The Medicinal Chemistry of Dengue Virus - ACS Publications

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The Medicinal Chemistry of Dengue Virus Mira A.M. Behnam, Christoph Nitsche, Veaceslav Boldescu, and Christian D. Klein J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01653 • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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Journal of Medicinal Chemistry

The Medicinal Chemistry of Dengue Virus

Mira A. M. Behnam,† Christoph Nitsche,† Veaceslav Boldescu,†§ Christian D. Klein*,†

†

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg

University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany §

Laboratory of Organic Synthesis, Institute of Chemistry of the Academy of Sciences of

Moldova, Academiei 3, 2028 Chisinau, Moldova

1 ACS Paragon Plus Environment


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Abstract The dengue virus and related flaviviruses are an increasing global health threat. In this perspective, we comment and review medicinal chemistry efforts aimed at the prevention or treatment of dengue infections. We include target-based approaches aimed at viral or host factors, and results from phenotypic screenings at cellular assay systems for viral replication. This perspective is limited to the discussion of results that provide explicit chemistry, SAR, or appear to be of particular interest to the medicinal chemist for other reasons. The discovery and development efforts discussed here may at least partially be extrapolated towards other emerging flaviviral infections, such as West Nile virus. Therefore this perspective, although not specifically aimed at flaviviruses in general, should also be able to provide a general overview of the medicinal chemistry of these closely related infectious agents.

Introduction The dengue virus1 (DENV) is a growing global health concern, with an estimated 390 million infections occurring annually worldwide.2 Since neither specific antiviral drugs nor vaccines are available, the global disease burden caused by DENV infections is considerable. This perspective strives to give an overview of medicinal chemistry aimed at the treatment of DENV infections. It includes target-based and phenotypic screening approaches reported until October 2015. The present work is restricted to results that appear relevant or at least interesting from the perspective of a medicinal chemist. Not included are reports of experimental or virtual screening that lack experimental validation or did not result in hits with significant activity and no subsequent SAR exploration. We did not consider publications in which the chemical


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structures of the active materials remain undisclosed. We were also critical with respect to natural product screening and will not discuss screening results of raw extracts and of compounds – such as natural polyphenols – with a strong potential for assay interference and promiscuity. In general, we noticed numerous reports of initial hits from high-throughput screening campaigns or theoretical docking studies which showed weak or moderate activity at their respective targets but were not followed up by SAR work or cellular studies. This currently appears to be a major shortcoming within the field of anti-flaviviral drug research. It is to be hoped that future efforts will have the necessary endurance to continue studying and optimizing antiviral compounds, although this may be associated with difficulties in the academic settings where this type of research is often conducted. Following a general overview of the medical and biological aspects of DENV, the literature covered in this perspective is organized with respect to the initial screening method or target. We discuss here compounds acting at viral targets and host targets as well as antiviral compounds identified by phenotypic screening for which the relevant target remains unclear. The aim is to provide the reader with a commented overview of the current status and to allow the identification of particularly promising routes towards novel dengue therapeutics.

Transmission and ecology of DENV DENV is transmitted by arthropod vectors,3 in particular mosquitoes such as Aedes aegypti 4 and to a lesser extent Aedes albopictus, and is therefore classified as an "arbovirus" (= arthropodborne). Vector control is the major preventive measure currently employed to control dengue outbreaks. The mosquito vectors are present in most tropical and subtropical regions, but are spreading towards higher latitudes. Dengue viruses persist within nonhuman primates in a



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"sylvatic" lifecycle,5 from which novel serotypes may emerge in the future. The recent major epidemic outbreaks, affecting millions of people, are caused by the four dengue serotypes for which a continuous inter-human transmission is established. Recently, a fifth serotype of dengue was reported.6

Clinical aspects of dengue virus infection The severity of dengue infections varies over a surprisingly wide range, and only about one quarter of infected persons develop symptomatic disease. Most of these patients, while experiencing highly unpleasant symptoms such as high fever and pain, recover without severe sequelae. A relatively small fraction of patients develops aggravated disease in the form of dengue hemorrhagic fever (DHF), recently also denoted as "severe dengue", with a potentially lethal outcome. The therapy of all clinical manifestations remains, up to now, symptomatic. Importantly, a previous DENV infection, resulting in the presence of antibodies against that serotype, appears to put patients at higher risk of developing severe disease upon infection with another serotype.7 The underlying mechanism, termed antibody-dependent enhancement (ADE),8-9 leads to an increase of viral load in experimental animals by a factor of up to 100.10-11

Vaccine development Attempts at the development of dengue vaccines started about 90 years ago,12-13 but success has, so far, been limited. The major impediments towards vaccination are: Vaccines should have a tetravalent effect, that is, result in immunogenic response against all four serotypes of DENV. If protection against one or more serotypes remains insufficient, then the infection with one of these serotypes may result in aggravated disease in "vaccinated" persons, due to ADE as


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discussed above. The recent reports on a fifth DENV phenotype6 put the tetravalent vaccines currently in development under additional scrutiny. Recently published stage-three clinical trials of a tetravalent dengue vaccine came up with mixed results,14 with an expert commenting that "The bumpy road to a vaccine-based solution for dengue continues".15

Genetics and replication mechanism DENV belongs to the genus Flavivirus within the family of Flaviviridae. The genus Flavivirus encompasses several other pathogenic viruses, among them the namesake species yellow-fevervirus (YFV), the West-Nile-virus (WNV) and the tick-borne-encephalitis virus (TBEV). Within the Flaviviridae family, the genus Hepacivirus contains another prominent pathogen, the hepatitis C virus (HCV). The Flaviviridae are positive-single-strand RNA viruses, containing a relatively small genome of around 10 kilobases. This genome codes for a polyprotein which is split by host proteases (furin, signalase) and by a viral protease into non-structural (NS) and structural proteins, which are subsequently assembled to form new virions. The structural proteins of DENV are located at the N-terminus of the viral polyprotein and encompass an envelope (E), capsid (C) and membrane-protein precursor (prM) protein. Towards the Cterminus, the non-structural proteins follow, which are numbered in accordance to their location within the polyprotein. A functional role could be assigned to some of the non-structural proteins: NS3 contains a helicase and a protease domain, and NS5 contains RNA polymerase and methyltransferase domains. A subdomain of the NS2 protein participates in the formation of the substrate recognition site of the viral protease (NS3). The other nonstructural proteins have, so far, been described to participate in the formation of the viral replication complex at the



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endoplasmatic reticulum and in the escape of host immune response. The replication mechanism of flaviviruses is depicted in Figure 1.

Figure 1. Key steps of flavivirus replication. Indicated are the target sites for pharmacological interaction. Targets that are, to our knowledge, not extensively exploret yet are parenthesized. The replication complex (5) is a structure composed of membranes and vesicles along with viral and probably also host proteins. It forms after viral infection and appears to harbor several steps


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of flaviviral replication. Further processing and assembly of progeny virions in the trans-Golgi network relies heavily on host factors.

For the viral enzymes, in particular the protease, in vitro assay systems have been established that are amenable to high-throughput screening and consequently allowed the discovery of screening hits. However, the success of follow-up medicinal chemistry has apparently been limited, since very few high-affinity compounds or leads that were derived from these hits have been reported so far. Phenotypic screening has resulted in several promising hits that act on nonstructural proteins whose function remains unclear.

Phenotypic screening and animal models The replication of dengue virus in cell culture was reported as early as 195016-17 and has since been performed in a large variety of mammalian and insect cells. Today, the preferred in vitro systems for the study of DENV infections are Huh-7 and Vero cells, and detailed antiviral screening protocols, amenable for high-throughput screening, are available.18-19 Reporter gene20 and subgenomic replicon assays21 were also developed. Scheme 1 provides an overview of cellular assay systems for biological studies and the characterization of antiviral agents. Resistance breeding experiments allow the identification of viral targets for compounds identified in phenotypic screens.



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Scheme 1: Schematic classification of cell culture assays for dengue infection and replication. With respect to animal studies of DENV infection and pathology, a variety of primate and rodent models exist and were recently reviewed by Vasudevan et al.22 Most of these animal models, however, suffer from the fact that key pathogenic steps such as ADE cannot be reproduced. Considerable efforts have therefore been made to develop mouse models that allow the study of this – for humans, but not for other organisms – highly relevant pathogenic mechanism of DENV. In spite of these efforts, the human DENV infection model remains essential, particularly for DENV vaccine research.23

Host targets DENV and other flaviviridae have a very small genome and therefore rely extensively on the exploitation of numerous host factors to achieve replication. Such host factors represent attractive target sites for antiviral agents, in particular because the development of resistance is hindered considerably. Furthermore, antiviral agents acting at host factors are more likely to 8 ACS Paragon Plus Environment

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have pan-antiviral activity, acting not only against DENV but also against related flaviviruses such as WNV. Phenotypic screening in cell culture resulted in the discovery of several promising antiviral hits that act on host targets, which we will comment and review in this perspective. High-throughput RNAi screening technology, in combination with cell culture models of virus replication, can aid in the identification of host targets for antiviral agents. In an early report, performed using DENV-infected insect cells, (notional) mammalian host factors were "extrapolated" from these results.24 Not unexpectedly, we were unable to identify any significant follow-up on this study in the direction of drug-discovery or target validation. Since the early days of RNAi screening, however, the methodology (as well as the scrutiny applied to the results) has ripened, and we expect it to identify host factors with higher validity in the near future. A more promising route towards the identification of host factors, at least for those with potential druggability, appears to be the screening of compound libraries and the subsequent identification of their target proteins. As reviewed below, this resulted in the discovery of various promising host targets.

Antiviral agents against dengue virus DENV entry inhibitors (EI) Infection of a cell by DENV starts with adsorption, internalization, and fusion of the virus with the host cells. The DENV envelope (E) glycoprotein is responsible for receptor recognition and attachment to the cell, clathrin mediated endocytosis, and involved in fusion of viral and cellular membranes. The E glycoprotein comprises several domains, of which the stem domain, the hydrophobic pocket (β-OG pocket), and the receptor binding domain III have been pursued as



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drug targets.25 It is noteworthy that majority of developed inhibitors of DENV adsorption target viral proteins and not host cell surface proteins. This can be explained through a high variety of receptors to which DENV binds depending on the type of the cells it is trying to infect. These include: heparan sulfate (hepatocytes, epithelial cells), DC-SIGN (dendritic cells), mannose receptor (monocytes, macrophages), CD12-associated protein (monocytes, macrophages), TIM and TAM receptors (epithelial cells, astrocytes), and glycosphingolipids (lymphoblasts).26

EI targeting the stem domain of the E glycoprotein Several research groups have demonstrated that short peptides derived from the E glycoprotein inhibit DENV replication in cell culture,27-29 some of them (peptides 1 and 2) being able to inhibit antibody-enhanced DENV infection.30 Inhibition occurs as a result of protein binding to the E protein trimer intermediate, induction of structural changes in the surface of DENV virions, and interference with virus-cell binding.27-29 1 (FWFTLIKTQAKQPARYRRFC) is a peptide designed to displace regions in the domain II hinge and the first domain I/domain II connection of E glycoprotein. It shows IC50 value of 7 µM in a focus forming unit (FFU) assay.29 2, a 33 amino acid peptide (MAILGDTAWDFGSLGGVFTSIGKALHQVFGAIY) mimicking the stem region of DENV-2 (residues 412-144), has been shown to reduce the infectivity of all four dengue virus serotypes by 50% at concentrations of 2-5 µM in a FFU assay in LLC-MK2 cells.31 Stem-derived peptides mimicking sequences 419-447 from DENV-1 to DENV-4 demonstrated cross-reactive antiviral activity (IC90 0.1-6.0 µM). Besides, it was revealed that residues 442-444 of the stem are determinants of inhibition and an increase of hydrophobicity in this region enhances inhibitory strength.32 Later, Schmidt et al.33 developed a fluorescence-


