened compounds that may lead to false-positive results must be considered for FRET-based assays,81 and correction factors should be applied.82 In summary, highly diverse substrates of the dengue protease were described over the last years. The benefits and disadvantages of these substrates vary and must be considered and addressed with respect to the particular biochemical or bioanalytical question. 3.5. Assay Conditions

As mentioned above for the construct mostly used for assay purposes NS2B(H)-gly-NS3pro (CF40.gly.NS3pro), the inventors identified pH 9 and 20% glycerol as conditions for optimal catalytic activity.56 Yusof et al. reported for the NS2B(H)-NS3pro construct without permanent linkage, which was frequently used particularly in earlier works on dengue protease, a maximal enzymatic activity between pH 9 and 9.5.49 Consequently, activity and inhibition assays summarized in Table 2 use slightly basic conditions between pH 7.5 and 9.5, with a clear preference for pH 9.0. Yusof and co-workers found a better protease stability over longer incubation times with Tris buffer as compared to HEPES.49 Therefore, with two exceptions where HEPES was evaluated,56,72 all assay buffers used Tris for pH settings ranging from 10 to 200 mM. The addition of sodium chloride leads to a nonlinear decrease of dengue protease activity for different constructs, indicating a low ionic strength tolerance.38,49,56 However, a minority of reported assay procedures use sodium chloride in assay buffer ranging from 3 to 50 mM. Glycerol is a commonly used ingredient in enzymatic assays, moderating hydrogen bonds and lipophilic interactions between the protein, substrate, inhibitor, or solvent as well as for general protein stabilization by cosmotropic properties.118,119 Therefore, concentrations between 5% and 35% glycerol have been used in dengue virus protease assays. However, a minority of works also used glycerol-free conditions. Because glycerol can cause problems due to its high viscosity and possible aggregation supporting properties, various alternative polyols have been studied.109 It was found that ethylene glycol and propylene glycol are able to increase the protease activity in the same concentration-dependent manner up to 30% as similarly found for glycerol. However, the viscosity, especially for ethylene glycol, is significantly lower, resulting in a proper substitute for glycerol in high-throughput screenings. The use of 10% ethylene glycol in assay buffers was, however, only reported by one research group;81,109−114,134 all other groups used glycerol or even no polyol. In approximately one-half of the reported assay conditions, detergents have been used as additives. Because NS3pro is supposed to be membrane associated in the biological relevant model, various detergents have been studied to increase the proteolytic activity and to reduce the undesirable identifications of promiscuous inhibitors, for example, due to aggregation.120 Leung et al. analyzed the effect of the detergents Triton X-100, Brij 35, and CHAPS, all increasing the enzymatic activity.56 Steuer and co-workers studied various nonionic detergents with influence on the catalytic activity.109 At low concentrations well below the critical micelle concentration, a remarkable protease activity difference between the analyzed detergents was found. Brij 58, Lubrol, and Triton X-100 gave the most promising results. In combination with glycerol or ethylene glycol, the proteolytic activity was further improved.109 Although it was 11363

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factors complicate the comparison of affinity measures from different test series. Reported affinity values for aprotinin, a readily available reference inhibitor of dengue protease, vary significantly (see below). The interested reader is therefore advised to exercise caution with respect to interseries and interlaboratory comparisons. Inhibitors of dengue protease frequently bind to the enzyme− substrate complex or interfere with the interaction between the NS3pro and NS2B-cofactor domains. Therefore, the binding mode is often not strictly competitive but either uncompetitive, mixed (competitive/uncompetitive), or noncompetitive. In numerous publications, however, the binding mode is not analyzed and characterized with respect to uncompetitive or noncompetitive components. Whenever reported in the literature, we provide details on the binding mode and both Ki (competitive, noncompetitive) and Ki′ (uncompetitive) binding constants. Similar caveats must be applied to the results of docking simulations, where various dengue and West Nile virus protease constructs and homology models with significantly different tertiary and quaternary structures of the protease have been used as targets/receptors for docking. With respect to the assay systems employed, it must be emphasized that colored, fluorescent, or UV-absorbing test compounds are prone to interfere with homogeneous, fluorescence-based assays. These types of assays are frequently used as a first screening method, and it is strongly suggested to verify the results of promising hits by an orthogonal type of assay, for example, one that involves chromatographic separation of product, substrate, and inhibitor.81 Artifacts of this type are particularly frequent in the context of initial screening, when compounds are tested at relatively high concentrations in the 25−50 μM range. Verification by HPLC or other methods, however, remains unreported in a considerable number of publications. Because of space limitations, we cannot provide all details on the employed assay procedures along with all inhibitors and publications reviewed herein. An overview of assay conditions and substrates used for inhibitor screenings is provided in Table 2. We strongly suggest that the interested reader refers to the original literature if she or he is interested in a particular class of compounds, to verify the respective method(s) of protease expression, enzymatic screening, or antiviral activity assay in cell culture.

shown that nonionic detergents gave satisfying results, so far, in most assays the zwitterionic detergent CHAPS has been used. However, for inhibitor screening campaigns, ionic detergents may cause problems by interacting with charged inhibitor moieties, leading to inactivation and possible false-negative results. 3.6. Protease Constructs, Substrates, and Assays: Concluding Remarks

Significant efforts were made to study the substrate specificity of the dengue virus protease. Various substrate sequences have been identified, which can be used to study the activity of dengue protease with high selectivity. The minimal consensus sequence of most substrates consists of two basic residues in N-terminal direction (nonprime site) near the cleavage site. Other positions in the substrates show a less distinct specificity. In addition to peptide sequences, various chromogenic and fluorogenic systems have been evaluated for successful measurement of dengue protease activity with certain benefits and disadvantages. These two facts lead to a large pool of reported substrates for the dengue protease (Table 2). Additionally, various assay conditions and protease constructs have been reported, and it is therefore difficult to compare results from different screenings of substrates or inhibitors (Table 2). With respect to dengue protease constructs for in vitro studies, a clear preference toward NS2B-NS3 constructs with truncated cofactor domains has evolved in the past decade. The most popular constructs contain a permanent covalent linkage between the protease- and the cofactor domains (NS2B(H)gly-NS3pro) and show convincing stability and catalytic efficiency under different assay conditions. Recent constructs aim to integrate the hydrophobic, membrane-associated domains of NS2B, which were neglected in previous investigations but may have an essential role under in cellulo and in vivo conditions. As yet, it is not clear which of the dengue protease constructs are most suitable for screening campaigns with respect to correlation against antiviral activity in cellular screening systems or in vivo studies.

4. INHIBITORS OF DENGUE VIRUS PROTEASE 4.1. General Remarks

The choice of serotype and type of expression construct for the dengue protease can have a significant effect on the binding behavior, including affinity and binding mode of inhibitors. For the most frequently used serotype 2 protease, a variety of expression constructs are described in the literature and used for assay purposes (cf. section 3.1), and therefore the published affinity constants for inhibitors cannot always be compared. Wherever possible, the serotype number of the dengue protease used in the experiment is reported in this Review along with IC50 and Ki values. Similarly, as outlined in the section on dengue protease assays, the assay conditions vary significantly and assays are often performed under nonphysiological conditions. The buffer pH is usually in the basic range, and the assay buffer contains detergents and polyols. Glycerol is often present in dengue protease assays at concentrations in a range of 20−30%. The presence of glycerol ensures high catalytic activity of the protease, but will also influence the thermodynamics of ligand−target interactions. When inhibitors bind to the enzyme−substrate complex, as is sometimes the case for dengue protease inhibitors (see below), the choice of substrate can have a significant influence on the inhibitory constants. These (variable)

4.2. Peptidic and Peptidomimetic Inhibitors

This section covers inhibitors that resemble the substrate recognition sequence of dengue protease to varying degrees and which are peptides or contain peptidomimetic structures. The substrate recognition sequence of a protease is frequently used as a starting point for the design of protease inhibitors. The value of this approach is documented by numerous peptidic or peptidomimetic protease inhibitors in clinical practice that target the HIV and HCV proteases, thrombin, the proteasome, and numerous other proteases. Peptidic substrates and inhibitors are often employed in the early phase of the drug discovery process to study the molecular recognition properties of the active site and therefore guide the design of peptidomimetic compounds. An important advantage of this strategy is the comparatively straightforward generation of structural diversity that can be achieved with peptidic inhibitor candidates. Clinical candidates derived from such efforts often contain a modified backbone, incorporating urea or carbamate scaffolds, unnatural side chains, and structural features that mimick the transition state (e.g., 11364

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Figure 11. Peptidic inhibitors based on the NS3/NS4A cleavage site combined with an electrophilic α-ketoamide or aldehyde function to target the catalytically active serine of dengue virus protease.

Figure 12. Peptidic inhibitors with the common sequence Bz-nKRR-X and various electrophilic “warheads” or other groups (X).

region, which is not conserved in the West Nile virus protease.123 However, interactions between aprotinin and the cofactor cannot be observed in the dengue protease/aprotinin cocrystal structure and appear to be of minor relevance in the WNV protease/aprotinin structure. Recent NMR-based studies show that the “closed” conformation, with the C-terminal NS2B cofactor region wrapped around the active site of the dengue protease, is obtained in the presence and absence of aprotinin in solution (cf. section 2.3).37,40,41 Additionally, it is reported that aprotinin is able to stabilize the dengue protease and prevents degradation.40 The first synthetic, peptidic inhibitors of dengue protease were based on its recognition sequences, in particular the cleavage sites between the NS3 and NS4A and the NS4B and NS5 domains of the dengue polyprotein (Table 3).56 In these compounds, αketoamide moieties between P1 and P1′ or an aldehyde function at the C-terminal end of P1 are supposed to form a covalent bond to the nucleophilic serine in the dengue protease active site. Notably, the inhibitors were tested at pH 7.5 and not at the enzymatic pH optimum (pH 9), because the ketoamides were unstable at higher pH. Two peptides with the NS4B/NS5 cleavage sequence and an α-ketoamide functionality displayed poor affinity with Ki values over 200 μM. However, for two peptide-ketoamides based on the NS3/NS4B cleavage site, more promising affinities were observed. The ketoamide 1 has a Ki (DENV-2) of 47 μM. The inhibitor 2 achieves improved potency (Ki = 16 μM) by omitting the P1′ and P2′ residues and appending an aldehyde “warhead” to the C-terminal end of the peptide (Figure 11). Chanprapaph et al. studied peptidic inhibitors without any covalent modifiers.86 For the peptide Ac-RTSKKR-NH2, representing the N-terminal part of the NS2A/NS2B cleavage site, a Ki (DENV-2) of 12 μM was observed. The related tripeptide with N-terminal acetylation and C-terminal amidation

hydroxyl groups) or establish a covalent interaction with the nucleophilic residue of the protease (e.g., α-ketoamides). Given the preference of dengue protease for basic sequences, it was an obvious choice to study known inhibitors of proteases with similar recognition properties. This led to the identification of the basic/bovine pancreatic trypsin inhibitor (BPTI, aprotinin), a small protein comprising 58 amino acids with three internal disulfide bonds, as an inhibitor of the dengue protease.56 Aprotinin is a strongly basic protein that contains 10 surface-exposed lysine and arginine residues. Other protease inhibitors like benzamidine, leupeptin, pepstatin A, phenylmethanesulfonyl fluoride, or the soybean trypsin inhibitor were not active against dengue protease. Aprotinin has significant affinity toward dengue protease, with a reported IC50 (DENV-2) of 65 nM56 and Ki values (DENV-2) of 26 nM31 and 6.5 nM.41 Other authors report an even higher affinity with Ki values of 79, 25, 88, and 6.4 pM (!), determined by active-site titration, for protease from the dengue serotypes 1, 2, 3, and 4, respectively.58 The crystal structure of aprotinin in complex with the catalytically active protease of dengue serotype 3 shows that aprotinin binds to the active site in the “open” conformation (without NS2Bc being involved) with recognition elements in the P4′−P3 sites (PDB accession code 3U1J) (cf. section 2.2).23 In contrast, the complex of West Nile virus protease with aprotinin contains a “closed” active site with a well-defined βhairpin motif of NS2Bc located close to the active site (PDB code 2IJO).121 For the WNV protease, some authors reported a significantly lower affinity toward aprotinin than for the dengue protease (Ki (WNVpro) = 0.162 μM).28,31,121 Another report provides an affinity in the same range (Ki (WNVpro) = 26 nM) as for the dengue protease.122 Doan and co-workers suggested that a possible tighter binding of aprotinin to dengue protease might be caused by a stiffer hydrogen-bonding network formed by glutamic acid residues from the C-terminal NS2B cofactor 11365