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polarization HTS method for compounds that block glycopeptide E binding and thus may inhibit viral entry. An active compound 3 (IC90=16.9 µM (PFU assay), CC50>100 µM) detected as a hit and a number of its analogues were shown to reversibly inhibit DENV-2 infectivity, with the most active 4 having IC90 around 1.5 µM (PFU assay) with CC50 = 84.5 µM.33

EI targeting the hydrophobic (β-OG) pocket of the E glycoprotein In 2003, together with the crystal structure of the DENV E glycoprotein, Modis et al.34 described a hydrophobic pocket between the domains I and II and suggested this pocket as a promising ligand binding site. One of the identified ligands was a detergent from the crystallisation buffer, n-octyl-β-D-glucoside (β-OG), from which the pocket received an alternative name: β-OG pocket. This observation initiated a search for other ligands of this site. Thus, Poh et al.35 performed an in silico virtual screen aimed at this pocket and tested the candidate molecules in two newly developed cell-based fusion assays. The first assay quantifies propidium iodide stained mosquito cells with membranes damaged upon viral protein-induced fusion. The second assay measures trypsin activity upon fusion between DENV and trypsincontaining liposomes. The most active compound, 5, inhibited dengue fusion in both assays with IC50 =6.8 µM and reduced viral titers with an EC50 of 9.8 µM in BHK cells infected with DENV2 (CC50 = 48.7 µM).35 In another virtual screen performed by Wang et al.36 111 compounds were selected for the biological assay from which 2 pyrimidine derivatives were selected for further SAR analysis and structural modifications to obtain more potent derivatives. The pyrimidine derivative, 6, and the quinazoline derivative, 7, were identified as being most potent, with EC50 values of 0.09 ± 0.01 and 0.07 ± 0.01 µM respectively, determined in cell-based flavivirus immunodetection assay



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(CFI), and 0.041 and 0.198 µM, in the plaque reduction assay, with CC50 values >20 µM in both cases. Despite better performance of 6 in the plaque reduction assay, 7 was chosen for further analyses of its activity and selectivity. From these, it was detected that it is efficacious against all four dengue serotypes with EC50 values ranging from 0.068 to 0.496 µM, as well as against other flaviviruses, such as YFV, WNV, and Japanese encephalitis virus (JEV).36 An in silico screen of a chemical database targeted at the hydrophobic pocket of the glycoprotein E resulted in the identification of 8.37 This was found to have low micromolar inhibitory activity against DENV-2 in the plaque reduction assay, as well as WNV and YFV, with IC50 values respectively 1.2, 3.8, and 1.6 µM, and selectivity index (SI = CC50 / EC50) more than 83. The potential tox/PK liabilities of the hydrazone moiety of 8 were not investigated further. The cytotoxic drug doxorubicin was shown to have an antiviral effect against dengue in cell culture.38 A derivative of doxorubicin with lower cytotoxicity, 9, containing a squaric acid amide ester moiety at the carbohydrate group, was identified in a search for less cytotoxic analogues. The compound is active against DENV in a cellular assay (EC50 = 0.52 ± 0.31 µM (DENV-2), CC50 = 28 ± 2.7 µM, MW = 653). Interestingly, 9 is not effective against DENV-4,38 but against antibody-opsonized and immature DENV-2.39 Molecular modeling studies indicated that 9 fits into the hydrophobic pocket of the E glycoprotein, but experimental validation for this mode-ofaction is lacking. Recently, Jadav et al.40 reported the exploration and optimization of a novel class of β-OG pocket-binding hybrid inhibitors developed from two hits previously identified by in silico screening and evaluated in vitro for antiflaviviral activity by Zhou et al.41 and Li et al.42 The most active of the developed inhibitors was 10 (EC50 = 1.39 ± 0.06 µM, DENV-2, plaque reduction assay, CC50 = 125 ± 40.8 µM, MW = 344). A docking simulation of the studied


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compounds aimed at the β-OG pocket indicates the following key factors: hydrophobic interaction with Leu207, π→π interaction with either Phe193 or Phe297, and interactions at the surface by H-bonding and a hydrophobic interaction with either Ile270 or Leu277.

EI targeting receptor binding domain III and other targets on glycoprotein E E glycoprotein domain III appears to be responsible for the initial contact and accumulation of DENV on the surface of host cells by binding to glycosaminoglycan (GAG) receptors.43-45 GAGs are long unbranched sulfated polysaccharides linked to core cell surface proteins.46 DENV was shown to bind to heparan-sulfate proteoglycans or syndecans on the outer surface of the host cells43-45 and, as a consequence, numerous studies were aimed at the discovery of new entry inhibitors mimicking the heparan sulfate (HS) moiety of these receptors. Starting from heparin, these studies identified a variety of sulfated poly-sugars and glycosaminoglycans with different levels of activity. The following compounds have been reported among the most active: chondroitin sulfate E (EC50 = 0.3-3.8 µg/mL (DENV serotypes 1-4), MW approx. 55000),47 λand ι-carrageenan (EC50 = 0.14-1.1 µg/mL (DENV-2 and DENV-3),48 fucoidan (EC50 = 4.7 µg/mL (DENV-2), MW approx. = 20000),49 pentosane polysulfate (EC50 30 µg/mL (DENV-2), MW approx. = 5700),50 λ- and ι-carrageenan (EC50 = 0.14-1.1 µg/mL (DENV-2 and DENV-3) and suramin (EC50 = 40 µM (DENV-2), MW = 1429).43, 51 Investigation of the SAR of these compounds demonstrated that the specific carbohydrate structure, rather than the degree of sulfation, is directly related to E glycoprotein binding and therefore to inhibitory activity. Thus, a disaccharide, with no matter how many sulfate groups, does not bind to the relatively elongated polyanion-binding site in DENV glycoprotein E. A tetrasaccharide (sulfated lactobionic acid) and a hexasaccharide (sulfated β-cyclodextrin), while



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sufficiently large to interact with the E glycoprotein, still do not bind strong enough to its polyanion-binding site. At the same time, a decasaccharide analogue of heparin and suramin, which is comparable in size, bind to E glycoprotein with identical affinity.43, 51 It was concluded that the minimum molecular size required for a strong interaction between a polyanion and E glycoprotein is around 40 Å. At the same time, these compounds should preferably have a high charge density and high level of structural flexibility.51 Two major problems related to the use of HS mimetics are their anti-coagulant activity and low bioavailability due to binding to plasma proteins. As a result, the search for new, less toxic and more effective HS mimetics is continued. Among the promising compounds binding to other parts of glycoprotein E is curdlan sulfate (CS) that binds to the interface between domains II and III and shows a good level of activity in plaque forming assay (EC50 = 7.0 µg/mL (DENV-2), 10 µg/mL (DENV-3), CC50 >104 µg/mL, MW approx. 41000).52 The main advantage of CS is low anti-coagulant activity (in contrast to heparin) and good tolerability by patients detected in clinical trials against HIV1 and malaria.53-54 Interestingly, a peptide, MLH40, derived from the conserved ectodomain region of the DENV M protein has been reported to block viral entry in all four viral serotypes at EC50 in the range of 24-31 µM.55 Moreover, the inhibitory activity was observed in several cell lines, including Vero, A549, and Huh-7. Binding to the DENV E protein was suggested by docking simulations. Another interesting compound with suggested, but not fully confirmed, binding to E glycoprotein is a teicoplanin derivative, 11, that has been reported to inhibit DENV-2 replication in vitro at EC50 = 6.9 ± 2.9 µM with a SI > 15. Importantly, the compound also exerts its antiviral effect towards DENV particles opsonized with antibodies. This effect was not antibody specific and may therefore be particularly valuable in the prevention of severe dengue induced by ADE.


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Several authors suggested that domain III is strongly conserved within each DENV serotype and can serve as target for vaccines, since it contains epitopes recognized by neutralizing antibodies.56-57 Among the promising results on development of anti-dengue antibodies with binding sites on DENV E glycoprotein and high efficiency in vivo, most outstanding are the results on antibodies 2D2258-59 and Ab513.60 2D22 is a DENV-2 specific human monoclonal antibody that binds across all three main domains of the glycoprotein E and locks both ends of its dimers, thus preventing its reorganization required for virus fusion.58 Studies in vitro demonstrated that 2D22 has a potent neutralization capacity against DENV-2,59 while in vivo tests in an AG129 mouse model demonstrated that 2D22 administrated before or after infection protects against DENV2.58 The latest facts indicate that the antibody has a high potential as prophylactic and therapeutic agent.58 Another antibody, Ab513, was engineered to bind to domain III and demonstrated high potency in vivo in AG129 mice against all four dengue serotypes. It has been shown to increase animal survival, reduce viremia, shorten the duration of thrombocytopenia, and reduce vascular leakage.60 These latest findings demonstrate that antibody-based immunotherapy can be potentially developed into an effective treatment of DENV infection.



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Figure 2: DENV entry inhibitors.

Ligands of the capsid protein (C) DENV C is a highly positively charged homodimeric protein containing 100 amino acid residues, 25 of them being Arg and Lys. NMR studies of the protein 3D structure revealed that its monomer contains four α-helical regions, and an intrinsically disordered N-terminal domain.61 The monomer core includes helixes α1-α3, with a largely uncharged region suggested to interact with lipid systems, and the longest helix α4 that extends away from the core and is suggested to interact with the viral RNA due to high density of basic amino acid residues.61-62 N-terminal region, as well as pep14-23, a peptide partially corresponding to N-terminal region sequence, are also involved in the interaction with lipid systems and are capable of inhibition of the hydrophobic core of the C protein.63 Targeting the DENV C hydrophobic core and N-terminal region are two promising strategies for anti-dengue drug development. A small molecule inhibitor of DENV C, 12,64 was identified by a phenotypic HTS assay for virus-induced CPE and reduces virus titer for all four serotypes in the low micromolar to sub-micromolar range,


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with the highest potency against DENV-2 (EC50 = 0.016 µM and EC90 = 0.125 µM). The interaction of 12 with the capsid protein was confirmed by dose-dependent quenching of intrinsic fluorescence of the target and resistance mapping to a serine→leucine mutation in position 34 of the C-protein. Pharmacokinetic analyses of the compound in AG129 mice indicated limited oral bioavailability (9.1%). 12 displayed efficacy in vivo by an average decrease of peak plasma viremia by 52-fold and reduction of viral load in the spleen and liver by 3- and 20-fold, respectively, following BID (twice daily) treatment. The exact mode of action of 12 was further characterized by Scaturro and coworkers,65 who found dual antiviral effects on assembly/release and entry of DENV infectious particles. Contrary to the mechanism suggested previously,64 12 did not alter interaction between capsid protein and lipid droplets. Instead, it affected the intracellular distribution of C-protein, through a shift of abundance from the cytosol to the nucleoli that was detected by immunofluorescence. Antiviral effects were mediated by increased capsid self-interaction as observed in a bioluminescence resonance energy transfer-based (BRET) assay, thus making 12 a direct protein-protein interaction (PPI) stabilizer.65

Figure 3: ST-148, a ligand of the dengue virus capsid protein with antiviral effect.