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Table 7. Selected Peptidic Inhibitors of the Dengue Virus Protease

Ac-KKR-NH2 had a Ki of 22 μM, which compares favorably to the above-mentioned peptide derivatives that incorporate electrophilic “warheads” and demonstrates that significant activities can be achieved with relatively small and nonreactive inhibitors. Furthermore, these examples, which do not incorporate any prime-position residues or mimics thereof, demonstrate the predominance of the positively charged nonprime positions of peptidic substrates and inhibitors for molecular recognition by dengue protease. Yin and co-workers extended this sequence with the unnatural amino acid norleucine (n) in P4 capped with a benzoyl moiety, resulting in a Bz-nKRR peptide that was combined with various C-terminal electrophiles and other groups (Figure 12).29 A general review of covalent modifiers in drug discovery was given by Potashman and Duggan in 2009.124 For the analogue with a C-terminal amide function (4), a higher affinity (Ki (DENV-2) = 128 μM) in comparison to the free carboxylic acid (3, Ki > 500 μM) was found. The trifluoroacetylsulfonamide (5) had much lower activity as compared to the amide analogue 4. All other C-terminal

“capping” groups and electrophilic warheads in the study resulted in compounds with higher affinity to the protease. Whereas the benzoxazole and thiazole derivatives were modestly more potent than the amide analogue, a highly increased activity was found for the aldehyde (6) and trifluoromethylketone (7) derivatives. In both compounds, the activated carbonyl function is supposed to interact covalently with the nucleophilic serine in a reversible way by forming hemiacetal or hemiketal structures. However, 6 was reported to be a “highly potent” inhibitor of human proteases such as furin, trypsin, thrombin, and elastase,125 and similar cross-inhibition activity may be suspected for the other derivatives. For this compound, a certain selectivity toward the serotype 2 was observed with respect to reported IC50-values of 11.8, 8.9, 7.9, and 1.4 μM for DENV-1, DENV-2, DENV-3, and DENV-4, respectively.36 By far the best affinity at dengue protease was found for the boronic acid congener 8 with a Ki of 43 nM. It is likely that this derivative forms a tetrahedral boronate complex with a boron−oxygen bond between the active serine and the boronic acid as found in the cocrystal structure of other boronic acid derivatives and serine proteases.126 In contrast to 11366

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into 3-phenylpropanoyl-KRR-H (Ki > 300 μM) resulted in a total loss of activity. N-Terminal acetylation is significantly less favored than benzoylation. It is noteworthy that the studied compounds displayed significantly higher affinities toward the protease of West Nile virus. Cyclic peptides offer certain pharmacokinetic and pharmacodynamic advantages over open-chain peptides.129 Gao et al. investigated cyclopeptides inspired by the kalata B1 cystine-knot peptide as dengue protease inhibitors.117,128 Cleavage site residues of dengue protease were introduced into these cyclopeptides, and two inhibitors (17, 18) with Ki values (DENV-2) in a promising range (Ki = 1.4 and 3.0 μM) were identified. Tomlinson and Watowich screened a library of 2000 compounds and identified tyrothricin, a mixture of antimicrobial peptides, as competitive dengue virus protease inhibitor with a Ki (DENV-2) of 12 μM.91,130 Rothan et al. reported the peptides protegrin-1, recombinant retrocyclin-1, and plectasin (40 amino acids) as dengue protease inhibitors that reduced viral replication in cell culture.89,90,106 The cationic cyclic peptide protegrin-1 with two internal disulfide bonds was identified as a competitive inhibitor of the dengue protease with a Ki of 5.85 μM (DENV2).89 In cellulo assays of protegrin-1 in infected MK2 cells (DENV-2) showed significant inhibition of viral replication at 2.5 and 12.5 μM. However, the compound was cytotoxic at concentrations above 12.5 μM. Furthermore, the antiviral activity of protegrin-1 decreased with the incubation time, probably due to low intracellular stability. For the cyclic peptide retrocyclin-1 with three disulfide bonds, slightly different results were obtained.90 The in vitro inhibition of the protease was found to be temperature-dependent with an IC50 of 46.1 μM at 28 °C, 21.4 μM at 37 °C, and 14.1 μM at 40 °C (DENV-2). The cytotoxicity for this peptide was much lower than for protegrin-1 with 150 μM as the highest nontoxic concentration. At this concentration, an inhibition of viral replication in infected Vero cells (DENV-2) could be observed, without any loss of inhibition potential over longer incubation times, indicating improved intracellular stability as compared to protegrin-1. For plectasin, an antimicrobial cationic peptide with three intramolecular disulfide bonds, a Ki (DENV-2) of 5.03 μM with a noncompetitive inhibition mechanism was reported.106 This peptide inhibits 80% of the viral replication (DENV-2) in Vero cells at the highest nontoxic concentration of 20 μM. The pharmaceutical application of retrocyclin-1, protegrin-1, and plectasin as dengue virus protease inhibitors appears unlikely because of insufficient activity as well as cytotoxicity at low concentrations. This class of antimicrobial peptides has previously been shown to possess activity against other, completely unrelated viruses and may thus act by an alternative mechanism, for example, by targeting host factors or viral entry.131,132 In a very recent work, Rothan and co-workers described the combination of the protegrin-1 and plectasin peptides by fusion to MAP30. The resulting chimeric protein (37.7 kDa) was able to inhibit dengue protease (IC50 = 0.5 μM, DENV-2) and prevent binding and proliferation of dengue virus in cellular models. The maximalnontoxic dose against Vero cells was found to be quite low with 0.67 μM. However, DENV-2-challenged mice treated with 50 mg/kg of the fusion protein showed a survival rate of 100%.103 The conotoxin MrIA, a cyclopeptide isolated from cone snails with two intramolecular disulfide bonds, was reported to possess inhibitory activity against the dengue protease serotype 2.93 Inspired by this screening result, synthetic peptides containing a single disulfide bond were evaluated. These cyclopeptides were based on the MrIA loop sequence CGYKLC (19) (Ki = 12.5

peptide aldehydes, the boronic acid electrophile was already shown to be compatible with clinical usage.127 The tetrapeptidyl aldehyde Bz-nKRR-H (6) was cocrystallized with dengue protease of serotype 3, and the X-ray structure of the resulting complex allows a detailed analysis of its binding mode (PDB accession code 3U1I,23 cf. the crystallography section of this Review for details) (Table 1). In this structure, the peptidic inhibitor forms a hemiacetal with the S135 residue of the dengue protease NS3 domain. The two arginine residues of 6 interact with recognition elements of the S1 and S2 pockets. The P1 arginine interacts via charge−charge interaction with D129 and forms a hydrogen bond to the backbone of F130, and the P2 guanidine function is located near the backbone of G82 (NS2B domain). The lysine and norleucine side chains are arranged near the S3 and S4 pockets. The amine functionality of the P3 lysine forms a hydrogen bond to the backbone of M84 (NS2B domain). The backbones of P2 and P3 interact with G151 and G153 of the NS3 domain, respectively. The side-chain interactions of P2 and P3 with cofactor residues may stabilize the “closed” active protease conformation with an ordered β-hairpin of the NS2B cofactor wrapped around the active site. The NS2B cofactor is clearly involved in ligand recognition. The N-terminal benzoyl group and the norleucine residue of the inhibitor are involved in crystal packing interactions with protein and ligand residues from an adjacent unit cell. Thus, the position of these residues in the crystal structure may not properly reflect the “dilute” conditions under which enzymatic testing is performed. Starting from the tetrapeptidyl aldehyde Bz-nKRR-H (6), the effect of changes in the peptide sequence was further analyzed.30 A scan of alanine, phenylalanine, lysine, proline, D-, and N-methyl amino acids throughout the sequence was performed, and the P1 arginine was replaced by several unnatural amino acids. The norleucine in P4 can be replaced by other hydrophobic amino acids like alanine, phenylalanine, or D-norleucine without significant loss of affinity. The arginine and lysine residues in P1−P3 appear to be highly relevant, and their replacement usually results in compounds with decreased affinity toward dengue protease. However, peptide aldehydes with 4-methylphenylalanine (11) or tryptophan (12) in P1 nearly reached the same activity as the arginine derivative, with Ki values (DENV-2) of 6.0 and 7.5 μM, respectively. A 4-guanidinylphenylalanine moiety (13) in P1, acting as arginine mimetic, resulted in a peptide with a Ki of 2.8 μM. One of the most interesting findings from this study was the observation that the norleucine residue in P4 is less essential than previously assumed. The N-benzoylated peptide aldehyde Bz-KRR-H (14) is the most potent analogue in the study with a Ki value of 1.5 μM. This result was picked up by Schüller et al. in their investigation of KRR and KKR tripeptidyl aldehydes with a variety of N-terminal “cap” moieties.87 None of the alternative caps increased the affinity of the peptide inhibitor Cap-KRR-H. For the Cap-KKR-H series with a lysine in P2 instead of an arginine, the best result was achieved with a 4phenylphenylacetyl cap (15) (Ki (DENV-2) = 12.2 μM). Slightly higher affinity for this compound was reported with a Ki of 7.91 μM in another publication.123 For all other evaluated caps, increased affinities were found with the sequence Cap-KRR-H, indicating that the dengue virus protease has a preference for an arginine residue in P2. Although in the cocrystal structure of the tetrapeptidyl aldehyde Bz-nKRR-H (3U1I) no specific interaction of the capping group with the target could be identified, it seems that the character of the cap has a significant influence on the affinity of the tripeptidyl aldehydes. For example, homologation of 2-phenylacetyl-KRR-H (16) (Ki = 6.7 μM) 11367

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Figure 13. Pro-Met dipeptide derivatives as dengue protease inhibitors with moderate activity.

Figure 14. Peptide hybrids of the sequence Cap-RKn-NH2 with various N-terminal cap moieties.