Inhibitors of the NS3 helicase Flavivirus helicase plays an important role in viral infection by separating dsRNA during viral replication. Helicase activity is an intrinsic property of the C-proximal domain of NS3 starting between residues 160 and 180.66-67 The structure of the DENV2 helicase catalytic domain was



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reported by Xu et al.68 and reveals a three-lobed flattened topology with a large number of loops with overall dimensions of about 60:60:35 Å and a significant structural feature - a long tunnel that runs across the center of one face of the protein. Residues Arg-457 and Arg-458 were shown to be involved in coupling of helicase and NTPase enzymatic activities and their substitution with Ala residues led to total loss of the first.68-69 Using in silico docking to explore the single-strand RNA access site within WNV helicase, ivermectin, a broad spectrum antiparasitic drug, was identified as potential inhibitor of NS3 helicase.70 Ivermectin displayed uncompetitive inhibition of flaviviral helicase unwinding activity in biochemical assays in the upper nanomolar range for WNV and DENV, but did not affect the ATPase activity of the NS3 helicase domain. In cell culture, ivermectin caused virus titer reduction with an EC50 of 0.7 µM for DENV and 4 µM for WNV.70 It should be noted, however, that the effect on DENV replication could also be attributed to the inhibition of DENV protease.71 Byrd et al. performed a high-throughput assay based on inhibition of DENV-induced CPE leading to the discovery of 13.72 The compound selectively inhibited DENV replication for all four serotypes in cell culture with EC50 in the range of 0.203-0.272 µM. Resistance to 13 was linked to a single point mutation (A263T) in the NS3 helicase domain. As for ivermectin, 13 inhibitory activity was limited to the unwinding activity of the helicase. When administered intraperitoneally to AG129 mice, 13 had minimal effect on peak viremia reduction, which could be potentially caused by its poor pharmacokinetic profile in vivo. Another HTS using a molecular beacon helicase assay identified suramin as inhibitor of DENV helicase. The compound had a non-competitive mode of inhibition, with a Ki of 0.75 µM.73 Considering the negligible permeability of suramin across cell walls, any effects that it may have


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in cellular systems of viral replication must be attributed to its effect on viral entry (cf. the corresponding section of this Perspective). A screen of drug-like fragments by thermal-shift assay (TSA) failed to confirm any hit for DENV helicase, suggesting that the target may not be suitable for fragment-based drug discovery.74 The same strategy was, however, successful at DENV methyltransferase.74 In addition to the aforementioned inhibitors acting on the DENV NS3-catalyzed RNA unwinding, reports included also inhibitors of the RNA-dependent ATPase activity of NS3 helicase: A series of HCV helicase inhibitors, synthesized by Li et al.,75 were further assessed by Ndjomou et al.76 revealing in vitro inhibitors of DENV helicase ATPase activity. The compounds are derived from primuline, a yellow fluorescent dye, and contain a dimeric benzothiazole core scaffold. The highest activity at DENV helicase was observed for 14, with an IC50 of 0.5 µM.76 The same compound inhibited DENV helicase unwinding activity with an IC50 of 1.5 µM, and had an EC50 value of 7.1 µM in a DENV replicon assay.77 Sweeney et al. established a DENV NS3 ATPase HTS assay,77 and combined this with DENV RNA unwinding assays to characterize the ability of the identified inhibitor to interfere with one or both NS3catalyzed functions. The HTS identified two chemotypes: pyrrolone derivatives with weak but specific effect on DENV ATPase, and the primuline analogues targeting both RNA unwinding and ATPase activities of NS3. The pyrrolone analogues inhibited WNV and DENV replication in cell culture (EC50 = 36 µM at DENV for the best derivative 15), but displayed poor selectivity (CC50 / EC50 = 4.5).77



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OH O S O

O O S O

HO

N N

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N O

O

O S

HO

13

HN

O

O NH

O HN

O

HN Suramin

HO

O O

NH

O H

O O

O H

O O

O

H

NH

O O OH

Ivermectin

O S O HO

O H

O

OH

O O S OH O S OH

Cl Cl

N

NH4

H N

O O S O

NH2

O

S

N O

O

S

N

S

O

N

HO

N 14

O

15

Figure 4: Inhibitors of dengue virus NS3 helicase.

Inhibitors of the NS2B-NS3 protease The biochemical and structural properties of dengue protease and the previous efforts aimed at inhibitor discovery have recently been extensively reviewed.78 Among the non-structural viral proteins, the NS2B-NS3 protease is one of the best studied targets for the development of antiinfective therapeutics against dengue virus. The dengue protease is responsible for the posttranslational cleavage of the viral polyprotein and therefore essential for viral replication. In the past, other viral proteases, such as those of HCV and HIV, have been successfully established as targets with therapeutic relevance. Numerous publications within the last decade report the characterization of DENV NS2B-NS3 protease and inhibitor discovery. Therefore, the interested reader is kindly referred to the recent review78 for a more detailed analysis of the target and the comprehensive history of assay and substrate development and inhibitor discovery. The present work focuses on those reports which we consider particularly promising.


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The so far developed inhibitors can approximately be classified as peptidic, peptide-derived or non-peptidic (small molecular) compounds. Aprotinin (bovine pancreatic trypsin inhibitor, BPTI), a polypeptide comprising 58 amino acids with three internal disulfide bonds, was found to have high affinity, sometimes reported in the very low nanomolar range, to DENV protease.7981

Because of its commercial availability, aprotinin is frequently used as a reference compound in

protease assays. It is also one of the few inhibitors for which an X-ray co-crystal structure with the protease could be solved.82 One of the first reported shorter peptide inhibitors 16 (Ac-RTSKKR-NH2) showed a Ki value (DENV-2) of 12 µM.83 Promising in vitro activity (Ki = 1.4 µM, DENV-2 protease) was also found for the small cyclic peptide 17 (CGKRKSC), cyclized by a disulfide bond.84 However, recent retrospective studies revealed no antiviral activity in cell culture for both candidates.85 A short peptide without basic side chain residues 18 (WYCW-NH2) was reported with Ki values of 4.2, 4.8, 24.4, and 11.2 µM for the protease from serotypes 1, 2, 3, and 4, respectively.86 Cellular data for this compound are so far unavailable. Small peptides with a C-terminal electrophilic trap for additional covalent interactions with the catalytically active serine of the protease of DENV-2 have also been reported.87-89 Within a series of peptide aldehydes, 19 (Bz-KRR-H) reached a Ki of 1.5 µM. For the analogue 20 (Bz-nKRR-H, where n = norleucine) with a Ki of 5.8 µM, a highly important co-crystal structure, revealing the placement of a covalent ligand in the substrate binding site, has been reported (pdb code: 3U1I).82 However, a similar analogue, 21 (Bz-AKRR-H), with comparable in vitro activity (Ki = 5.3 µM) showed neither antiviral activity nor sufficient permeability (PAMPA).85 The value of peptide aldehydes appears to be, not quite unexpectedly, limited to in vitro and co-crystallization studies.



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The combination with a boronic acid electrophile turned out to be very promising with a 135fold affinity increase compared to the aldehyde analogue. The resulting compound 22 (BznKRR-B(OH)2) reached a Ki of 43 nM. However, further studies (including cellular data) are lacking.87 In summary, peptidic inhibitors are useful tools for the generation of X-ray crystal structures and revealing the substrate specificity and binding sites. Some of them reach high affinities, however those few examples for which cellular data have been reported do not show relevant antiviral activity. Figure 5 shows peptide-derived inhibitors of DENV-2 protease. These tripeptides with nonpeptidic N-terminal caps were derived from the moderately affine 23 (Ki = 15.6 µM),90 which is lacking antiviral activity in cells.85 The evolution of the N-terminal cap led to 24 with increased in vitro affinity (Ki = 4.9 µM),90 and subsequently to compounds 25 and 26, which showed promising antiviral activity in cell culture with EC50 values of 44.9 and 16.7 µM, respectively.91 Noteworthy, 25 (Ki = 1.8 µM, Ki‘ = 7.9 µM) and its analogues showed more pronounced in vitro activity with dominating competitive binding mechanisms compared to 26 (Ki = 9.3 µM, Ki‘ = 4.4 µM) and its analogues with mixed inhibition modes, which, however, performed better in the cellular assay.91 The enhanced cellular activity of 26 can be explained by improved permeability91 or off-target effects of the N-terminal rhodanine cap. The potential promiscuity of this group and its activity at unrelated targets is discussed in a later chapter (kindly refer to DENV NS5 methyltransferase inhibitors). Recently, an additional study found that a C-terminal phenylglycine residue increases affinity significantly, resulting in compound 27 with a Ki of 0.4 µM.92 An additional extensive screening regarding hydrophobic substituents at the N-terminal thiazolidinedione domain provided a series of dual inhibitors of DENV-2 and WNV proteases in


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vitro.93 While the nature of the N-terminal capping group had only limited influence on the inhibitory activity at the protease from DENV-2, a nearly 3-fold higher activity in comparison to 35 could be achieved at the WNV protease.93 The compounds mostly retained submicromolar potency at DENV-2 protease with 28 showing the lowest Ki value between 0.1 and 0.4 µM (depending on the calculation method). A combined search for replacements of the arginine side chain and smaller N-terminal caps yielded a number of compounds with increased potency, for example 29 with a benzamidine side chain and a 4-trifluoromethylbenzoyl cap.94 This compound showed promising in vitro activity (Ki = 0.14 µM) and 38% reduction of virus titer at 50 µM in a plaque assay. Recently, 4-benzyloxy-D-phenylglycine and numerous congeners thereof were combined as nonnatural C-terminal residues with the sequence (Bz-Arg-Lys), for example in compound 30.95 The modification enhanced the in vitro activity at both DENV-2 and WNV proteases (IC50 = 0.367 µM and 0.728 µM, respectively). A fragment merging strategy generated a class of highly affine dual-inhibitors of DENV-2 and WNV proteases, with remarkable selectivity against thrombin and trypsin. The most active analogue, compound 31, incorporated a 4trifluoromethylbenzyl ether and a 2-thiazole cap. This derivative reached a Ki value of 12 nM at DENV-2 and 39 nM at WNV proteases and showed cellular activity in DENV-2 and WNV plaque assays (EC50 = 20.3 ± 2.5 µM at DENV-2, and 23.3 ± 3.3 µM at WNV) without cytotoxicity (EC50 = 20.35 ± 2.50 µM, (CC50 >100 µM).

Compound 32, with a 3-

methoxybenzyl ether and a bithiophene cap, (IC50 = 0.176 µM and 0.557 µM, at DENV-2 and WNV respectively) displayed the highest activity in cell-based assay at DENV-2 (EC50 = 3.42 ± 1.30 µM, CC50 >100 µM, SI >25) and WNV (EC50 = 15.5 ± 0.9 µM, CC50 >100 µM, SI >7)..95 The lower activity in cellular assays could be explained by the insufficient permeability of these



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compounds as observed in PAMPA assay, despite the enhanced metabolic stability. Specific interaction of 31 and 32 at both viral targets was demonstrated by tryptophan quenching assays.95 Several approaches towards non-peptidic small-molecular inhibitors of dengue protease have been reported during the last decade.78 A considerable proportion of these screening efforts remained unfruitful mostly due to cytotoxicity or inadequate permeability, stability and antiviral activity of in vitro hits.78,

85

A significant number of compounds that failed in cellular assays

contain multiple phenolic, aromatic amine or guanidine moieties with PK/Tox liabilities. Some successful examples from previous studies are shown in Figure 6. 33 was reported as DENV-2 protease inhibitor with moderate affinity (39% inhibition at 50 µM) but good ligand efficiency and significant antiviral activity at concentrations higher than 10 µM, without cytotoxic effects at 100 µM.96 The amphiphilic quaternary ammonium salts 34 and 35 were independently identified from two screening campaigns.97-98 Both compounds inhibit DENV-2 protease in vitro (IC50 = 15.4 µM for 34, IC50 = 22.6 µM for 35) and viral replication of all four serotypes in cellular assays with moderate cytotoxicity for 34 (CC50 = 29.3 µM) and without detectable cytotoxic effects for 35. Resistance breeding experiments confirmed protease inhibition in cell culture. Noteworthy, antiviral activity in cell culture is relatively high in comparison to DENV protease affinity (EC50 = 0.17 µM for 34, EC50 = 1.03 µM for 35), which may be explained by the artificial character of the isolated protease construct, accumulation in the host cells or compartments, or binding to non-protease targets. Compound 36 was developed from a virtual screening hit, followed by a scaffold hopping approach (IC50 = 9.45 µM).99 It shows antiviral activity in a dengue replicon assay (EC50 = 24.7 µM) without relevant cytotoxicity (CC50 > 100 µM).