μM), which was supposed to be responsible for the interaction with the protease. Introduction of cleavage-site inspired di- or tribasic subsequences into this sequence gave cyclic peptides with increased affinity, for example, CGKRKLC (20) (Ki = 2.5 μM) and CGKRKSC (21) (Ki = 1.4 μM). Cyclization via peptide

instead of disulfide bond resulted in cyclic compounds like CAGKRKSG (22) with similar activities (Ki = 2.2 μM, amide bond between N- and C-terminus). The noncyclic analogue of this peptide is significantly less active (Ki = 42 μM), indicating an improvement of affinity by cyclization and rigidization. Five 11368

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Figure 15. Peptide hybrids with selected C-terminal substituents that have a remarkable influence on the activity profile of the compounds. The phenylglycine moiety was found to be most promising, leading to the highly affine compound 57.

modifications like inversion of the proline stereocenter (29), oxidation of the thioether (30), extension of the linker between amide bond and phenyl residue (31), or protection of the Nterminal amine (26) resulted in loss of activity. Docking simulations of 25 at the 2FOM dengue protease structure (serotype 2) suggest a binding close to the active site, with hydrogen-bond interactions of the proline carbonyl with S135 and of the N-terminal amine with G151 and N152. An ambiguous role of the aromatic nitro group can be deduced from the structure−activity data. The absence of the nitro group in the most active 4-nitroanilide 25 leads to a crucial loss of activity (27). The opposite result was found for the moderately active benzylamide derivative 32, where a significant loss of activity was observed by inserting a 4-nitro substituent to the aromatic moiety (31). Therefore, a clear structure−activity relationship cannot be derived from the data presented for this compound class. Recently, peptide hybrids using a combination of short peptide sequences with small-molecular synthetic scaffolds have been investigated as dengue protease serotype 2 inhibitors (Figure 14).113,114 These peptides are based on the substrate sequence explored by Yin et al.29,30 for the tetrapeptidic inhibitors BznKRR-X (X = electrophile), but without the C-terminal electrophiles. For the retro sequence Cap-RRKn-NH2 (Ki = 15 μM), with an arylcyanoacrylamide scaffold as a cap moiety, a much higher affinity was observed than for the substrate-based sequence Bz-nKRR-NH2 (Ki = 128 μM).29 More surprisingly, the removal of one of the arginine residues led to even smaller compounds (Cap-RKn-NH2, Figure 14) with promising affinities (Ki < 10 μM). Different dibasic sequences without the norleucine residue unexpectedly featured a much lower affinity and less selectivity between the dengue and West Nile virus proteases. Other variations of the sequence did not result in compounds with higher affinity, leading to the sequence Cap-RKn-NH2 as the most promising one. For the cap moiety, arylcyanoacrylamides had been identified as the most active alternative in initial studies.112,113 Five-membered thiazolidinone heterocycles were recently investigated as replacements for the cyanoacrylamide moiety.114 With hydrophobic substituents at the nitrogen in position 3 of the heterocycle, highly affine rhodanine- and 2,4thiazolidinedione-based peptide hybrids were obtained. For the thiazolidinedione derivatives, the highest in vitro activity with a Ki value of 1.5 μM and a competitive inhibition mode was observed for compound 52, whereas the rhodanines possess a slightly higher membrane permeability (PAMPA) and antiviral activity in cell culture. The derivative with the most promising performance in cell culture (44) showed antiviral activity with an EC50 value of 16.7 μM in a Renilla luciferase reporter gene assay

selected peptides show a significant loss of activity after 2 h of incubation with dengue protease, indicating digestion of the peptides by the protease, with the peptide cyclized via amide bond being the most stable analogue (25% loss of inhibition after 2.5 h). This compound showed also good permeability in BHK21 and Vero cells. However, cell culture-based data for antiviral activity are not reported. Prusis et al. analyzed substrate-based octapeptides bearing four amino acids each in the nonprime (P1−P4) and prime positions (P1′−P4′) using proteochemometric models for the Michaelis and the cleavage rate constants.79 Notably, several octapeptides with a tetra-Arg sequence in P1−P4 had affinities in the upper nanomolar range, for example, Ac-RRRRHWCW-NH2 (23) with reported Ki values for DENV-1, DENV-2, DENV-3, and DENV-4 of 0.4, 0.3, 0.6, and 2.7 μM, respectively. Starting with peptides of the composition RRRRXXXX, the authors successively removed arginine residues from the nonprime positions, resulting in the sequence WYCW-NH2 (24), which has a promising Ki against the protease from dengue serotypes 1 (Ki = 4.2 μM) and 2 (Ki = 4.8 μM). For the dengue virus protease of the serotypes 3 and 4, lower affinities were found with Ki values of 24.4 and 11.2 μM, respectively. Although by the design considerations one would expect an interaction with prime site recognition residues of the protease, docking simulations suggest that the tetrapeptide inhibitor interacts with the S1−S4 pockets near the catalytic triad, and its amino terminus is located close to D75 of the S2 pocket. Thus, the amino terminus may be regarded as a replacement of the basic P2 side chain. However, the docking simulations should not be overinterpreted because the docking target structure (3U1I) is from serotype 3, for which the analyzed tetrapeptide showed the lowest inhibition as compared to the other three serotypes. Two interesting consequences can be drawn from these findings. First, to obtain promising dengue virus protease inhibitors as possible drug candidates, it is important to consider all four viral serotypes in an early stage of the drug development process, because the affinity of a compound toward the serotypes can differ considerably. Second, there is no absolute requirement for basic side chains in dengue protease inhibitors; these may be replaced by other basic functionalities such as the N-terminus of the discussed tetrapeptide inhibitor WYCW investigated by Prusis and coworkers. Smaller peptide analogues in the form of methionine-proline anilides were reported as dengue protease serotype 2 inhibitors with moderate activity (Figure 13).133 The highest affinity was found for a C-terminal 4-nitroaniline-substituted methionineproline dipeptide with nonsubstituted N-terminal amine function (Ki = 4.9 μM) as competitive inhibitor (25). Various 11369

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Figure 16. Nonpeptidic inhibitors of the dengue virus protease with guanidine groups and structure of the alkaloid piperine.

Figure 17. Panduratin derivatives, anthracene derivatives, and azo compounds identified as screening hits.

4.3. Nonpeptidic Inhibitors

and reduced viral replication by 66% in a virus titer determination assay (plaque assay) at 50 μM. 44 exerted cytotoxic effects at concentrations between 100 and 200 μM. The interaction of the peptide hybrids with the protease was analyzed by docking simulations targeted at the crystal structure of dengue serotype 3 protease (3U1I). The simulations suggest that the two basic side chains of the inhibitor (49) interact with residues of the S1 and S2 pockets, similar to the two arginine residues in the cocrystal structure of the tetrapeptide Bz-nKRR-H.23 The norleucine residue is located near the prime site, and the hydrophobic cap interacts with residues of the NS3 and NS2B domains near the S3 pocket, possibly stabilizing the “closed” protease conformation. Further development of these peptide hybrids may involve replacement of the basic side chains or their modification into pro-drugs. Noteworthy is that these compounds combine the benefits, as well as the drawbacks, of peptide-based and smallmolecular inhibitors. Behnam and co-workers studied various C-terminal residues for N-terminally benzoyl capped tripeptides of the sequence BzRKX-NH2.134 They found a remarkable influence of derivatizations at this position on the in vitro inhibition of dengue protease (Figure 15). For example, compound 56 with a C-terminal proline substituent has an IC50 of 192 μM, whereas the phenylglycine analogue 55 has a promising activity with an IC50 of 3.3 μM (58-fold increase of activity). Combination of the phenylglycine-containing peptide moiety with one of the most promising N-terminal caps from the previous work resulted in the most active peptide hybrid 57 with a Ki value of 0.4 μM at DENV serotype 2 protease.

The dengue virus protease is remarkably resistant toward inhibition by small molecules. This can be exemplified by the early work of Leung and co-workers,56 who studied a diverse panel of serine protease inhibitors, including irreversible inhibitors such as 4-amidinophenylmethanesulfonyl fluoride (APMSF) and N-tosyl-L-lysine chloromethylketone (TLCK) in the millimolar (!) concentration range, none of which inhibited dengue protease. In another noteworthy study, diisopropyl fluorophosphate (DFP) was described as practically inactive against dengue protease.31 Nevertheless, some progress has been made in recent years, and numerous nonpeptidic inhibitors of the dengue virus protease have been identified, in particular from high-throughput screening campaigns and virtual screening approaches. The affinity of the nonpeptidic inhibitors currently remains inferior to most of the peptide-based inhibitors. We are not aware of any successful optimization of lead compounds or screening hits that resulted in nonpeptidic inhibitors with affinities in the lower nanomolar range. However, some of the nonpeptidic series of dengue protease inhibitors appear to be significantly more druglike than the peptides. The chemical diversity of the nonpeptidic inhibitors is very high, and, so far, a structural classification does not appear possible. An early report of nonpeptidic inhibitors was based on a screening of compounds bearing guanidino groups as a mimetic for the arginine residues in P1 (Figure 16).95 Three compounds were found to have Ki values (DENV-2) below 50 μM (58, 59, 60). The most active compound 60 (racemic) showed a promising Ki value of 14 μM. The drug-likeness of 60, featuring a guanidino group and a hypophosphorous acid substituent, 11370

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Figure 18. In vitro and in cellulo inhibition potential of doxycycline and mefenamic acid as compared to ribavirin.

Figure 19. Selected bis-substituted phthalazine derivatives with activity against the proteases from all four dengue serotypes.

however, remains questionable. Anyhow, in comparison to other inhibitors of the dengue protease, the size of these compounds is small, and consequently their ligand efficiency is relatively high. No further optimization of these lead-like compounds toward derivatives with higher drug-likeness was reported. Another screening campaign resulted in the identification of guanidinylated 2,5-dideoxystreptamines, which were analyzed toward their inhibition potential against all four serotypes of dengue virus protease (Figure 16).85 The two compounds 61 and 62 were the most active derivatives and displayed a certain degree of serotype-selectivity. No cytotoxic or antiviral effects were observed for compound 62 at concentrations up to 100 μM, which may be due to inadequate membrane permeability or sequestration by components of the cell culture medium. The natural alkaloid piperine (Figure 16) was identified as an inhibitor of the dengue virus protease serotype 2 with moderate activity (Ki = 22.2 μM).135 No further studies on piperine were reported. Two cyclohexenyl chalcone derivatives isolated from fingerroot (Boesenbergia rotunda) plant extracts were reported as competitive inhibitors of the dengue virus protease serotype 2 (Figure 17): 4-hydroxypanduratin A (63) and panduratin A (64), with Ki values of 21 and 25 μM, respectively.96 Phenolic and polyphenolic functionalities were also found as important structural features in a compound series reported by Tomlinson et al.88,130,136 Initially, a virtual screening simulation using two early structures of the DENV protease as targets led to the identification of the nitro-anthraquinone 65, which inhibits dengue virus (DENV-2) replication in LLC-MK2 cells with an EC50 value of 4.2 μM and is only moderately cytotoxic (CC50 = 69 μM, selectivity 1:16).136 Additional studies with 65 showed a more pronounced cytotoxic effect in Vero cells (CC50 = 20.8 μM) and promising antiviral activity against all four serotypes (EC50: DENV-1: 5.96 μM, DENV-2: 0.44 μM, DENV-3: 3.08 μM, DENV-4: 0.88 μM). The replication of Japanese encephalitis virus was less affected by 65, indicating a denguespecific effect (EC50 = 30.1 μM).102 However, 65 was inactive at the isolated protease (Ki value = 432 μM), indicating that other targets are relevant for the antiviral effect of this compound.88,130

65 was also identified as an uncompetitive inhibitor of trypsin (Ki′ = 13 μM).88,130 The derivatives 6688,130 and 6788,130 showed more promising in vitro results against the protease (DENV-2), with a mixed (competitive/uncompetitive) inhibition mechanism of dengue protease and absence of trypsin inhibition for both compounds. In general, the highest inhibition results were associated with phenolic substituents that supposedly interact with H51 and D75 of the catalytic triad. However, no experimental evidence is provided that these compounds bind close to the active site. It should be noted that due to a variety of reasons (aggregation, promiscuity, pharmacokinetic liabilities), polyphenolic compounds are usually not considered as suitable leads for drug discovery efforts. A verification of the protease inhibition by an orthogonal in vitro assay system (cf. ref 81) is not described for the anthracene and anthraquinone derivatives. An interaction of these strongly UV/vis absorbing compounds with the fluorogenic readout of the employed protease assay cannot be ruled out and is discussed by the authors as an “explanation” for the partially uncompetitive binding mode.88 Assay artifacts related to the absorption of the excitation or emission wavelengths by test compounds or other assay components are of general concern in the use of fluorogenic or FRET-based substrates for protease screening assays and other drug discovery applications. Another phenolic inhibitor of dengue virus protease is the antibiotic doxycycline, which was found to inhibit the replication of all four dengue serotypes in cell culture (Figure 18).104,105 The IC50 of doxycycline at dengue serotype 2 protease was found to be temperature-dependent with 52.3 and 26.7 μM at 37 and 40 °C (“fever” temperature), respectively. A noncompetitive binding mode with a Ki value of 55.6 μM was observed. The selectivity of doxycycline in cell culture is limited, with an EC50 of 40 μM (determined from reduced viral RNA for DENV-2) and a CC50 of 125 μM in Vero cells. The antiviral activity of doxycycline may also result from an interaction with the dengue envelope protein and subsequent reduction of virus binding to the host cell.105,137 In addition to doxycycline, the antiinflammatory drug mefenamic acid was identified as an inhibitor with moderate activity from a screening of eight selected 11371

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Figure 20. Quaternary ammonium salts with moderate in vitro affinity toward dengue protease and promising antiviral activity in cell culture.

drugs.104 For this compound, a Ki of 32 μM, a CC50 of 150 μM, and an EC50 of 32 μM were reported. Noteworthy, the cytotoxic and antiviral measurements were performed in relation to the well-known viral replication inhibitor ribavirin, which showed an EC50 value in the same range of 30 μM, but a much better selectivity index (9.3) with a CC50 of only 280 μM. Bodenreider and co-workers described phthalazine derivatives as inhibitors of dengue protease (Figure 19), which were thoroughly characterized by various orthogonal methods and assays.35,36 High-throughput screening yielded a hit whose structure was not reported, and which proved to be unsuitable for further development due to low solubility and difficult chemical accessibility. The initial hit was therefore not pursued any further, but a search for structurally related compounds resulted in 68 as a promising lead with an IC50 value of 6 μM against the dengue virus protease serotype 2. Binding to the active site was confirmed by NMR spectroscopy. Analysis of the 15N-HSQC NMR spectrum of the protease after addition of the ligand indicates a conformational change from an “open” to a “closed” conformation, accompanied by decreased flexibility of the cofactor domain. However, this conformational change might be an artifact from degraded protein.37 Unspecific interactions of 68 with the protease were excluded using a FRET-based aprotinin competition assay (Kd = 7.2 μM), indicating a competitive binding behavior of 68 and aprotinin at the active site. Kd-values determined by isothermal titration calorimetry (7.3 μM) and surface plasmon resonance (3.7 μM) were in the same range. On the basis of 68 as lead compound, approximately 130 analogues were synthesized. None of these derivatives showed activity in the nanomolar range, and, with a few exceptions, the structure−activity relationships remained “flat”, although there is evidence for interaction with the active site, and the phthalazine derivatives do not appear to act by a “promiscuous” mechanism. However, the structure−activity relationships indicate that minor structural changes can lead to a significant modulation of target affinity, a phenomenon that is sometimes described as “activity cliff”. Derivative 69, with two cyclohexano-imidazoline moieties, was identified as the compound with the highest activity in the series, whereas the structurally similar compound 70 has much lower affinity. Another indicator of specific inhibitor−target interactions is the subtype-selectivity of the phthalazine analogues. At the time of these efforts, no X-ray crystal structures of the catalytically active dengue protease in complex with an inhibitor were available. The authors therefore used an X-ray crystal structure of the closely related West Nile virus protease (2FP7) as a target for ligand docking simulations. The docking poses for 68 suggest a π−πinteraction of the phthalazine ring with Y161 in the S1 pocket, salt bridges between the two positively charged imidazole rings and the protease residues G159, D129, and N84, and an interaction of the secondary amine linker with the backbone carbonyl oxygen of P131. Unfortunately, these compounds did not show

antiviral activity in a cell culture-based assay system, probably due to insufficient permeability through biological membranes.125 In another high throughput screening, 41 600 compounds were analyzed toward their potential of dengue virus protease inhibition (serotype 2).84 Three hits with IC50 values below 40 μM were identified, two of which were rejected because of toxicity (BHK-21 cells). 71 as the only remaining compound had an IC50 value of 15.4 μM and moderate cytotoxicity (Figure 20). Antiviral activity against all four dengue serotypes was observed in a plaque reduction assay, with a more than 10 000-fold reduction of serotype 2 dengue virus replication by 71 at 12 μM. In a luciferase reporter gene assay, the EC50 value was determined (0.17 μM), giving a selectivity ratio between reporter suppression and cytotoxicity of approximately 170:1 (CC50 = 29.3 μM), a remarkable and promising result. It was further discovered that 71 does not interfere with translation of the viral polyprotein early in the replication process, but inhibits viral RNA synthesis at later stages. The authors employed resistance breeding experiments to pinpoint residues in NS2B and NS3 that are crucial for the effect of 71. The results indicate that 71 disturbs the interaction between these two domains. A remarkable discrepancy is evident between the relatively low affinity of 71 against the isolated protease in vitro and its considerable effect against dengue replication in cell culture. The authors explain this discrepancy by the artificial protease construct and unphysiological assay conditions, which differ substantially from the cellular conditions under which viral replication occurs. Recently, screening of 60 000 small-molecular compounds using a luciferase replicon assay of subgenomic dengue virus serotype 2 resulted in the identification of another quaternary ammonium salt, 72, as an inhibitor of dengue virus replication with an EC50 value of 1.03 μM in BHK-21 cells, which was confirmed in a virus yield reduction assay (Figure 20).83 Without detectable cytotoxicity, this compound was able to inhibit the viral replication of all four serotypes. Selectivity was further confirmed by a counterscreen using Japanese encephalitis virus with no relevant activity against this related flavivirus. Resistance breeding experiments indicated that an E66G mutation in the central region of the NS3 protease domain was crucial for resistance of the mutants (15.2-fold resistance). This was interpreted as evidence for an interaction of 72 with the NS3 protease as a mechanism of antiviral activity, which was further confirmed by a dengue virus infection assay with the same mutation (17.2-fold resistance) and in vitro with a similar mutant of the NS2B-NS3 protease construct (3.1-fold resistance). The IC50 value against the wild-type protease was found to be only 22.6 μM and therefore much lower than the antiviral activity in cellulo, in analogy to the findings for the analogous, bisquaternary compound 71. Quaternary ammonium salts with aliphatic side chains may be privileged structural features for dengue virus protease inhibitors. Additional research on the 11372

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Figure 21. Compounds identified by high-throughput screening as inhibitors of the dengue and West Nile virus proteases. The affinity of the 8hydroxyquinoline derivatives 77 and 78 as well as amodiaquine for dengue protease was not reported, although these compounds are active against dengue virus replication in cell culture.

Figure 22. Aminobenzamide, triazole, and benzisothiazolinone derivatives as inhibitors of dengue protease identified from high-throughput screenings.

mode of inhibition and structural information on the inhibitor− target complex would be desirable for this class of compounds, whose highly amphiphilic character is suggestive of aggregation, interaction with biomembranes, and nonspecific binding. Further studies will hopefully address these aspects and clarify the suitability of this compound class for potential pharmaceutical applications.

73, 74, and 75 were identified in a screening campaign directed at the protease of West Nile virus and were shown to be moderately active against dengue virus protease (DENV-2).94,138 The 8-hydroxyquinoline/aminobenzothiazole derivatives such as 76 were studied as promising West Nile virus protease138,139 as well as dengue virus protease inhibitors.98 Derivative 76 has a Ki (DENV-2) of 2.36 μM and appears to be a good basis for further 11373

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Figure 23. α-Ketoamides, cyanoacrylamides, and five-membered heterocycles combined with arylidene moieties as inhibitors of the dengue protease with moderate activity and high ligand efficiency. The derivatives with the highest reported in vitro activity of each compound series are shown (87, 88, 89).

mode of inhibition with suggested π−π interactions of the fluorobenzyl substituent and Y161 as well as hydrophobic interactions between the phenoxyphenyl moiety and V72, indicated by docking simulations based on the 3U1I structure. The aminobenzamide moiety is supposed to be arranged near the catalytic triad. Data on the antiviral activity of this compound class were not published. An additional series of compounds based on the benzisothiazolinone scaffold, identified as inhibitors of the dengue and West Nile virus proteases by screening of a compound library, was published by the same research group.97,101,141 Synthetic derivatizations of the initial hit resulted in several analogues with IC50 values (DENV-2) at dengue protease below 5 μM, and the structure−activity relationships were analyzed with respect to the substituents R3−R5 (Figure 22). The highest in vitro affinities were found for compounds 85 (IC50 = 3.48 μM, Ki = 4.77 μM) and 86 (IC50 = 3.75 μM). The inhibitory potencies against dengue and West Nile virus protease appear to be highly correlated within the 79 and 86 series of compounds. Docking simulations targeted at crystal structures and homology models suggest interactions with the active site of the protease. The antiviral activity of these compounds was not reported. A screening of small-molecular aldehydes as covalent inhibitors of the dengue virus protease resulted in cinnamaldehyde as the sole “hit” with moderate activity, leading to the identification of the cinnamoyl moiety as privileged structure for dengue protease inhibitors.110 On the basis of this initial success, electrophiles with higher drug-likeness and other scaffolds were combined with the cinnamoyl moiety (Figure 23).110−112 The evaluation of the ketoamide and nitrile functions as covalent modifiers of the catalytic serine led to compounds with moderate affinity, but the SAR remained flat, with IC50 values around 50 μM for the best derivatives (DENV-2). However, the ligand efficiency of these compounds is significantly higher than for numerous other screening hits and lead compounds. A covalent interaction with the target could not be confirmed. For αketoamide derivative 87, antiviral activity at concentrations above 10 μM was found in a reporter gene assay in Huh-7 cell culture with no cytotoxic effects at the highest assayed concentration of 100 μM. Cyclization of the acrylamide moiety led to derivatives with highly functionalized five-membered heterocycles like rhodanines (X = S, Y = S), 2,4-thiazolidinediones (X = O, Y = S), thiohydantoines (X = S, Y = NH), or

derivatizations. Unfortunately, neither antiviral activity data nor information on optimization attempts or the activity of the enantiomers of 76 were published. For the simplified analogues 77 and 78, an inhibition of DENV-2 replication in cell culture with only moderate to low cytotoxicity was reported.138 However, to our knowledge, no in vitro data for the protease of any dengue virus serotype are available for these compounds, rendering data interpretation and SAR analysis difficult. The available SAR for 76 and analogues is limited to the observation that a number of modifications are detrimental for activity, such as removal of the benzyl ether function, arranging the benzyl ether in ortho position to the amine residue, or replacing the benzothiazole with a thiazole heterocycle. The metal-chelating properties of the 8-hydroxyquinoline moiety are potentially associated with pharmacokinetic and toxicological liabilities. However, the authors addressed this caveat by studying the inhibitory potential of 76 against dengue protease in the presence and absence of 40 μM ZnCl2 and did not find a significant influence of the metal ion.98 It would be highly desirable to obtain further information on the stereospecific activity of these compounds (for example, to rule out nonspecific effects) and the essentiality of the hydroxyquinoline moiety. Docking simulations were done on the dengue serotype 3 crystal structure (3U1I) and suggest that the eutomer is S-configured. The docking simulations suggest an interaction of the 8-hydroxyquinoline moiety with the aromatic residues F130, Y150, and Y161 of the S1 pocket and interactions of the benzyl ether group with recognition elements of the S2 subsite. The benzothiazole moiety is supposed to be located at the prime positions of the substrate recognition region. The S3 and S4 binding sites are unoccupied in the model. Recently, an extensive study of closely related amodiaquine derivatives toward their inhibition of dengue virus serotype 2 replication and infectivity was published.140 For amodiaquine (Figure 21), EC50 values of 1.08 and 7.41 μM were reported for viral infectivity and replication, respectively. However, no in vitro data are available, and from their experimental data the authors suppose that it is unlikely for the viral protease to be the relevant target of amodiaquine. Triazole- and aminobenzamide-substituted urea derivatives were investigated as dengue virus protease inhibitors, with a moderate activity for the best derivative 79 (Ki = 8.77 μM, DENV-2) (Figure 22).100 This compound shows a competitive 11374

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Figure 24. Identification of quinoline-based dengue protease inhibitors by functional group modification and scaffold hopping, starting from hits identified by screening.