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Compounds 37 and 38 were identified from independent virtual screening campaigns.100-101 37 was reported as non-competitive inhibitor of DENV-2 protease with promising antiviral activity against all four serotypes (EC50 = 0.97, 0.98, 2.43, 0.74 µM for DENV-1, 2, 3, 4, respectively) and moderate cytotoxicity (CC50 = 67.3 µM).100 38 was obtained from a WNV-inspired screening campaign, but shows also inhibition of DENV-2 protease (IC50 = 2.75 µM) along with moderate phenotypic antiviral activity (EC50 = 39.9 µM) and low cytotoxicity (CC50 = 213 µM). Other recent results in the development of small-molecular dengue protease inhibitors are highlighted in Figure 7. Viswanathan and coworkers identified 39 by a virtual screening approach. Experimentally, the compound turned out to be a mixed non-competitive inhibitor (Ki = 7 µM, Ki’ = 15 µM at DENV-2 protease).102 However, cellular data were not reported, the compound appears to be relatively lipophilic, and it is therefore difficult to assess the real potential of this hit. Compound 40 shows DENV-2 protease inhibition with an IC50 value of 2.24 µM.103 The direct interaction with the enzyme was confirmed by surface plasmon resonance spectroscopy (Kd = 2.07 µM). A screening from an in-house library by Raut and coworkers revealed 41 as DENV-2 protease inhibitor with an IC50 value of 5.95 µM and significant reduction of viral titers for all four serotypes at 30 µM (no cytotoxicity at 100 µM).104 It is noteworthy that the arylcyanoacrylamide fragment reported before (compound 24, Figure 5) re-appears in 40 and 41.105 Compound 42 was obtained by a computational screening approach and shows only moderate inhibition of DENV-2 protease (85% at 300 µM), but promising antiviral results in cell culture (BHK-21 cells: EC50 = 5.0 µM, CC50 > 300 µM; Huh-7 cells: EC50 = 5.0 µM, CC50 = 55 µM).106



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The obvious discrepancy between promising viral inhibition in cell culture and moderate protease affinity of 42 suggests an alternative mode of action. Wu et al. reported compound 43 as inhibitor of DENV protease of the serotypes 2 (IC50 = 4.2 µM) and 3 (IC50 = 0.99 µM).107 A cell-based protease assay revealed an IC50 of 3.2 µM. This compound also showed promising antiviral activity in Vero cells (EC50 = 0.8 µM), but had cytotoxic effects at concentrations above 10 µM.

Figure 5: Peptide-derived inhibitors of dengue virus protease.


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Figure 6: Small-molecular inhibitors of dengue protease with antiviral activity in cell culture.

Figure 7: Recently published small-molecular inhibitors of dengue virus protease.

Inhibitors of NS4B NS4B is one of the small hydrophobic NS proteins of dengue virus, consisting of 248 amino acid residues. It was shown to interact in its full length with helicase domain of NS3 and dissociate it from ss-RNA, enhancing thus helicase activity of NS3 and modulating viral replication.108 Later, Kakumani et al.109 identified NS4B protein as one of the DENV proteins inhibiting RNAi machinery involved in controlling viral replication in mammalian cells. Besides, it was demonstrated that NS4B domain located between amino acid residues 77 and 125 is involved in interferon signaling inhibition.110 While Grant et al.111 demonstrated that Phe at position 52 in 27 ACS Paragon Plus Environment


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NS4B confers virulence to two independent DENV-2 strains in AG129 mice through enhancement of viral RNA synthesis. A relatively large number of NS4B ligands were discovered by phenotypic screening and were recently reviewed.112 The first ligands of flaviviral NS4B were identified using a replicon-based HTS assay, where replication was monitored through expression of a Renilla luciferase gene by YFV pseudoinfectious particles. Two small-molecule compounds, 45 and 46, inhibited YFV genome replication by EC50 values of 0.4 and 1.48 µM, respectively. A single-residue K128R mutation in the cytoplasmic region of NS4B was identified in escape mutants.113 Lycorine, a plant alkaloid, suppressed flaviviral RNA replication for WNV, DENV and YFV. For WNV, a V9M substitution in the 2K peptide spanning the endoplasmic reticulum (ER) membrane between NS4A and NS4B conferred resistance to lycorine.114 Lycorine derivatives were synthesized and tested for their inhibitory effect on DENV in CFI assay. The most active analogue 1-acetyllycorine, 46, had an EC50 of 0.4 µM and an improved selectivity index of more than 750. However, no confirmation of the mechanism of action for this analogue was reported.115 For DENV, the first NS4B inhibitor 47, an aminothiazole derivative, was identified from a HTS of 1.8 million structurally diverse compounds using a luciferase replicon of DENV2.116 The antiviral activity was further confirmed in DENV-2 CFI and titer reduction assays with EC50 of 1–1.6 µM. 47 selectively inhibited all DENV serotypes (EC50 = 1–4.1 µM), while being inactive against other RNA viruses (EC50 > 40 µM). Resistance against 47 is caused by two mutations (P104L, A119T) in the region of NS4B that spans the ER membrane. 47 was suggested to reduce viral RNA synthesis through potential disruption of NS3-NS4B complex formation, based on the previously published effect of P104L mutation on abolishing NS3-NS4B interaction.108 The poor pharmacokinetic profile of 47 – putatively due to its high lipophilicity –


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impeded testing in animal models. Attempts to modulate the lipophilicity caused loss of activity.117 A screen of the NIH clinical collection (NCC) in a DENV-2 replicon assay revealed 49, a naltrindole analogue and potent δ opioid receptor antagonist, as inhibitor of DENV RNA replication (EC50 = 1.9 µM). 49-resistant replicons harbored a F164L mutation in the cytoplasmic loop of NS4B. Remarkably, resistance against 49 antiviral effects could be achieved also through the P104L substitution in the NS4B trans-membrane domain 3, as described above for the 48-resistant replicon. Despite the localization of the mutated residues in different subcellular compartments, they both caused resistance to 49, suggesting that they can induce similar conformational changes to NS4B. It was postulated that 49 interferes with NS4Bmediated antagonism of interferon (IFN)-signaling, because the compound did not display antiviral effects in non-mammalian C6/36 mosquito cells lacking IFN responses.118 Inhibitors of c-Scr/Abl tyrosine kinase, dasatinib and AZD0530, in addition to Fyn RNAi, were found to inhibit DENV-2 replication through a mechanism linked to the NS4B viral protein, as revealed by resistance breeding experiments.119-120 These compounds are discussed in the section on host targets. A potent DENV inhibitor possessing a spiropyrazolopyridone scaffold (compound 49) was identified from a DENV-2 and HCV replicon-based screening of a 1.8 million-compound library.121 The hit compound was active against DENV-2 and -3 (EC50 = 10-80 nM), but not against serotypes 1 and 4 (EC50 > 20 µM) or HCV replicon (EC50 > 5 µM). The R enantiomer was found to be at least 83-fold more active than the S enantiomer. Resistance in escape mutants was linked to exchange of Val63 in NS4B to several other amino acids. The exact mechanism behind the antiviral effects of the compound could not be elucidated.121 Although the [3H]-



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labeled compound binds to NS4B, it could not block any of the known actions mediated by NS4B. This suggests interference with an unknown PPI between NS4B and host proteins.121-122 Optimization of the physicochemical properties such as aqueous solubility yielded compound 50. The efficacy of 50 was demonstrated in a DENV-2/AG129 mouse model by suppression of viremia, even when the treatment was initiated post-infection.121, 123

CH3

O H3C

O

S

HN

S

OH RO

N NH H Cl HN Cl Cl O

O

H O N

O

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OH

R=H, Lycorine R=Ac, 1-acetyllycorine or 46

O 45

44

Cl X N HN N

O

O OH

O OH 47

N N

Cl

S N H

O

H N

48

O N H X=CH, 49 R isomer X=N, 50 R isomer

Figure 8: Inhibitors of dengue virus NS4B.

Inhibitors of NS5 methyltransferase DENV NS5 containing 900 amino acid residues is a bifunctional protein with the S-adenosyl methionine transferase activity expressed within its N-terminal domain, and RNA-dependent RNA polymerase (RdRp) activity characteristic for residues 270 to 900. Egloff et al.124 reported a crystal structure of N-terminal domain of the DENV-2 NS5 (NS5MTaseDENV) in complex with S-adenosyl-L-homocysteine (SAH) and a guanosine triphosphate analogue at 2.4Å resolution. The structural analysis demonstrated that the closest to the α-phosphate amino acid residues are Lys29 and Ser150, while the specificity to ribonucleotides is provided by two specific hydrogen


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bonds involving Lys14 and Asn18 side chains. At the same time, the purine base of the nucleotide is situated against the aromatic ring of Phe25, which plays an essential role in guanine binding.124

Inhibitors of DENV NS5 methyltransferase and guanylyltransferase showed limited progress in the last years. Among the earliest inhibitors identified are two methyltransferase competitive inhibitors: SAH, a product inhibitor of the methylation reaction; and the natural product sinefungin, a nucleoside analogue of the methyl donor S-adenosyl-L-methionine (SAM).125-126 Both compounds are non-selective inhibitors with activity on a large variety of viral and eukaryotic methyltransferases. Sinefungin demonstrated higher inhibitory potential against both the guanine N-7 and the ribose 2’-O MTase activities (IC50 values = 0.030 µM and 0.041 µM, respectively) in comparison to SAH (IC50 values of 1.77 µM and 0.49 µM).126 This difference in inhibitory activity could be explained by the high binding affinity of sinefungin to DENV MTase, as it displayed a 6-fold higher Kd-value in comparison to SAM (136 nM vs. 826 nM).127 One of the major challenges in designing specific inhibitors of flaviviral MTase is its similarity to human RNA and DNA MTases. A hydrophobic pocket near the SAM-binding site, first identified in WNV MTase,128 may be helpful in achieving selectivity for the viral enzyme: Consequently, derivatives of SAH with an extended hydrophobic substituent at the N-6 position of the adenine ring were synthesized.129 In addition to the enhanced potency in comparison to SAH, the inhibitors achieved higher selectivity against human MTases in vitro. Occupation of the hydrophobic cavity was confirmed by crystallization of 51, one of the most active derivatives (Ki of 0.82 µM and 0.17 µM for N7 and 2’-O DENV-3 MTase activities, respectively), in enantiopure form with DENV-3 MTase.129



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A series of potent HIV-1 inhibitors characterized by a 1,2,3-triazole scaffold derived from 3’azidothymidine (AZT), a nucleoside reverse transcriptase inhibitor, were repurposed as DENV and WNV MTase inhibitors by incorporation of a bulky silyl protecting group at the 5’ position.130 The 5’-TBS group was initially introduced to the structure to provide similarity to TSAO-T, a known HIV non-nucleoside reverse transcriptase inhibitor. Unexpectedly, none of the 5’-protected derivatives inhibited HIV-1. A promising analogue, 52, reduced DENV titer by 98% at 10 µM (EC50 = 7.4 µM at DENV and 8.4 µM at WNV).130 However, the selectivity of the compounds in viral titer-reduction assays was poor. A SAM competition assay verified the SAM-pocket in MTase as binding site, and docking studies suggested binding of the 5’-TBS group to the hydrophobic cavity near the SAM binding site.130 A HTS revealed 2-thioxothiazolidin-4-ones as potent inhibitors of DENV NS5 GTP-binding and guanylyltransferase activity.131 The most promising derivative, 53, inhibited NS5 guanylation with an IC50 of 7.3 µM and displayed an EC50 of 30.8 µM in DENV replicon assay. 53 displayed promising antiviral effects against WNV in vitro but had a modest selectivity index of 6 in cell culture assays. The compound lacked efficacy in vivo, due to a poor pharmacokinetic profile.132 Interestingly, combining N-terminal caps based on the 2-thioxothiazolidin-4-one scaffold to a substrate mimicking tripeptide was reported to inhibit NS3 protease activity and DENV replication in replicon and plaque assays.91 However, 2-thioxothiazolidin-4-one scaffolds are often associated with promiscuous effects in biochemical assays and were reported to exert activity in the low micromolar range at other unrelated targets; some examples include Schistosoma mansoni purine nucleoside phosphorylase,133 Escherichia coli MurD ligase,134 and the cellular enzyme tyrosyl-DNA phosphodiesterase I.135


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NS5 MTase was pursued as target for fragment-based drug discovery.74 Thermal shift assay and X-ray crystallographic screening at DENV-3 MTase identified fragment hits, displaying micromolar to low millimolar inhibitory potential for MTase enzymatic functions in biochemical assays. Analysis of fragments interactions revealed three novel binding sites.74 Mechanistically related to MTase inhibitors are the isoneplanocins, reported in the section on host targets as inhibitors of the S-adenosylhomocysteine hydrolase enzyme (SAHase).