Figure 25. Hits from an experimental screening of compounds that were preselected by a computer-based virtual screening campaign. The compounds show activity against the dengue virus protease from serotype 4.

hydantoines (X = O, Y = NH). Combined with a 5-arylidene moiety, rhodanines and thiohydantoines had some activity against the dengue virus protease with IC50 values (DENV-2) around 50 μM. The substituents on the arylidene moiety did not appear to have a significant influence on the SAR, although substitution by an aliphatic chain led to a substantial decrease of activity. In general, compounds without an arylidene substituent remained inactive, irrespective of the heterocycle. In a follow-up effort, the arylcyanoacrylamide, rhodanine, and thiazolidinedione moieties were combined with short peptide sequences to yield peptide−hybrid inhibitors with increased affinity toward dengue protease, indicating a certain structural preference for these scaffolds.113,114 A virtual screening of approximately 600 000 compounds from the ACD database against the crystal structure of dengue virus protease (2FOM) led to the identification of 90 as the most active and promising compound from an in vitro analyzed set of 27 selected in silico hits (Figure 24).80 90 has an IC50 value (DENV-2) of 13.1 μM and reduces replication of viral replicons (DENV-2) in a luciferase reporter gene assay, which is accompanied by cytotoxic effects in BHK cells (CC50 = 12.6 μM). Variation of the residues R1, R2, R3, and R4 of the central phenyl scaffold of 90 did not result in higher in vitro activity. Therefore, the ZINC library was virtually screened for an alternative drug-like core scaffold using different parameters. This scaffold hopping approach resulted in the identification of the quinoline moiety as a suitable replacement for the central phenyl scaffold in 90. Quinoline derivatives with optimized

properties could be synthesized, with 91 as the most promising compound with an improved IC50 value of 9.45 μM, an EC50 value of 24.7 μM in the dengue replicon assay, and no cytotoxic effects at the highest assayed concentration of 100 μM. While scaffold hopping was successful in identifying novel, optimized leads with improved activity, the desired nanomolar activity range was missed. The observed activity in the dengue replicon assay system appears promising, but no report was made on the activity of the compounds against dengue virus in cell culture, which would have been a highly significant, additional indicator of in vivo activity. Suggestions for the interaction of 91 with dengue protease were generated by molecular modeling/docking simulations. The central quinoline scaffold is expected to form hydrophobic interactions with the L85 and L149 residues. The hydrazone moieties in ortho-position to the quinoline nitrogen form hydrogen bonds to N152 and K73, and the 3-bromophenyl substituent is arranged near the S4 subsite in the poses obtained by docking simulations. The amide-hydrazine moiety in paraposition to the quinoline nitrogen is involved in interactions with K74 and I165, and the phenyl substituent is arranged between the E88 and A66 residues of the protease. Another virtual screening of 300 000 compounds (Grid Application Platform Virtual Screening Service) resulted in a preselection of 36 compounds, which were assayed in vitro against the protease of dengue serotype 4.99 Seven compounds had IC50 values below 100 μM, and three of these were identified as screening hits with inhibition constants below 5 μM (92, 93, 94, Figure 25). These inhibitors are structurally diverse and 11375

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inhibitors.92 About 13 000 compounds from the ZINC database were subjected to docking simulations against several homology models of dengue protease. Notably, no restrictions were made on the docking site of the ligands. Thus, potential binding sites on the whole target macromolecule(s) were explored in this approach. Subsequent in vitro studies of some compounds indicated relatively minor affinities of the identified hits. The most active compound 98 had a Ki value (DENV-2) of 69 μM with a noncompetitive binding mode (Figure 27). Pambudi et al. also discovered a noncompetitive inhibitor (99), which is supposed to interfere with the interaction of NS2B cofactor and NS3 protease.102 A virtual screening of more than 660 000 smallmolecular compounds from the Molecular Operating Environment lead-like database and a subsequent evaluation of a set of 39 selected compounds in a cell-based antiviral activity assay led to identification of 99. This compound inhibited replication of all four dengue serotypes with EC50 values below 2.5 μM. Good selectivity as compared to Japanese encephalitis virus (EC50 = 29.8 μM) and only moderate cytotoxicity (CC50 = 67.3 μM) was found for 99. In vitro studies with isolated dengue protease (DENV-2) confirmed the noncompetitive inhibition mechanism; however, no quantitative information on the in vitro affinity constant was published. Molecular modeling simulations suggest that 99 occupies the NS2B-binding site. The targeting of allosteric binding sites, particularly at the interface of protease and cofactor, by small-molecular inhibitors is an interesting, alternative, but highly challenging approach to the discovery of protease inhibitors. Pambudi et al. provide evidence, in particular the cell-culture data, that this is a valid approach. However, a quantification of the binding affinity of 99 to the protease in vitro and structural evidence for the proposed binding mode would be highly desirable. Searching for inhibitors that bind to the cofactor-binding site of the West Nile virus protease in a noncompetitive way, Shiryaev and co-workers performed a virtual screening of approximately 275 000 compounds from the NCI database.144 The supposed allosteric inhibitors were also evaluated against the dengue protease in vitro (DENV-2), leading to compounds 100 and 101 as the most promising hits with IC50 values of 2.75 and 2.04 μM, respectively (Figure 27). Both compounds have moderate antiviral activity in a DENV-2 replicon assay in BHK-21 cells (EC50 = 39.9 μM for 100 and 59.5 μM for 101) as well as relatively low cytotoxicity (CC50 = 213 μM for 100 and 117 μM for 101). Determination of kinetic parameters against WNV

relatively large in size. Synthetic derivatizations to optimize the identified hits toward more promising lead structures with higher affinity were neither reported nor were these compounds characterized in cell culture. Docking simulations indicated that the relatively high affinity of the compounds is due to the formation of hydrogen bonds with catalytic residues and hydrophobic interactions in the active site of the protease. Knehans and co-workers reported a virtual screening of 14 million compounds from the ZINC database using a homology model of dengue virus protease.142 After filtering for druglikeness, substructure/neighborhood searching, and focusing on the S1/S2 interactions, 23 compounds were selected for in vitro studies (DENV-2). Compounds 95 and 96 showed inhibition with a supposed competitive mechanism in a promising range with Ki-values of 2.0 and 31.1 μM, respectively (Figure 26). The

Figure 26. Guanidine-substituted phenyl benzoates as dengue protease inhibitors identified from an in silico-inspired screening campaign.

closely related derivative 97 was identified as ligand for West Nile and dengue virus proteases using NMR studies.35,143 However, to our knowledge no data from enzymatic assays for this compound were reported for the dengue protease. Although a competitive inhibition mechanism is reported, the authors suggest that a covalent interaction of the activated ester bond with the catalytic serine may be possible. The presented data do not clearly support a competitive binding mode. A timedependent inhibition study would be suitable to clarify whether this class of inhibitors binds covalently and is subsequently degraded by the protease. For compound 95 it is most likely that the two guanidinium groups interact with residues of the S1 and S2 pockets, in lieu of the two corresponding lysine or arginine side chains of the substrate. Docking simulations confirm this binding mode and suggest electrostatic interactions with D129 and F130 (S1) as well as N152 (S2).142 Whereas most other virtual screenings were aimed at the discovery of competitive inhibitors, Heh and co-workers pursued a rational approach to find noncompetitive dengue protease

Figure 27. Noncompetitive and allosteric inhibitors identified in independent screening campaigns. 11376

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protease (IC50 = 0.26 μM) revealed a mixed inhibition mode for 100. A rational approach to the discovery of allosteric inhibitors was pursued by Yildiz and co-workers.32 The surface of the protease was “scanned” by introducing cysteine mutants, which were subsequently reacted with thiol-reactive probes. This led to the identification of a region around the A125 residue, which appeared sensitive toward the binding of covalent inhibitors. The DTNB probe could, however, not be observed in X-ray crystal structures of the mutant enzymes. Whereas the pursuit of allosteric inhibitors carries a certain attractiveness, its practical usefulness for the discovery of dengue protease inhibitors remains questionable.

catalytic serine. We therefore believe that a covalent interaction with the catalytic serine, which is protease-specific and yields a significant enthalpic contribution to ligand-target recognition thermodynamics, will probably be included in successful future series of dengue or WNV protease inhibitors. Several series of inhibitors presented here have in cellulo activities in the higher nanomolar range (e.g., 65, 71, 72, 99) but only mediocre in vitro activity against the isolated protease. This may either be due to interaction with unrelated targets, such as host proteins, but can also be explained with the artificial nature of the in vitro protease assay. As outlined in the section on assays (section 3), the protease constructs may not properly reflect the state of the viral protease in the replication complex. Furthermore, the assay conditions complicate in vitro/in cellulo correlations by altering the protonation states of ligands and target (pH) and modulation of the hydrogen-bonding and hydrophobic interactions (polyol additives). A promising alternative to in vitro assays of dengue protease acitivity would be cell-based assays, in which the binding of inhibitors to the enzyme is studied under native conditions.

4.4. Inhibitors: Concluding Remarks

The first generation of dengue virus protease inhibitors was derived from the cleavage sites of the viral polyprotein. Peptidic compounds that combine these peptide-based inhibitors with highly reactive electrophiles can target the catalytically active serine and occupy all important recognition sites of the protease as confirmed by X-ray crystal structure analysis. Only a few druglike peptidic inhibitors reached these highly promising affinities up to now. None of the nonpeptidic inhibitors reported to date were able to achieve inhibition constants in the nanomolar range. Both experimental and virtual high-throughput screenings resulted in the identification of highly diverse compound classes with in vitro activity in the lower micromolar range, but subsequent optimizations did not yield significant successes or remained unreported. The binding of highly diverse compounds with only moderate affinity implies a relatively promiscuous and nonspecific ligand recognition profile of dengue protease. This leads to the conclusion that dengue protease is, similar to the related HCV protease, a difficult target for drug discovery. In comparison to HCV protease, the dengue protease may even be a more difficult target, given the highly charged nature of the natural substrate and the corresponding binding pockets. This will probably require appropriately charged and hydrophilic inhibitors, significantly complicating DMPK aspects. Additionally, and in contrast to HCV, in the case of the dengue protease no product inhibition has been observed after substrate cleavage, making the development of suitable inhibitors much more challenging. However, interest in the dengue protease as a target for medicinal chemistry has increased significantly since 2011, giving hope that the next years will witness the discovery and development of promising, novel lead structures. It is noteworthy that several small-molecular compounds identified as hits in dengue-virus-related screenings contain one or more (poly-) phenolic substructures, indicating that this structural feature is either a preferred chemotype for molecular recognition by the protease, or particularly prone to give falsepositive assay results. In any case, the drug-likeness of (poly-) phenolic compounds and their tractability as lead compounds, suitable for further optimization, is questionable. We find it remarkable that only two of the small-molecular inhibitor series target the catalytically active S135 by a drug-like electrophilic moiety, although the most promising results for peptide-based inhibitors were achieved with the inclusion of covalent warheads such as boronic acid or trifluoromethylketone. This can be put in relation to other successful drug discovery efforts targeted at viral serine proteases, in particular the HCV protease inhibitors. Especially the first generation of HCV protease inhibitors, which recently entered the market, contain a ketoamide functionality that establishes a covalent bond with the

AUTHOR INFORMATION Corresponding Author

*Phone: ++49-6221-544875. E-mail: [email protected]. Author Contributions

C.N. wrote the sections on constructs, substrates, assays, and inhibitors. C.D.K. wrote the sections on structural biology and the introduction. T.S. and S.H. wrote the section on mutagenesis, and checked all facts and citations. Notes

The authors declare no competing financial interest. Biographies

Christoph Nitsche studied chemistry at the University of HalleWittenberg and business administration at the Universities of Wales and Hagen. He received his diploma in chemistry in 2010 and his MBA in 2013. He had worked in the field of bioorganic chemistry at the Leibniz Institute of Plant Biochemistry and the University of Halle-Wittenberg before he started his doctoral studies in 2010 in the area of medicinal chemistry at Heidelberg University with a fellowship of the German National Academic Foundation. He worked with Prof. Gottfried Otting at the Australian National University in 2013 and finished his Ph.D. at Heidelberg University in the group of Prof. Christian Klein in early 2014. Since then, he has continued his postdoctoral research in the same group. His current work is focused on the synthesis and biochemical evaluation of potential antiviral drugs. 11377

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Christian Klein studied pharmacy and obtained a Ph.D. in Pharmaceutical Chemistry in 2000 under the guidance of Profs. Ulrike Holzgrabe (University of Bonn) and A. J. Hopfinger (UIC, Chicago). Following postdoctoral work at ETH Zürich, he became an Emmy Noether junior group leader. Since 2007 he is professor of Pharmaceutical Chemistry at Heidelberg University. Currently, his main research interests are antiviral compounds and fundamental questions in medicinal chemistry, such as the study of unusual binding modes and structural motifs.

Steven Holloway, born in Hannover in 1983, studied pharmacy at the University of Würzburg from November 2003 until April 2009, when he passed his Second State Exam. After one year training in public pharmacies from May 2009 until April 2010, he passed his Third State Exam in June 2010 and received his qualification license as a pharmacist in July 2010. From October 2010 until March 2014, Steven Holloway worked as a Ph.D. student in the research group of Prof. Dr. Tanja

ACKNOWLEDGMENTS C.N. and C.D.K. appreciate support by the Deutsche Forschungsgemeinschaft, KL-1356/3-1. Hongmei Wu provided help for the table of X-ray structures.

Schirmeister, first at the University of Würzburg until September 2011, afterwards at the University of Mainz. Currently, he is working in a public pharmacy near Mainz and preparing for the oral defense of his

ABBREVIATIONS Abz amino benzoic acid Aca 4-aminocyclohexylalanine ACD Available Chemicals Directory ACMC 7-amino-3-carbamoylmethyl-4-methylcoumarin Ama trans-4-aminomethylcyclohexylalanine AMC 7-amino-4-methylcoumarin Amf 4-aminomethylphenylalanine APMSF 4-amidinophenylmethanesulfonyl fluoride Boc tert-butoxycarbonyl BPTI bovine pancreatic trypsin inhibitor BHK baby hamster kidney C capsid CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate Dansyl 5-(dimethylamino)naphthalene-1-sulfonyl Dabcyl 4-(dimethylamino)azobenzene-4′-carboxylic acid DENV dengue virus DFP diisopropyl fluorophosphat DHF dengue hemorrhagic fever DMPK drug metabolism and pharmacokinetics DTNB 5,5′-dithiobis(2-nitrobenzoic acid) DSS dengue shock syndrome E envelope Edans 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid EDDnp N-(ethylenediamine)-2,4-dinitrophenyl amide ER endoplasmic reticulum FRET Förster resonance energy transfer GST glutathione S-transferase HCV hepatitis C virus

Ph.D. thesis.

Tanja Schirmeister received her Ph.D. degree in Medicinal Chemistry from Freiburg University in 1993. From 1993 to 1999 she was Assistant Professor at the Institute of Pharmacy of the University of Freiburg. In 2000 she received the position of a full professor at the Institute of Pharmacy and Food Chemistry of the University of Wuerzburg. Since 2011 she holds the Chair of Pharmaceutical and Medicinal Chemistry at the Institute of Pharmacy and Biochemistry of the Johannes-Gutenberg University of Mainz. Her research interests include protease inhibitors, the development of new drugs for neglected diseases, peptide chemistry, and the chemistry of small heterocycles. 11378

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(12) Romano, C. M.; Lauck, M.; Salvador, F. S.; Lima, C. R.; VillasBoas, L. S.; Araujo, E. S.; Levi, J. E.; Pannuti, C. S.; O’Connor, D.; Kallas, E. G. PloS One 2013, 8, e70318. (13) Jessie, K.; Fong, M. Y.; Devi, S.; Lam, S. K.; Wong, K. T. J. Infect. Dis. 2004, 189, 1411. (14) Limon-Flores, A. Y.; Perez-Tapia, M.; Estrada-Garcia, I.; Vaughan, G.; Escobar-Gutierrez, A.; Calderon-Amador, J.; Herrera-Rodriguez, S. E.; Brizuela-Garcia, A.; Heras-Chavarria, M.; Flores-Langarica, A.; Cedillo-Barron, L.; Flores-Romo, L. Int. J. Exp. Pathol. 2005, 86, 323. (15) Vaughn, D. W.; Green, S.; Kalayanarooj, S.; Innis, B. L.; Nimmannitya, S.; Suntayakorn, S.; Endy, T. P.; Raengsakulrach, B.; Rothman, A. L.; Ennis, F. A.; Nisalak, A. J. Infect. Dis. 2000, 181, 2. (16) Bäck, A. T.; Lundkvist, Å. Infect. Ecol. Epidemiol. 2013, 3, 19839. (17) Acosta, E. G.; Kumar, A.; Bartenschlager, R. Adv. Virus Res. 2014, 88, 1. (18) Guzman, M. G.; Halstead, S. B.; Artsob, H.; Buchy, P.; Farrar, J.; Gubler, D. J.; Hunsperger, E.; Kroeger, A.; Margolis, H. S.; Martinez, E.; Nathan, M. B.; Pelegrino, J. L.; Simmons, C.; Yoksan, S.; Peeling, R. W. Nat. Rev. Microbiol. 2010, 8, S7. (19) Wan, S. W.; Lin, C. F.; Wang, S.; Chen, Y. H.; Yeh, T. M.; Liu, H. S.; Anderson, R.; Lin, Y. S. J. Biomed. Sci. 2013, 20, 37. (20) Wendt, A.; Adhoute, X.; Castellani, P.; Oules, V.; Ansaldi, C.; Benali, S.; Bourliere, M. Clin. Pharmacol. 2014, 6, 1. (21) Gordon, C. P.; Keller, P. A. J. Med. Chem. 2005, 48, 1. (22) Lin, J. C.; Lin, S. C.; Chen, W. Y.; Yen, Y. T.; Lai, C. W.; Tao, M. H.; Lin, Y. L.; Miaw, S. C.; Wu-Hsieh, B. A. J. Immunol. 2014, 193, 1258. (23) Noble, C. G.; Seh, C. C.; Chao, A. T.; Shi, P. Y. J. Virol. 2012, 86, 438. (24) Erbel, P.; Schiering, N.; D’Arcy, A.; Renatus, M.; Kroemer, M.; Lim, S. P.; Yin, Z.; Keller, T. H.; Vasudevan, S. G.; Hommel, U. Nat. Struct. Mol. Biol. 2006, 13, 372. (25) Luo, D.; Xu, T.; Hunke, C.; Gruber, G.; Vasudevan, S. G.; Lescar, J. J. Virol. 2008, 82, 173. (26) Luo, D.; Wei, N.; Doan, D. N.; Paradkar, P. N.; Chong, Y.; Davidson, A. D.; Kotaka, M.; Lescar, J.; Vasudevan, S. G. J. Biol. Chem. 2010, 285, 18817. (27) Chandramouli, S.; Joseph, J. S.; Daudenarde, S.; Gatchalian, J.; Cornillez-Ty, C.; Kuhn, P. J. Virol. 2010, 84, 3059. (28) Noble, C. G.; Shi, P.-Y. Antiviral Res. 2012, 96, 115. (29) Yin, Z.; Patel, S. J.; Wang, W.-L.; Wang, G.; Chan, W.-L.; Rao, K. R. R.; Alam, J.; Jeyaraj, D. A.; Ngew, X.; Patel, V.; Beer, D.; Lim, S. P.; Vasudevan, S. G.; Keller, T. H. Bioorg. Med. Chem. Lett. 2006, 16, 36. (30) Yin, Z.; Patel, S. J.; Wang, W.-L.; Chan, W.-L.; Ranga Rao, K. R.; Wang, G.; Ngew, X.; Patel, V.; Beer, D.; Knox, J. E.; Ma, N. L.; Ehrhardt, C.; Lim, S. P.; Vasudevan, S. G.; Keller, T. H. Bioorg. Med. Chem. Lett. 2006, 16, 40. (31) Mueller, N. H.; Yon, C.; Ganesh, V. K.; Padmanabhan, R. Int. J. Biochem. Cell Biol. 2007, 39, 606. (32) Yildiz, M.; Ghosh, S.; Bell, J. A.; Sherman, W.; Hardy, J. A. ACS Chem. Biol. 2013, 8, 2744. (33) Wu, P. S.; Ozawa, K.; Lim, S. P.; Vasudevan, S. G.; Dixon, N. E.; Otting, G. Angew. Chem., Int. Ed. 2007, 46, 3356. (34) Su, X. C.; Ozawa, K.; Qi, R.; Vasudevan, S. G.; Lim, S. P.; Otting, G. PLoS Neglected Trop. Dis. 2009, 3, e561. (35) de la Cruz, L.; Nguyen, T. H. D.; Ozawa, K.; Shin, J.; Graham, B.; Huber, T.; Otting, G. J. Am. Chem. Soc. 2011, 133, 19205. (36) Bodenreider, C.; Beer, D.; Keller, T. H.; Sonntag, S.; Wen, D.; Yap, L.; Yau, Y. H.; Shochat, S. G.; Huang, D.; Zhou, T.; Caflisch, A.; Su, X.-C.; Ozawa, K.; Otting, G.; Vasudevan, S. G.; Lescar, J.; Lim, S. P. Anal. Biochem. 2009, 395, 195. (37) de la Cruz, L.; Chen, W. N.; Graham, B.; Otting, G. FEBS J. 2014, 281, 1517. (38) Kim, Y. M.; Gayen, S.; Kang, C.; Joy, J.; Huang, Q.; Chen, A. S.; Wee, J. L.; Ang, M. J.; Lim, H. A.; Hung, A. W.; Li, R.; Noble, C. G.; Lee le, T.; Yip, A.; Wang, Q. Y.; Chia, C. S.; Hill, J.; Shi, P. Y.; Keller, T. H. J. Biol. Chem. 2013, 288, 12891. (39) Bi, Y.; Zhu, L.; Li, H.; Wu, B.; Liu, J.; Wang, J. Biomol. NMR Assignments 2013, 7, 137.