Figure 9: Inhibitors of dengue virus NS5 methyltransferase and guanylyltransferase.

Inhibitors of NS5 polymerase The crystal structure of the RdRp catalytic domain was resolved at 1.85-angstrom resolution by Yap et al.136 The following structural analysis revealed two conserved cavities (A and B) suitable for development of allosteric inhibitors.137 Later, Zou et al.138 demonstrated that in cavity B out of four selected amino acid residues, Leu-328, Lys-330, Trp-859, and Ile-863, all are critical for viral replication, being involved in initiation of RNA synthesis and interaction with helicase domain. At the same time, in cavity A only one (Lys-756) of the seven selected amino acid



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residues has been detected to be critical for viral replication. Therefore, namely cavity B was proposed for rational drug design.138 Compounds acting at DENV NS5 RdRp can be classified into nucleoside inhibitors (NI) and non-nucleoside inhibitors (NNI). NI act by interfering with the polymerase binding site by acting as RNA chain terminators. The second category includes compounds acting at allosteric sites of NS5, thus inhibiting its enzymatic activity. NIs of the DENV (or HCV) polymerases require an initial conversion to the biologically active triphosphate form by cellular kinases. Therefore the synthesis of the corresponding nucleoside triphosphate (NTP) is essential to study their activity in biochemical assays. In addition, the obtained activity in cell-culture is often influenced by factors affecting the phosphorylation efficiency in the cell type used for the assay and the suitability of the modified nucleoside as substrate for kinase enzymes.139 With the lack of sufficient selectivity, NIs may have off-target effects on other polymerases such as mitochondrial DNA polymerase-gamma, resulting in mitochondrial toxicity.140 Initial efforts for targeting DENV RdRp involved the evaluation of known HCV NIs at DENV. 7-Deaza-2’-C-methyl-adenosine (54), a potent and selective inhibitor of HCV,141 showed efficacy when orally dosed to AG129 mice infected with dengue.142 54 originally showed promising antiviral activity against bovine viral diarrhea virus (BVDV) and WNV (EC50 < 5 µM at both viruses), and to a lesser extent against DENV-2 (EC50 = 15 µM).141 Mice treated with 54 showed reduction in viremia, inflammatory cytokine levels and splenomegaly, whereas ribavirin was inactive.142 55, a phosphoramide prodrug of 6-O-methyl-2’-C-methyl-guanosine, is another HCV NI that was tested at DENV (EC50 = 14.2 nM at DENV-2).143 The drug was discontinued for HCV


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following cardiac and renal adverse effects in clinical trials.144 A combination therapy was performed in cell-culture by using INX-08189 as nucleoside analogue with ribavirin or brequinar as inhibitors of nucleoside de novo biosynthesis.145 Ribavirin is a broad-spectrum antiviral agent acting on cellular inosine monophosphate dehydrogenase (IMPDH), thus inhibiting the synthesis of guanine nucleotides; brequinar inhibits uracil nucleotide synthesis by targeting cellular dihydooroate dehydrogenase (DHODH; for more details cf. the section on host targets). The use of drug combinations aims to lower the dose needed from each drug and hence improve the therapeutic window. Only the combination with ribavirin displayed synergistic antiviral activity, which still requires further evaluation in a DENV mouse model or clinical trials.145 56 is a 7-deaza-2’-C-ethynyl analogue of adenosine that was identified to inhibit DENV serotypes 1-4 in cell-culture with micromolar to sub-micromolar potency. The compound also inhibited HCV, as well as other flaviviruses, such as WNV and YFV.146 The biologically active triphosphate form competes with the natural substrate resulting into subsequent chain termination in RdRp assay.147 Efficient phosphorylation by host kinases was verified by in vivo (mice and rats) metabolic studies and chemical modifications were associated with either lower activity or higher cytotoxicity.148 Despite the promising pharmacokinetic profile of 56 and efficacy in dengue mouse models, the compound was not further pursued due to the resulting toxicity following 2-weeks oral administration in rats.117, 146, 148 A prodrug strategy was used to develop 57, an ester of a 7-carbamoyl derivative of 56. 57 showed similar activity profile, mechanism of action, pharmacokinetic properties and efficacy in mouse models as the parent 56. However, as for 56, the compound failed during in vivo toxicity studies.149



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A methyl analogue of the natural product tubercidin, 58, showed nanomolar inhibition of DENV and poliovirus (PV) in cell-culture.150 Tubercidin is a broad-spectrum antiviral and antineoplastic agent but suffers from high cytotoxicity due to interference with cellular processes. Although 58 didn’t show cytotoxicity following treatment for 7 h, extended treatment for 24 or 48 h revealed comparable toxicity to tubercidin.150 A class of nucleoside analogues containing a benzo-fused 7-deazapurine scaffold revealed micromolar to sub-micromolar antiviral activity against DENV in cell-culture.151-152 Among the 6-chloro derivatives bearing a 4-heteroaryl substituent, the 2-furyl analogue (59) showed highest activity and toxicity (EC50 = 0.238 µM in Huh-7, CC50 = 0.175 µM in HepG-2). Optimization to the bioisosteric 2-thienyl derivative (60) almost retained the potency, and yielded a higher selectivity index (EC50 = 0.335 µM in Huh-7, CC50 = 19.92 µM in HepG-2).151 Further modifications of the scaffold achieved moderate activity against HCV, but lower potency against DENV and mostly higher cytotoxicity. This is exemplified by the most active analogue in this series, 61, a 4-amino-5-chloro nucleoside (EC50 = 0.85 µM, CC50 = 1.14 µM in Vero cells).152 An interesting example for the limitations of NI is balapiravir, an ester prodrug of the nucleoside analogue 4’-azidocytidine. The prodrug was initially developed for HCV and was discontinued following a phase II clinical trial for safety concerns because of its severe hematological side effects.153 Balapiravir was repurposed for the treatment of DENV in a clinical trial, where it failed to achieve efficacy, despite its in vitro inhibition of DENV RdRp154 and established activity against three DENV serotypes (1-2 and 4; 3 was not tested) in various cell lines (EC50 = 1.3-11 µM).155 Investigation of the in vitro / in vivo discrepancy indicated insufficient conversion to the corresponding NTP in activated peripheral blood mononuclear cells (PBMCs), a prime DENV host cell type. PBMC activation was manifested by higher levels


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of cytokines (TNF-α, IL-10, and INF-β) following DENV infection154 and was previously reported to influence phosphorylation and hence activity of HIV-1 NI.156 Noteworthy, the adenosine-based NI 56 was not significantly influenced by PBMC activation, which would suggest that the observed effect is either molecule- or scaffold-specific. Another suggested explanation was the lower potency of balapiravir in DENV-infected hepatocytes, one of the major sites of replication.154 The first reported allosteric inhibitor of DENV RdRp, 62, was obtained following a two-step optimization process.157 First, a hit identified from HTS using a primer extension-based assay (IC50 = 7.2 µM) was modified to the N-sulfonylanthranilic acid lead 63 (IC50 = 0.7 µM). Further optimization gave the more potent 62 (IC50 0.26 µM), which displayed selectivity against HCV and human DNA polymerase enzymes.157 The parent lead 63 suppressed RNA elongation and to a lesser extent de novo RNA synthesis.158 A benzophenone derivative (64) was used for photoaffinity labeling and pinpointed a methionine residue (M343) in the allosteric binding site between the finger and thumb region of NS5 RdRp.157-158 This amino acid is located at the entrance of the RNA template tunnel and therefore this compound class is expected to block template entry. 64 – the photolabel compound – was the only derivative of this class reported to show effect in a cell-based plaque assay, where it reduced DENV titer by 3.4- and 4.5-fold at 6 and 17 µM, respectively. The main hit and lead structures possessed no activity in cell-culture and no further results were published for remaining members of this class.158



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Figure 10: Nucleoside inhibitors of dengue virus NS5 polymerase.

Figure 111: Non-nucleoside inhibitors of dengue virus NS5 polymerase and inhibitors of NS5 nuclear translocation.

Compounds blocking NS5 nuclear localization Recently a new class of compounds was reported to prevent DENV NS5 nuclear localization by blocking NS5 interaction with host nuclear transport proteins, without interfering with 38 ACS Paragon Plus Environment

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polymerase enzymatic reaction. This process has been implicated to play a role in DENV infectivity.159 Two NNI, ivermectin and fenretinide, were identified by an alpha-screen assay as inhibitors of the NS5 interaction with importin (IMP) α/β1, hence blocking NS5 nuclear localization.160-162 Ivermectin is also a rather weak inhibitor of DENV NS3 protease71 and a relatively potent inhibitor of the DENV helicase unwinding activity.70 Ivermectin was also reported to inhibit HIV replication by blocking integrase (IN) transport by IMP-α/β1.163 At DENV, ivermectin prevented NS5 binding to IMP-α/β1 with an IC50 of 17 µM,160 and inhibited DENV 1-4 with EC50 of 1.2-1.6 µM in CFI assay.161 Fenretinide or 4-HPR (N-(4hydroxyphenyl)-retinamide) is a synthetic retinoid compound widely investigated for cancer chemoprevention and with clinical potential for treatment of diet-induced obesity, glucose intolerance, and insulin resistance.164 4-HPR mediates its effects through modulation of a number of pathways including retinoid homeostasis, reactive-oxygen species (ROS) and dihydroceramide generation, activation of stress kinases and autophagy.164 4-HPR prevented the association of NS5 to IMP-α/β1 and IMPα∆IBB in an alpha-screen assay with IC50 values of 1–1.5 µM and reduced viral replication in cell culture for DENV serotypes 1-4 and ADE-infection by comparable EC50 values (0.8–2.6 µM). Additionally, the compound was protective against DENV infection in peripheral blood mononuclear cells and the ADE-infection mouse model. When orally dosed at 180 mg/kg BID daily for 10 days, 4-HPR caused significant reduction (1.73log10) in peak viremia in comparison to control.162, 165 Antiviral effects of 4-HPR were found to be not related to the previously listed pathways. Instead, 4-HPR affected steady-state DENV genome replication165 and was reported to modulate the unfolded protein response (UPR) by activating the protein kinase R-like endoplasmic reticulum (PERK) arm.162 Interestingly, activation of the three branches (PERK,



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IRE-1 and ATF-6) of the UPR cascade by DENV infection was reported previously.166-167 The antiviral effects of 4-HPR extended to related viruses including WNV (Kunjin), Modoc and HCV. Considering the established safety and tolerability of 4-HPR, it is a promising candidate for repurposing as pan-antiflaviviral drug.165 The interaction of DENV NS5 with NS3 in the replication complex is another target site for antiviral drugs. Compound 65, a purine derivative, was identified by virtual screening of Src kinase inhibitors at an allosteric pocket of NS5 (cavity B),138 that interferes with RNA synthesis and NS5-NS3 complex formation. An alpha-screen assay168 verified compound 16i as weak inhibitor of this interaction (33% inhibition at 50 µM).169 The compound inhibited DENV replication and CPE (EC50 = 5.3 ± 6.6 µM). 16i is also an inhibitor of the c-Scr and Fyn kinases, two known host targets, with ID50-values of 4.9 µM and 3.6 µM,169 respectively. These host kinases may therefore be considered as the targets with actual responsibility for the phenotypic effect. (Host kinase inhibitors are discussed in the host targets section.)