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC high-performance liquid chromatography HSQC heteronuclear single quantum coherence Itz iminothiazolidine LMPC lyso-myristoylphosphatidylcholine MAP30 Momordica Anti-HIV Protein (30 kDa) mstX membrane-integrating mistic protein NCI National Cancer Institute NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NS nonstructural NS2Bc C-terminal NS2B domain NS2B(FL) full length NS2B domain NS2B(H) hydrophilic core sequence of NS2B NS3pro protease domain of NS3 PDB protein data bank pNA para-nitroanilide prM membrane protein-precursor SAR structure−activity relationship SBzl thiobenzyl ester TAMRA carboxytetramethylrhodamine TBEV tick-borne-encephalitis virus TLCK N-tosyl-L-lysine chloromethylketone Tris tris(hydroxymethyl)aminomethane Triton X-100 polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether TRX thioredoxin WNV West Nile virus WT wild type YFV yellow fever virus ZINC ZINC is not commercial

REFERENCES (1) WHO. Fact sheet no. 117, http://www.who.int/mediacentre/ factsheets/fs117/en/, 2014. (2) Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A. W.; Moyes, C. L.; Drake, J. M.; Brownstein, J. S.; Hoen, A. G.; Sankoh, O.; Myers, M. F.; George, D. B.; Jaenisch, T.; Wint, G. R.; Simmons, C. P.; Scott, T. W.; Farrar, J. J.; Hay, S. I. Nature 2013, 496, 504. (3) Kuno, G.; Chang, G. J.; Tsuchiya, K. R.; Karabatsos, N.; Cropp, C. B. J. Virol. 1998, 72, 73. (4) Bonizzoni, M.; Gasperi, G.; Chen, X.; James, A. A. Trends Parasitol. 2013, 29, 460. (5) Becker, N.; Geier, M.; Balczun, C.; Bradersen, U.; Huber, K.; Kiel, E.; Kruger, A.; Luhken, R.; Orendt, C.; Plenge-Bonig, A.; Rose, A.; Schaub, G. A.; Tannich, E. Parasitol. Res. 2013, 112, 1787. (6) Vasilakis, N.; Ooi, M.; Rabaa, M.; Mayer, S.; Widen; Perera, D.; Hickey, A.; Travassos Da Rosa, A.; Bossart, K.; Tesh, R.; Holmes, E.; Cardos, J., III. International Conference on Dengue and Dengue Haemorrhagic Fever, Bangkok, 2013. (7) Whitehorn, J.; Simmons, C. P. Vaccine 2011, 29, 7221. (8) Wu, S. J.; Grouard-Vogel, G.; Sun, W.; Mascola, J. R.; Brachtel, E.; Putvatana, R.; Louder, M. K.; Filgueira, L.; Marovich, M. A.; Wong, H. K.; Blauvelt, A.; Murphy, G. S.; Robb, M. L.; Innes, B. L.; Birx, D. L.; Hayes, C. G.; Frankel, S. S. Nat. Med. 2000, 6, 816. (9) Kyle, J. L.; Harris, E. Annu. Rev. Microbiol. 2008, 62, 71. (10) Sangkawibha, N.; Rojanasuphot, S.; Ahandrik, S.; Viriyapongse, S.; Jatanasen, S.; Salitul, V.; Phanthumachinda, B.; Halstead, S. B. Am. J. Epidemiol. 1984, 120, 653. (11) Thai, K. T.; Henn, M. R.; Zody, M. C.; Tricou, V.; Nguyet, N. M.; Charlebois, P.; Lennon, N. J.; Green, L.; de Vries, P. J.; Hien, T. T.; Farrar, J.; van Doorn, H. R.; de Jong, M. D.; Birren, B. W.; Holmes, E. C.; Simmons, C. P. J. Virol. 2012, 86, 835. 11379

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Chemical Reviews

Review

(40) Chen, W.-N.; Loscha, K. V.; Nitsche, C.; Graham, B.; Otting, G. FEBS Lett. 2014, 588, 2206. (41) Li, H.; Zhu, L.; Hou, S.; Yang, J.; Wang, J.; Liu, J. FEBS Lett. 2014, 588, 2794. (42) Falgout, B.; Pethel, M.; Zhang, Y. M.; Lai, C. J. J. Virol. 1991, 65, 2467. (43) Cahour, A.; Falgout, B.; Lai, C. J. J. Virol. 1992, 66, 1535. (44) Falgout, B.; Miller, R. H.; Lai, C. J. J. Virol. 1993, 67, 2034. (45) Clum, S.; Ebner, K. E.; Padmanabhan, R. J. Biol. Chem. 1997, 272, 30715. (46) Champreda, V.; Khumthong, R.; Subsin, B.; Angsuthanasombat, C.; Panyim, S.; Katzenmeier, G. J. Biochem. Mol. Biol. 2000, 33, 294. (47) Khumthong, R.; Angsuthanasombat, C.; Panyim, S.; Katzenmeier, G. J. Biochem. Mol. Biol. 2002, 35, 206. (48) Khumthong, R.; Niyomrattanakit, P.; Chanprapaph, S.; Angsuthanasombat, C.; Panyim, S.; Katzenmeier, G. Protein Pept. Lett. 2003, 10, 19. (49) Yusof, R.; Clum, S.; Wetzel, M.; Murthy, H. M.; Padmanabhan, R. J. Biol. Chem. 2000, 275, 9963. (50) Phong, W. Y.; Moreland, N. J.; Lim, S. P.; Wen, D.; Paradkar, P. N.; Vasudevan, S. G. Biosci. Rep. 2011, 31, 399. (51) Niyomrattanakit, P.; Winoyanuwattikun, P.; Chanprapaph, S.; Angsuthanasombat, C.; Panyim, S.; Katzenmeier, G. J. Virol. 2004, 78, 13708. (52) Salaemae, W.; Junaid, M.; Angsuthanasombat, C.; Katzenmeier, G. J. Biomed. Sci. 2010, 17, 68. (53) Zuo, Z.; Liew, O. W.; Chen, G.; Chong, P. C.; Lee, S. H.; Chen, K.; Jiang, H.; Puah, C. M.; Zhu, W. J. Virol. 2009, 83, 1060. (54) Iempridee, T.; Thongphung, R.; Angsuthanasombat, C.; Katzenmeier, G. Biochim. Biophys. Acta 2008, 1780, 989. (55) Wu, C. F.; Wang, S. H.; Sun, C. M.; Hu, S. T.; Syu, W. J. J. Virol. Methods 2003, 114, 45. (56) Leung, D.; Schroder, K.; White, H.; Fang, N. X.; Stoermer, M. J.; Abbenante, G.; Martin, J. L.; Young, P. R.; Fairlie, D. P. J. Biol. Chem. 2001, 276, 45762. (57) Arakaki, T. L.; Fang, N. X.; Fairlie, D. P.; Young, P. R.; Martin, J. L. Protein Expression Purif. 2002, 25, 241. (58) Li, J.; Lim, S. P.; Beer, D.; Patel, V.; Wen, D.; Tumanut, C.; Tully, D. C.; Williams, J. A.; Jiricek, J.; Priestle, J. P.; Harris, J. L.; Vasudevan, S. G. J. Biol. Chem. 2005, 280, 28766. (59) Huang, Q.; Li, Q.; Joy, J.; Chen, A. S.; Ruiz-Carrillo, D.; Hill, J.; Lescar, J.; Kang, C. Protein Expression Purif. 2013, 92, 156. (60) Choksupmanee, O.; Hodge, K.; Katzenmeier, G.; Chimnaronk, S. Biochemistry 2012, 51, 2840. (61) Huang, Q.; Chen, A. S.; Li, Q.; Kang, C. Protein Expression Purif. 2011, 80, 169. (62) Valle, R. P.; Falgout, B. J. Virol. 1998, 72, 624. (63) Chambers, T. J.; Hahn, C. S.; Ricardo, G.; M, R. C. Annu. Rev. Microbiol. 1990, 44, 649. (64) Falgout, B.; Markoff, L. J. Virol. 1995, 69, 7232. (65) Speight, G.; Coia, G.; Parker, M. D.; Westaway, E. G. J. Gen. Virol. 1988, 69, 23. (66) Nowak, T.; Farber, P. M.; Wengler, G.; Wengler, G. Virology 1989, 169, 365. (67) Stadler, K.; Allison, S. L.; Schalich, J.; Heinz, F. X. J. Virol. 1997, 71, 8475. (68) Lescar, J.; Luo, D.; Xu, T.; Sampath, A.; Lim, S. P.; Canard, B.; Vasudevan, S. G. Antiviral Res. 2008, 80, 94. (69) Preugschat, F.; Yao, C. W.; Strauss, J. H. J. Virol. 1990, 64, 4364. (70) Preugschat, F.; Lenches, E. M.; Strauss, J. H. J. Virol. 1991, 65, 4749. (71) Shiryaev, S. A.; Ratnikov, B. I.; Aleshin, A. E.; Kozlov, I. A.; Nelson, N. A.; Lebl, M.; Smith, J. W.; Liddington, R. C.; Strongin, A. Y. J. Virol. 2007, 81, 4501. (72) Bera, A. K.; Kuhn, R. J.; Smith, J. L. J. Biol. Chem. 2007, 282, 12883. (73) Melino, S.; Paci, M. FEBS J. 2007, 274, 2986. (74) Shiryaev, S. A.; Kozlov, I. A.; Ratnikov, B. I.; Smith, J. W.; Lebl, M.; Strongin, A. Y. Biochem. J. 2007, 401, 743.