Host cell targets Targeting host factors for the development of antiviral agents is an attractive strategy. Host factors have a higher genetic barrier towards mutations and are thus less prone to resistance development in comparison to viral targets. If the host target is required for replication by several related viruses, then broad-spectrum antivirals may be developed. Additionally, known inhibitors – possibly even drugs in clinical use – of a cellular pathway involved in viral replication can be repurposed, offering significant advantages with respect to clinical drug development. Putative host targets against dengue were reviewed by Fischl and Bartenschlager,170 Krishnan et al.24 and


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Acosta et al.171 The present chapter will focus on the most promising host factors, for which ligands have already been described.

Inhibitors of Host Cell Nucleoside Biosynthesis A relatively straightforward approach for the development of antiviral drugs is to target host enzymes involved in the de novo biosynthesis of nucleosides, since the virus relies on their supply from the native metabolism of the host cell. One of the first classes of broad-spectrum antiviral agents targeting DENV and other flaviviruses are inhibitors of IMPDH, an enzyme required for guanine nucleotides biosynthesis. Compounds listed in this category include ribavirin,172 and its 5’-ethynyl analogue EICAR, in addition to the non-nucleoside mycophenolic acid (MPA).173 Ribavirin displayed the weakest activity among the three examples and its potency varied, depending on the cell line.174-175 Studied by indirect immunofluorescent flow cytometry, ribavirin had an EC50 of 40 µM against DENV-2 in Huh-7 cells, while MPA showed an EC50 of 1.9 µM. Both compounds were active in a plaque assay and the antiviral effects were reversed by exogenous supply of guanosine.174 The main mode of action is by inhibition of IMPDH and thus depletion of the intracellular guanine nucleotide (GTP) pool, although other mechanisms were also proposed for ribavirin.175 As discussed previously, combining ribavirin with another nucleoside analogue showed synergistic antiviral effects. Ribavirin-treated cells weakly induced interferon-stimulated response elements (ISRE) activation and did not result in higher activation when combined with beta-interferon.145 Noteworthy, despite the higher antiviral effects of MPA compared to ribavirin, its immunosuppressive activity makes it unsuitable as therapeutic candidate for DENV infections.174



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Another cellular target is DHODH, an enzyme required for pyrimidine biosynthesis. Brequinar is an anti-neoplastic agent that acts by inhibition of DHODH. It exhibits antiviral activity against a broad spectrum of viruses, including the flaviviruses (DENV, WNV, and YFV). The compound causes potent inhibition of DENV-2 in CFI-assay with an EC50 of 78 nM.176 In a DENV-2 luciferase replicon assay, supplementing pyrimidines (cytidine or uridine) but not purines (adenine or guanine) reverses the effect of brequinar. Resistance breeding experiments on DENV-2 resulted in two main mutations; a methionine → valine substitution at position 260 (M260V) located at the helix of domain II of the envelope protein, and a glutamic acid → glutamine mutation at position 802 (E802Q) of the priming loop of the polymerase domain in DENV-2 NS5. Analysis of the mechanism of action suggests that brequinar depletes the intracellular pyrimidine pool, causing inhibition of RNA synthesis, in addition to interfering with viral assembly and release. Interestingly, the (E802Q) mutation in DENV-2 is the wild-type sequence for DENV serotypes 1, 3 and 4, and hence DENV-1 is less sensitive to brequinar.176 The combination of brequinar and a nucleoside analogue did not have a synergistic antiviral effect. In contrast to ribavirin, brequinar-treated cells showed pronounced activation of ISREs.145 The compound 66 was reported by researchers from two teams of the Novartis Institute for Tropical Disease (NITD), in publications with partially overlapping authorships,177-178 and later reviewed by some of the co-authors.179 The first two publications on this compound were submitted within a few days and describe two similar phenotypic assay systems, employing different concentrations of the screening compounds and different host cell lines. Several inconsistencies surround this compound: First, 66 (NITD-982)178 was presented in one publication178 as an analogue to itself in another publication (compound 6b).177 Second, while in


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two of the publications 66 was reported to have insufficient solubility for isothermal titration calorimetry (ITC),178-179 one publication177 presents a Kd value obtained by ITC. Third, the activity of 66 on isolated DHODH enzyme described in one publication178 reproduces the results described in another.177 Notwithstanding these peculiarities, 66 was described to have broad spectrum antiviral activity in plaque assays, with the highest potency against WNV followed by DENV-2, with EC90 values of 0.31 nM and 5.2 nM, respectively. The compound showed cross-resistance with brequinarresistant mutants, was less effective at DENV serotypes 1, 3, and 4. Moreover, uridine addition restored DENV-2 replication in cells treated with 66. Regarding the pharmacokinetic profile, oral dosing was associated with high plasma protein binding, and better exposure could be achieved by subcutaneous administration, which was chosen for animal studies. When tested in a DENV viremia mouse model (AG129 mice), however, plasma uridine levels were unaffected, indicating an insufficient effect on host DHODH, and no effect on viremia was observed. Similar to brequinar, 66 enhanced exogenous IFN-induced ISRE activation.145

Figure 12: Host nucleoside biosynthesis inhibitors with anti-DENV activity. The compound 66 is denoted as "6b" (and as an analogue of itself) in one of the publications.177



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Host cell lipid biosynthesis inhibitors Biosynthesis of cholesterol and fatty acids, as well as transport of cholesterol are cellular processes found to be associated with replication of DENV and other flaviviruses. The severity of DHF is associated with variations in the plasma lipid profile.180-181 Research on the cholesterol requirements for DENV-2 infection revealed two observations: first, depletion of cholesterol by methyl-β-cyclodextrin (MβCD) or filipin III reduced DENV-2 infection mainly at the replication steps and to a lesser extent at the entry stage.182 In contrast to this observation, pretreatment of DENV virions of serotypes 1-4 with MβCD or nystatin was reported to prevent viral fusion and uncoating, resulting into impaired RNA and protein synthesis.183 The second observation contrasts to findings in other viruses, where supplementation of excess amounts of cholesterol blocked an early step of DENV-2 infection, putatively viral entry and RNA uncoating. Furthermore, when cholesterol was added after viral entry, an as-yet-unidentified intracellular step of viral infection was also inhibited resulting in reduced viral replication.182 A similar finding was also described for the virucidal effect exerted by treatment of DENV virions with excess cholesterol.183 This stands in contrast to the results by Rothwell and coworkers, where supply of cholesterol caused partial restoration of DENV replication in Renilla luciferasereplicon cells grown in a delipidated medium.184 With these somehow contradictory findings about the effect of cholesterol depletion or supply on DENV infection, the exact mechanism remains in need of further elucidation. Especially in the reports by Lee et al.182 and Carro et al.,183 the fact that both exogenous supply of cholesterol and its depletion cause similar phenotypic outcomes, makes it rather difficult to provide validation for the proposed mechanisms. In this regard, Soto-Acosta and colleagues suggested that the cholesterol effects on DENV might be cell type-specific.185 For this purpose, they studied the effects of MβCD, filipin


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and nystatin in hepatic cells (Huh-7). For both DENV-2 and DENV-4, the tested compounds reduced viral yield, confirming the role of cholesterol and lipid rafts in the entry phase. For 2h pre-treatment before DENV-4 infection, nystatin (20 µg/mL, MW=926), filipin (7 µg/mL, MW=655) caused a half log reduction of virus yield, while MβCD (3.6 mM) caused one full log reduction.185 An siRNA screen revealed mevalonate decarboxylase (MVD), an enzyme of the cholesterol biosynthesis pathway, to be involved in DENV replication. Additionally, hymeglusin, an HMG-CoA synthase inhibitor and zaragozic acid, a squalene synthase inhibitor, caused significant inhibition of DENV-2 NGC live virus replication in plaque assays (EC50 = 4.5 µM and 8.3 µM, respectively). Statins, such as fluvastatin, lovastatin, mevastatin, and simvastatin, were shown to have an inhibitory effect in a dengue replicon assay in A549 epithelial cells (EC50 = 1.82-8.75 µM). However, they displayed high cytotoxicity which was explained by the sensitivity of the used cells for HMGR inhibition.184 Lovastatin was also reported to cause a half-log reduction of virus yield and RNA replication in Huh-7 cells at 50 µM, without influencing cell viability.185 However, the effect of lovastatin in AG129 mice infected with DENV-2 did not yield promising results.186 67, a blocker of intracellular cholesterol trafficking and an inhibitor of cholesterol biosynthesis, causes an accumulation of cholesterol in the late endosome/lysosome and inhibited DENV infection (EC50 = 2.9 µM and CC50 = 34.4 µM in DENV-2 Huh-7 replicon), probably due to inhibition of viral fusion or uncoating.187 67 also displayed synergy with 68, a fatty acid synthase (FASN) inhibitor with an EC50 value of 5.7 µM (CC50 > 100 µM) at the DENV-2 Huh-7 replicon, highlighting the need of both cholesterol and fatty acids for DENV replication.187 FASN was identified in a siRNA screen as critical factor for DENV replication; this could be further confirmed by the use of pharmacological inhibitors, cerulenin and 68, which resulted in a dose-dependent suppression of



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DENV replication. The inhibitory effect of 68 extended also to WNV and YFV. It has been observed that the NS3 protease colocalizes and interacts with FASN in infected cells, possibly recruiting FASN to the sites of DENV replication and promoting fatty acid synthesis.188 This interaction between DENV NS3 and FASN is mediated by Rab18. Rab or Ras-related proteins in the brain are a class of membrane trafficking GTPases. Rab18 is located on lipid droplets (LD), which are known to be implicated in the DENV life cycle.189

Figure 213: Compounds affecting lipid metabolism with antiviral effect against DENV.