(75) Gouvea, I. E.; Izidoro, M. A.; Judice, W. A.; Cezari, M. H.; Caliendo, G.; Santagada, V.; dos Santos, C. N.; Queiroz, M. H.; Juliano, M. A.; Young, P. R.; Fairlie, D. P.; Juliano, L. Arch. Biochem. Biophys. 2007, 457, 187. (76) Keelapang, P.; Sriburi, R.; Supasa, S.; Panyadee, N.; Songjaeng, A.; Jairungsri, A.; Puttikhunt, C.; Kasinrerk, W.; Malasit, P.; Sittisombut, N. J. Virol. 2004, 78, 2367. (77) Niyomrattanakit, P.; Yahorava, S.; Mutule, I.; Mutulis, F.; Petrovska, R.; Prusis, P.; Katzenmeier, G.; Wikberg, J. E. Biochem. J. 2006, 397, 203. (78) Prusis, P.; Lapins, M.; Yahorava, S.; Petrovska, R.; Niyomrattanakit, P.; Katzenmeier, G.; Wikberg, J. E. Bioorg. Med. Chem. 2008, 16, 9369. (79) Prusis, P.; Junaid, M.; Petrovska, R.; Yahorava, S.; Yahorau, A.; Katzenmeier, G.; Lapins, M.; Wikberg, J. E. S. Biochem. Biophys. Res. Commun. 2013, 434, 767. (80) Deng, J.; Li, N.; Liu, H.; Zuo, Z.; Liew, O. W.; Xu, W.; Chen, G.; Tong, X.; Tang, W.; Zhu, J.; Zuo, J.; Jiang, H.; Yang, C. G.; Li, J.; Zhu, W. J. Med. Chem. 2012, 55, 6278. (81) Nitsche, C.; Klein, C. D. Methods Mol. Biol. 2013, 1030, 221. (82) Liu, Y.; Kati, W.; Chen, C. M.; Tripathi, R.; Molla, A.; Kohlbrenner, W. Anal. Biochem. 1999, 267, 331. (83) Yang, C. C.; Hu, H. S.; Wu, R. H.; Wu, S. H.; Lee, S. J.; Jiaang, W. T.; Chern, J. H.; Huang, Z. S.; Wu, H. N.; Chang, C. M.; Yueh, A. Antimicrob. Agents Chemother. 2014, 58, 110. (84) Yang, C. C.; Hsieh, Y. C.; Lee, S. J.; Wu, S. H.; Liao, C. L.; Tsao, C. H.; Chao, Y. S.; Chern, J. H.; Wu, C. P.; Yueh, A. Antimicrob. Agents Chemother. 2011, 55, 229. (85) Cregar-Hernandez, L.; Jiao, G. S.; Johnson, A. T.; Lehrer, A. T.; Wong, T. A.; Margosiak, S. A. Antiviral Chem. Chemother. 2011, 21, 209. (86) Chanprapaph, S.; Saparpakorn, P.; Sangma, C.; Niyomrattanakit, P.; Hannongbua, S.; Angsuthanasombat, C.; Katzenmeier, G. Biochem. Biophys. Res. Commun. 2005, 330, 1237. (87) Schüller, A.; Yin, Z.; Brian Chia, C. S.; Doan, D. N. P.; Kim, H.-K.; Shang, L.; Loh, T. P.; Hill, J.; Vasudevan, S. G. Antiviral Res. 2011, 92, 96. (88) Tomlinson, S. M.; Watowich, S. J. Antiviral Res. 2011, 89, 127. (89) Rothan, H. A.; Abdulrahman, A. Y.; Sasikumer, P. G.; Othman, S.; Rahman, N. A.; Yusof, R. J. Biomed. Biotechnol. 2012, 2012, 6. (90) Rothan, H.; Han, H. C.; Ramasamy, T. S.; Othman, S.; Rahman, N. A.; Yusof, R. BMC Infect. Dis. 2012, 12, 314. (91) Tomlinson, S. M.; Watowich, S. J. Antiviral Res. 2012, 93, 245. (92) Heh, C. H.; Othman, R.; Buckle, M. J. C.; Sharifuddin, Y.; Yusof, R.; Rahman, N. A. Chem. Biol. Drug Des. 2013, 82, 1. (93) Xu, S.; Li, H.; Shao, X.; Fan, C.; Ericksen, B.; Liu, J.; Chi, C.; Wang, C. J. Med. Chem. 2012, 55, 6881. (94) Mueller, N. H.; Pattabiraman, N.; Ansarah-Sobrinho, C.; Viswanathan, P.; Pierson, T. C.; Padmanabhan, R. Antimicrob. Agents Chemother. 2008, 52, 3385. (95) Ganesh, V. K.; Muller, N.; Judge, K.; Luan, C.-H.; Padmanabhan, R.; Murthy, K. H. M. Bioorg. Med. Chem. 2005, 13, 257. (96) Kiat, T. S.; Pippen, R.; Yusof, R.; Ibrahim, H.; Khalid, N.; Rahman, N. A. Bioorg. Med. Chem. Lett. 2006, 16, 3337. (97) Lai, H.; Dou, D.; Aravapalli, S.; Teramoto, T.; Lushington, G. H.; Mwania, T. M.; Alliston, K. R.; Eichhorn, D. M.; Padmanabhan, R.; Groutas, W. C. Bioorg. Med. Chem. 2013, 21, 102. (98) Lai, H.; Sridhar Prasad, G.; Padmanabhan, R. Antiviral Res. 2013, 97, 74. (99) Nguyen, T. T.; Lee, S.; Wang, H. K.; Chen, H. Y.; Wu, Y. T.; Lin, S. C.; Kim, D. W.; Kim, D. Molecules 2013, 18, 15600. (100) Aravapalli, S.; Lai, H.; Teramoto, T.; Alliston, K. R.; Lushington, G. H.; Ferguson, E. L.; Padmanabhan, R.; Groutas, W. C. Bioorg. Med. Chem. 2012, 20, 4140. (101) Tiew, K.-C.; Dou, D.; Teramoto, T.; Lai, H.; Alliston, K. R.; Lushington, G. H.; Padmanabhan, R.; Groutas, W. C. Bioorg. Med. Chem. 2012, 20, 1213. (102) Pambudi, S.; Kawashita, N.; Phanthanawiboon, S.; Omokoko, M. D.; Masrinoul, P.; Yamashita, A.; Limkittikul, K.; Yasunaga, T.; Takagi, T.; Ikuta, K.; Kurosu, T. Biochem. Biophys. Res. Commun. 2013, 440, 393. 11380

dx.doi.org/10.1021/cr500233q | Chem. Rev. 2014, 114, 11348−11381

Chemical Reviews

Review

(137) Yang, J. M.; Chen, Y. F.; Tu, Y. Y.; Yen, K. R.; Yang, Y. L. PloS One 2007, 2, e428. (138) Padmanabhan, R.; Pattabiraman, N.; Mueller, N.; Nagarajan, K. Patent WO 2010/039538 A2, 2010. (139) Ezgimen, M.; Lai, H.; Mueller, N. H.; Lee, K.; Cuny, G.; Ostrov, D. A.; Padmanabhan, R. Antiviral Res. 2012, 94, 18. (140) Boonyasuppayakorn, S.; Reichert, E. D.; Manzano, M.; Nagarajan, K.; Padmanabhan, R. Antiviral Res. 2014. (141) Padmanabhan, R.; Groutas, W. C. Patent WO 2010/039534 A2, 2010. (142) Knehans, T.; Schuller, A.; Doan, D. N.; Nacro, K.; Hill, J.; Guntert, P.; Madhusudhan, M. S.; Weil, T.; Vasudevan, S. G. J. Comput.Aided Mol. Des. 2011, 25, 263. (143) Ekonomiuk, D.; Su, X.-C.; Ozawa, K.; Bodenreider, C.; Lim, S. P.; Otting, G.; Huang, D.; Caflisch, A. J. Med. Chem. 2009, 52, 4860. (144) Shiryaev, S. A.; Cheltsov, A. V.; Gawlik, K.; Ratnikov, B. I.; Strongin, A. Y. Assay Drug Dev. Technol. 2011, 9, 69. (145) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.

(103) Rothan, H. A.; Bahrani, H.; Mohamed, Z.; Rahman, N. A.; Yusof, R. PloS One 2014, 9, e94561. (104) Rothan, H. A.; Buckle, M. J.; Ammar, Y. A.; Mohammadjavad, P.; Shatrah, O.; Rahman, N. A.; Rohana, Y. Trop. Biomed. 2013, 30, 681. (105) Rothan, H. A.; Mohamed, Z.; Paydar, M.; Rahman, N. A.; Yusof, R. Arch. Virol. 2014, 159, 711. (106) Rothan, H. A.; Mohamed, Z.; Suhaeb, A. M.; Rahman, N. A.; Yusof, R. Omics 2013, 17, 560. (107) Lai, H.; Teramoto, T.; Padmanabhan, R. Methods Mol. Biol. 2014, 1138, 345. (108) Manzano, M.; Padia, J.; Padmanabhan, R. Methods Mol. Biol. 2014, 1138, 331. (109) Steuer, C.; Heinonen, K. H.; Kattner, L.; Klein, C. D. J. Biomol. Screening 2009, 14, 1102. (110) Steuer, C.; Gege, C.; Fischl, W.; Heinonen, K. H.; Bartenschlager, R.; Klein, C. D. Bioorg. Med. Chem. 2011, 19, 4067. (111) Mendgen, T.; Steuer, C.; Klein, C. D. J. Med. Chem. 2012, 55, 743. (112) Nitsche, C.; Steuer, C.; Klein, C. D. Bioorg. Med. Chem. 2011, 19, 7318. (113) Nitsche, C.; Behnam, M. A. M.; Steuer, C.; Klein, C. D. Antiviral Res. 2012, 94, 72. (114) Nitsche, C.; Schreier, V. N.; Behnam, M. A.; Kumar, A.; Bartenschlager, R.; Klein, C. D. J. Med. Chem. 2013, 56, 8389. (115) Kang, C.; Gayen, S.; Wang, W.; Severin, R.; Chen, A. S.; Lim, H. A.; Chia, C. S.; Schuller, A.; Doan, D. N.; Poulsen, A.; Hill, J.; Vasudevan, S. G.; Keller, T. H. Antiviral Res. 2013, 97, 137. (116) Alves, F. M.; Hirata, I. Y.; Gouvea, I. E.; Alves, M. F.; Meldal, M.; Bromme, D.; Juliano, L.; Juliano, M. A. J. Comb. Chem. 2007, 9, 627. (117) Gao, Y.; Cui, T.; Lam, Y. Bioorg. Med. Chem. 2010, 18, 1331. (118) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4667. (119) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4677. (120) Ryan, A. J.; Gray, N. M.; Lowe, P. N.; Chung, C. W. J. Med. Chem. 2003, 46, 3448. (121) Aleshin, A. E.; Shiryaev, S. A.; Strongin, A. Y.; Liddington, R. C. Protein Sci. 2007, 16, 795. (122) Shiryaev, S. A.; Ratnikov, B. I.; Chekanov, A. V.; Sikora, S.; Rozanov, D. V.; Godzik, A.; Wang, J.; Smith, J. W.; Huang, Z.; Lindberg, I.; Samuel, M. A.; Diamond, M. S.; Strongin, A. Y. Biochem. J. 2006, 393, 503. (123) Doan, D. N.; Li, K. Q.; Basavannacharya, C.; Vasudevan, S. G.; Madhusudhan, M. S. Protein Eng. Des. Sel. 2012, 25, 843. (124) Potashman, M. H.; Duggan, M. E. J. Med. Chem. 2009, 52, 1231. (125) Lim, S. P.; Wang, Q. Y.; Noble, C. G.; Chen, Y. L.; Dong, H.; Zou, B.; Yokokawa, F.; Nilar, S.; Smith, P.; Beer, D.; Lescar, J.; Shi, P. Y. Antiviral Res. 2013, 100, 500. (126) Bone, R.; Shenvi, A. B.; Kettner, C. A.; Agard, D. A. Biochemistry 1987, 26, 7609. (127) Aghajanian, C.; Soignet, S.; Dizon, D. S.; Pien, C. S.; Adams, J.; Elliott, P. J.; Sabbatini, P.; Miller, V.; Hensley, M. L.; Pezzulli, S.; Canales, C.; Daud, A.; Spriggs, D. R. Clin. Cancer Res. 2002, 8, 2505. (128) Cui, T.; Puah, C. M.; Liew, O. W.; Lee, S. H. Patent WO 2007/ 149052 A1, 2007. (129) Giordanetto, F.; Kihlberg, J. J. Med. Chem. 2014, 57, 278. (130) Watowich, S. J.; Tomlinson, S. M.; Gilbertson, S. Patent WO2013/134135 A1, 2013. (131) Steinstraesser, L.; Tippler, B.; Mertens, J.; Lamme, E.; Homann, H. H.; Lehnhardt, M.; Wildner, O.; Steinau, H. U.; Uberla, K. Retrovirology 2005, 2, 2. (132) Cole, A. M.; Hong, T.; Boo, L. M.; Nguyen, T.; Zhao, C.; Bristol, G.; Zack, J. A.; Waring, A. J.; Yang, O. O.; Lehrer, R. I. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1813. (133) Zhou, G.-C.; Weng, Z.; Shao, X.; Liu, F.; Nie, X.; Liu, J.; Wang, D.; Wang, C.; Guo, K. Bioorg. Med. Chem. Lett. 2013, 23, 6549. (134) Behnam, M. A.; Nitsche, C.; Vechi, S. M.; Klein, C. D. ACS Med. Chem. Lett. 2014, 5, 1037. (135) Rahman, A.; Yusof, R.; Tat, T. K. Patent PI 20060021, 2006. (136) Tomlinson, S. M.; Malmstrom, R. D.; Russo, A.; Mueller, N.; Pang, Y.-P.; Watowich, S. J. Antiviral Res. 2009, 82, 110. 11381

dx.doi.org/10.1021/cr500233q | Chem. Rev. 2014, 114, 11348−11381