Alpha-glucosidase inhibitors Alpha-glucosidase inhibitors are interlinked with targeting of viral proteins that depend on Nglycosylation for correct folding and activity. These include DENV glycoproteins such as prM/M and E, in addition to NS1.190 For the E glycoprotein of some enveloped viruses, processing in the ER by N-glycosylation is described to be required for secretion. However, the initially attached glucose-mannose oligosaccharide must subsequently be "shortened" by glucosidases: these enzymes partially or completely remove the terminal glucose units in a process termed "glucose trimming". Inhibition of glucose trimming had no influence on the 46 ACS Paragon Plus Environment

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formation of prME heterodimer, but the generated complex was less stable and had reduced folding efficiency.191 As for NS1, it is the main diagnostic marker for DENV infection and plays a role in viral RNA replication.192 The folding, stability, secretion and activity of NS1 depend on its N-glycosylation at N130 and N207, which makes the responsible cellular glucosidase a potential host target.192-193 Three-dimensional structures of human α-glucosidases are usually obtained through homology modeling based on the available crystal structures of other organisms.194 Among available are three-dimensional structures of the following α-glucosidases: S. solfataricus α-glucosidase,195 αglycosidase YicI,196 etc. The three-dimensional structures of processing α-glucosidases I and II have not yet been reported. Iminosugar derivatives such as castanospermine (CST) and N-nonyl-deoxynojirimycin (NNDNJ) are inhibitors of α-glucosidase that showed antiviral effects against DENV.192 Treatment of a mouse neuronal model of DENV infection (DENV-1) with CST or NN-DNJ generated a dose-dependent reduction of infectious virus. The mechanism was linked to impaired folding and assembly of the envelope protein in mouse neuroblastoma cells.191 Wu et al. also evaluated NNDNJ at DENV-2 and YFV and found a reduction in intracellular levels and secretion of the flaviviral glycoproteins E, prM, and NS1.197 The effect of treatment of BHK-21 infected cells was more pronounced on DENV-2, which displayed lower levels of RNA replication and viral titers. The influence of the compound on nonglycosylated proteins such as NS3 was negligible. A potential role of the ER chaperone calnexin in the mechanism of action was suggested, based on the interaction of viral E and prM proteins with this chaperone. This interaction was affected by DNJ.197 Further analysis of CST was carried out by Whitby et al.:198 The effect of this indolizine alkaloid was demonstrated on all DENV serotypes (1-4) and is mainly due to



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impairment of viral secretion and infectivity, and to a lesser extent RNA replication or translation. WNV and YFV were less susceptible to CST actions. In DENV-2 infected mice, CST improved survival rates and was protective against mortality even with intracranial viral inoculation but gastrointestinal toxicity was observed with higher doses.198 Celgosivir (6-Obutanoyl-CST) is an oral prodrug of CST. This prodrug caused strong inhibition of DENV 1-4, with EC50 values in the range of 0.22-0.68 µM in a CFI assay in BHK-21 cells, and reduced viral titer for DENV-2 ADE infection.199 For the CFI assay, the effect on DENV-2 was about 100-fold higher than the parent CST (EC50 = 0.2 µM versus 28 µM), but the in vivo potency was later found to be only 2-fold higher than CST.199-200 The mode of action of celgosivir involves blocking E protein and NS1 transport from the ER to Golgi. Intracellular accumulation of misfolded NS1 occurred following a defect in its N-glycosylation events. Celgosivir effects could be also linked to a modulation of the UPR host machinery. Consistent with the proposed mechanism, the activity of the nonglycosylated proteins NS3 and NS5 were unaffected by this drug.199 Both celgosivir and NN-DNJ are efficacious in vivo, by lowering viremia and cytokine levels, in addition to promoting survival in DENV infection mouse models, even with delayed treatment.142, 199 Due to its rapid metabolism, efficacy of celgosivir in mice increased with higher doses or with twice-a-day schedule of administration compared to a once-a-day dosing regimen.200 Despite its promising activity and safety profiles, celgosivir failed to be efficacious in patients with dengue fever.201 Besides CST and its prodrug, other iminosugar derivatives also displayed promising activity against DENV. DNJ modifications range from the optimization of the DNJ head group and the tail N-alkyl chain to increase potency and reduce cytotoxicity.190


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Liang et al. screened a series of five-membered iminocyclitols against a panel of glycosidases in situ. Potent α-glucosidase inhibitors were further tested in plaque assays against DENV-2 and JEV.202 The most potent transition state analogue, 69 with a nanomolar Ki (53 nM) at αglucosidase, features a bicyclic head group. Following this step, N-alkyl side chains of variable length were introduced. Although the N-alkylated analogues were more active at DENV-2 than JEV, they did not achieve higher activities than NN-DNJ in plaque assays, where the best derivative at DENV-2, 70, showed an EC50 of 4.7 µM.202 Six-membered DNJ derivatives with a conformationally restricted cyclohexyl group in the Nside chain displayed a favorable safety and antiviral profile with one-digit micromolar EC50 values against DENV (best derivative: compound 71, EC50 = 1.5 µM), WNV and BVDV.203 Introduction of a hydroxyl group and extension of the alkyl linker in compound 72 retained antiviral activity with an EC50 value of 2 µM at DENV, but no improvement in relation to the parent compound NN-DNJ (EC50 at DENV-2 =1µM).203 Building on the structure of 72, a class of imino sugars bearing an oxygenated side chain and a terminally restricted ring was developed.204 Structural variations resulted in compounds with lower toxicity and superior antiviral effects against DENV compared to WNV. The most active analogues 73 and 74 carry a hexyloxycyclohexyl side chain and reach submicromolar EC50 values (0.075-0.1 µM) against DENV-2 with CC50 values above 65 µM. Based on the correlation between lipophilicity and potency, it was suggested that the increased efficacy is linked to improved cellular uptake rather than increased inhibitory activity at α-glucosidase.204 75 is an open chain analogue of 73 and 74 with an EC50 value of 1.1 µM and a pharmacokinetic profile suitable for oral dosing. The compound was tested for efficacy in combination with ribavirin.205 The combined therapy showed an enhancement of inhibitory activity in A549 cells



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with predicted synergistic effect in vivo. In AG129 mice, the drug combination reduced viremia significantly, in contrast to the ineffective ribavirin monotherapy.205 To further explore the SAR of the tail group, an ether bond was introduced in the N-alkyl side chain.206 Following assessment of the antiviral profile and ADME properties, compounds 76 and 77 were identified as promising lead molecules, with EC50 values of 0.4 and 0.3 µM, respectively, at DENV-2 in BHK cells and selectivity indices higher than 1000. Derivative 76 displayed improved oral bioavailability (F = 92 ± 4 %) in rats.206 An NN-DNJ analogue with terminal methoxy group in the tail, 78, was pursued after it displayed efficacy in AG129 mice following an otherwise lethal DENV infection. The compound showed micromolar potency (EC50 = 17 µM) in plaque assay for DENV-2 infected Vero cells and favorable toxicity profile (CC50 > 500 µM).207 Pharmacokinetic evaluation of 78 revealed 84% bioavailability following oral administration but a rapid elimination half-life. For animal studies, the compound was dosed three times daily to overcome its short half-life. 78 reduced viremia and cytokine levels in mice in a dose-dependent manner. Whether the reduction of cytokines is a consequence of decreased viral load or a direct effect of 78 remains to be determined. No significant effect of the compound on the level of anti-DENV antibodies was observed and the therapeutic window of the drug covered the first 48h of infection.207 A follow-up of this work studied DENV in vivo response to the host-targeted 78.208 The findings were consistent with a high genetic barrier imposed by targeting a host factor with no evidence of an escape mutant. Sequencing of viral populations from treated and control mice revealed drug-specific mutations in the treated group and nonspecific mutations in both treated and control groups. The specific mutations were insufficient to provide escape and were used to putatively pinpoint the drug targets, these included glycosylated viral proteins and unexpectedly NS5.208


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Cyclization at the terminal side of 78 N-alkyl chain provided 79.209 This analogue inhibited αglucosidase in vitro and DENV-2 in a plaque assay (EC50 = 21.71 µM). 78 displayed a favorable drug-like profile regarding safety and oral bioavailability. Efficacy in mouse models was achieved with 100% protection against DENV infection upon pre-treatment with 79 one hour before infection. The effect of 79 on viral load was variable, depending on the tissue: no reduction was reported in serum, liver, and spleen. Cytokines levels were reduced following treatment with 79, except for KC which was elevated. A similar outcome was found for 78.208-209

Figure 314: Alpha-glucosidase inhibitors with anti-DENV activity.

Glycolysis inhibitors Recently the glycolytic pathway was revealed as potential host target against DENV infection.210 In the absence of exogenous glucose, DENV replication is impaired. Pharmacological inhibition of glycolysis using oxamate, a lactase dehydrogenase inhibitor, or 2-deoxy-D-glucose (2DG), an 51 ACS Paragon Plus Environment


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inhibitor of hexokinase, caused a dose-dependent reduction of viral RNA synthesis and release of infectious virus.210

Figure 415: inhibitors of glycolysis affecting DENV replication.

Host kinase inhibitors Through a phenotypic screening using an immunofluorescence image-based assay, two cScr/Abl tyrosine kinase inhibitors, dasatinib and AZD0530, were identified as potent inhibitors of DENV infection.119 Dasatinib and AZD0530 showed EC90 of 4.7 µM and 12.2 µM, respectively, against DENV-2 in virus titer reduction assays. A follow-up study attempted to identify the antiviral mechanism of action of these compounds.120 A mutation in the transmembrane domain 3 of the NS4B protein was identified in dasatinib-resistant mutants, which also displayed resistance to the inhibitory effects of AZD0530 and Fyn RNAi. Northern blot analysis revealed a decrease of steady-state RNA accumulation in presence of these kinase inhibitors, with an RNAi experiment indicating involvement of the host kinase Fyn. Dasatinib additionally inhibited assembly and secretion of DV particles by an unknown mechanism.120

Figure 516: Inhibitors of host kinases


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Inhibitors of S-adenosylhomocysteine hydrolase (SAHase) enzyme The human SAHase monomer is a 432-residue protein with cofactor (NADH) binding and catalytic domains. A 3D crystal structure of the enzyme was resolved as selenomethionylprotein in complex with NADH and the ligand (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1yl)adenine (pdb code 1A7A).211 The structural analysis suggested certain roles of amino acid residues in the catalytic site of SAHase: Lys186 serves as general proton donor/acceptor in the catalytic site, Glu156 serves in proton management of Lys186, maintaining its correct protonation state, Asp190 forms hydrogen bond to 2’-OH group of the substrate, Asn181 and Asn191 interact with Lys186 and hold its flexible side chain in an optimal position for its catalytic function.212 Since the moment when SAHase became an attractive target for the design of antiviral agents, three generations of its inhibitors have appeared, each having higher potency and specificity in action: From naturally occurring adenosine analogues to neplanocin A and arisetromycin analogues, and from these to specific prodrugs activated by the hydrolytic activity of the enzyme.213 Based on neplanocins, a group of naturally occurring carbocyclic nucleotides and known inhibitors of SAHase, derivatives of 1’,6’-isoneplanocin were synthesized by Schneller and Ye.214 The SAHase enzyme is essential in methylation reactions where SAM is used as methyl donor. The enantiomers displayed micromolar activity and moderate toxicity in cell culture (EC50 = 1-6 µM) against DENV. Higher potency was achieved against other viruses such as human cytomegalovirus, measles, and Ebola. Several of the compounds were potent inhibitors of SAHase from rabbit erythrocytes, with the lowest IC50 value at 0.9 nM.214



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Figure 617: Inhibitor of S-adenosylhomocysteine hydrolase enzyme.

Inhibitors of viral translation with unconfirmed target The first reported broad-spectrum inhibitor of flavivirus mRNA translation, 80, was identified by phenotypic screening of a chemical library using a WNV replicon assay.215 The compound belongs to a family of secondary sulfonamides and blocks the initial phase of viral RNA translation and subsequent replication of WNV, DENV and YFV with EC50 values in the low micromolar range and favorable selectivity indices (SI > 10). Another compound, 81, was identified using a HTS based on a DENV CPE-assay.216 The inhibitor has a benzomorphan nucleus and displayed a flavivirus-specific antiviral spectrum in viral titer reduction assays, with slightly higher activity against DENV-2 (EC90 1.7 µM) than WNV or YFV (EC90 4.5 µM and 4.9 µM, respectively). The metabolic stability of the initial hit was improved by replacing the hydroxy group, which is highly prone to glucuronidation, with a cyano group, leading to derivative 82. Only the S,R,S enantiomer showed inhibition of viral replication in a plaque assay. Further studies on the mode of action of 82 in a DENV-1 luciferase replicon assay confirmed that the compound inhibits viral replication in part by suppression of RNA translation. Additional validation was provided by testing the compound in vitro, in translation assays for DENV-1, where it showed an IC50 of 8.9 µM. The compound is not selective, as it inhibited RNA translation of both DENV-2 and host RNAs with comparable IC50 values of 4.1 µM and 4.5 µM, respectively. 82 showed efficacy in a DENV viremia mouse


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model when dosed daily at 25 mg/kg. However, at doses above 75 mg/kg, the compound caused severe adverse effects.216

Figure 718: DENV translation inhibitors with unconfirmed target

DENV inhibitors with unconfirmed mechanism of action Several compounds with other clinical indications have been screened for anti-dengue activity and revealed some interesting results without a deeper understanding of their mechanism of action. A known antimalarial 4-aminoquinoline, amodiaquine, has been reported to inhibit DENV-2 replication in vitro with EC50 values of 1.08 ± 0.09 µM measured by plaque assay and 7.41 ± 1.09 µM as measured by replicon assay, while the CC50 measured in BHK-21 cells was 52.09 ± 4.25 µM.217 It has also been demonstrated that the diethylaminomethyl group is crucial for the anti-dengue activity of amodiaquine, while exploration of potential target binding showed that neither DENV protease nor 5’-methyltransferase or RNA-dependent RNA polymerase are the main targets of the compound. In the same article, Boonyasuppayakorn et al. reported the anti-dengue activity of another known antimalarial 4-aminoquinoline derivative, chloroquine, which inhibited DENV-2 replication in BHK-21 cells (EC90 = 5.04 ± 0.72 µM) but had no effect in a DENV-2 replicon assay. Based on these results, the authors suggested that chloroquine acts either at the stage of viral entry or viral assembly.217 These findings were supported later in vivo, in a study against DENV-2 infection in Aotus azarai infulatus monkeys,



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which confirmed that chloroquine is more effective if administered before the infection occurred.218 A similar activity was detected for hydroxychloroquine, which was active against DENV-2 with IC50 values from 9.7-12.9 µM in three different cell lines: A549, Hepa1-6, WS-1 cells, and J774A.1 macrophages.219 Interestingly, anti-DENV-2 activity has been reported220 for a neomycin analogue – geneticin (G418, MW=497), which inhibits protein synthesis in prokaryotic and eukaryotic cells via binding to ribosomes. According to the published results, geneticin prevents cytopathic effects in BHK cells infected with DENV-2 (EC50 = 3 ± 0.4 µg/mL), lowers the yield of viral titers (EC50 = 2 ± 0.1 µg/mL), blocks viral RNA replication and viral translation. Lanatoside C, a cardiac glycoside, has anti-DENV (serotypes 1-4) activity in Huh-7 cells with IC50 values in the range of 0.5-1 µM. Activity against DENV-2 was confirmed for a wide range of cell lines. Anti-viral activity of this compound was also confirmed for other RNA viruses (WNV (Kunjin), Chikungunya virus, Sindbis virus, human enterovirus 71).221 Puig-Basagoiti et al. reported a triaryl pyrazoline compound, {[5-(4-chloro-phenyl)-3-thiophen2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} that inhibited DENV-1 (EC50 = 17 µM in a replicon assay) and other flaviviruses.222 EC50 for WNV determined by viral titer reduction assay was estimated to be 28 µM. At the same time, in the case of DENV-2, YFV, and Saint Louis encephalitis virus, viral titers were significantly reduced when infected cells were treated with 33 µM.222 A group of N-substituted acridone derivatives has been demonstrated to be active against Junin virus (JUNV) and DENV-2 in Vero cells, with the two most active compounds 83 and 84 having EC50 values in the range of 2.5-5.5 µM.223 Both compounds also inhibited DENV-1 and DENV-3 with EC50 values in the range of 8.3-15.9 µM, while the activity against DENV-4 is


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significantly lower for 83 and the EC50 value for 84 reaches 19.9 µM. Another derivative, compound 85, detected in this screening and further explored in a follow-up study,224 demonstrated anti-dengue activity in the virus yield reduction assay in Vero cells against all 4 serotypes with EC50 values range of 13.5-27.1 µM. A screening of 1,3,4-oxadiazole, 1,2,4-triazolyl-3-thione, and imidazo[2,1-b]thiazole derivatives substituted with a carbohydrate moiety showed that compound 86 inhibits replication of DENV2 and JUNV in Vero cells with EC50 values of 29.9 and 12.0 µM, respectively.225 The bromine substituent appears to be essential for activity, as the debrominated analogue was inactive even at concentrations > 100 µM. Aman et al.226 developed a quino[8,7-h]quinoline derivative 87 with broad activity against multiple, partially unrelated viruses including DENV (1-4) (EC50 = 0.4-0.9 µM, DC-Sign Raji cells). The compound demonstrated good pharmacokinetics in C57BL/6 mice with organ concentrations ranging from 19.5 (spleen) to 43.1 µg/g (kidney), as well as good survival parameters among Ebola infected BALB/c mice.226 An anti-DENV-2 replicon HTS of a series of 2,4-diaminoquinazoline derivatives reported by Chao et al.227 showed that 88 has high anti-DENV activity in BHK-D2RepT cells (EC50 = 0.15 µM, (DENV-2)). Starting with this lead, a series of 2,4-diaminoquinazolines was synthesized with the most active 89 having an excellent pharmacokinetic profile and EC50 values in the nanomolar range (EC50 = 2.8 nM, SI >1000, (DENV-2)). The bulky electron-donating substituent in C-5 position of the 2,4-diaminoquinazoline ring (e.g, the tert-butoxy group of 89) is crucial for anti-DENV activity of this class of compounds. Another anti-DENV-2 replicon HTS of a series of imidazole 4,5-dicarboxamide (I45DC) derivatives reported by Saudi et al.228 detected a compound 90 with dual activity against DENV



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and YFV in Vero cells (EC50 = 2.50 µM (DENV-2), EC50 = 3.37 µM (YFV)). Based on this lead, a class of pyrazine 2,3-dicarboxamides was synthesized. The highest levels of anti-dengue activity were detected for compounds 91 and 92 with an EC50 = 0.93 µM (DENV-2) for both. Within the imidazole series, a para attachment of the heterocycle to the ring C was shown to be important for biological activity, while in the pyrazine series the para-pyrimidin-4-yl substituent on the same ring appeared to be more advantageous.

Figure 819: Compounds with unconfirmed mechanism of action at DENV.

Conclusion Even though the present perspective covers only a selection of antiviral approaches against dengue, it becomes clear that the disease is not quite as neglected as it may appear to an outside observer. We believe that particularly the progress in assay systems on the cellular or replicon level will be a major catalyst in therapeutical dengue research because it facilitates the translation


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of target-based medicinal chemistry into animal models and allows the identification of new cellular targets with relevance to dengue. It is therefore to be assumed that new successes in the area of dengue-related medicinal chemistry will become visible in the next few years. We are fairly optimistic that our discipline will be able to repeat the recent breakthrough of medicinal chemistry in the HCV area in the fields of dengue and related flaviviral infections.

Abbreviations 2DG, 2-deoxy-D-glucose; 4-HPR, (N-(4-hydroxyphenyl)-retinamide); ADE, antibody-dependent enhancement; Arbovirus, arthropod-borne; AZT, azidothymidine; BID, twice daily; BPTI, bovine pancreatic trypsin inhibitor; BRET, bioluminescence resonance energy transfer; BVDV, bovine viral diarrhea virus; CFI, cell-based flavivirus immunodetection; CS, curdlan sulfate; CST, castanospermine; DHF, dengue hemorrhagic fever; DENV, dengue virus; DHODH, dihydooroate dehydrogenase; EI, entry inhibitors; ER, endoplasmic reticulum; FASN, fatty acid synthase; FFU, focus forming unit; GAG, glycosaminoglycan; GTP, guanosine triphosphate; HS, heparan sulfate; HCV, hepatitis C virus; IFN, interferon; IMP, importin; IMPDH, inosine monophosphate dehydrogenase; IN, integrase; ISRE, interferon-stimulated response elements; ITC, isothermal titration calorimetry; JEV, Japanese encephalitis virus; JUNV, Junin virus; LD, lipid droplets; MPA, mycophenolic acid; MVD, mevalonate decarboxylase; NCC, NIH clinical collection; NI, nucleoside inhibitors; NN-DNJ, N-nonyl-deoxynojirimycin; NNI, non-nucleoside inhibitors; NS, non-structural; NTP, nucleoside triphosphate; PAMPA, parallel artificial membrane permeability assay, PBMCs, peripheral blood mononuclear cells; PERK, protein kinase R-like endoplasmic reticulum; PPI, protein-protein interaction; PFU, plaque forming units; PV, poliovirus; RdRp, RNA-dependent RNA polymerase; ROS, reactive-oxygen species;



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SAH, S-adenosyl-L-homocysteine; SAHase, S-adenosylhomocysteine hydrolase; SAM, Sadenosyl-L-methionine; SI, selectivity index; TBEV, tick-borne-encephalitis virus; TSA, thermal-shift assay; UPR, unfolded protein response; WNV, West-Nile-virus; YFV, yellowfever-virus.

Acknowledgements Prof. Ralf Bartenschlager provided valuable comments on Scheme 1. We thank Dominik Graf for useful discussions on cell-based assays. Mira Behnam appreciates financial support from the German Academic Exchange Service. Veaceslav Boldescu appreciates funding provided by the Alexander von Humboldt Foundation. The work on dengue virus in the group of Christian Klein is sponsored by the Deutsche Forschungsgemeinschaft, KL-1356/3-1.

Biographical sketches Mira Behnam studied pharmacy and biotechnology at The German University in Cairo, where she obtained her B.Sc. degree (2009) and M.Sc. degree (2011) in pharmaceutical chemistry in collaboration with Würzburg University. Since 2013, she is DAAD scholarship holder and PhD candidate in the group of Prof. Christian Klein (Heidelberg University) working on the development of potent antiviral compounds against dengue and West Nile virus. Christoph Nitsche studied chemistry and business administration. He obtained his PhD on the development of dengue virus protease inhibitors under the guidance of Prof. Christian Klein at Heidelberg University with a scholarship from the German National Academic Foundation. He continues his postdoctoral work as a Feodor Lynen Fellow (Alexander von Humboldt-


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Foundation) in the laboratory of Prof. Gottfried Otting at the Australian National University. His current research interest is focused on novel NMR methods for drug discovery. Veaceslav Boldescu studied pharmaceutical technology and obtained a PhD in Technology of special products (pharmaceuticals) in 2008 under the guidance of Acad. Gheorghe Duca (Academy of Sciences of Moldova). He started his research pathway at the State University of Moldova and continued it at the Institute of Chemistry of the Academy of Sciences of Moldova, working in the Laboratory of Organic Synthesis lead by Prof. Fliur Macaev. His main research interests include development of new chemotherapeutic agents against infections such as tuberculosis and dengue. Christian Klein studied pharmacy and obtained a PhD 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. His main research interests are antiinfective compounds and fundamental questions in medicinal chemistry, such as the study of unusual binding modes and structural motifs.

Author information: Corresponding author: C.D. Klein, phone +49-6221-544875, e-mail [email protected]



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Figure 1. Key steps of flavivirus replication. Indicated are the target sites for pharmacological interaction. Targets that are, to our knowledge, not extensively exploret yet are parenthesized. The replication complex (5) is a structure composed of membranes and vesicles along with viral and probably also host proteins. It forms after viral infection and appears to harbor several steps of flaviviral replication. Further processing and assembly of progeny virions in the trans-Golgi network relies heavily on host factors. 254x533mm (300 x 300 DPI)

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The Medicinal Chemistry of Dengue Virus - ACS Publications

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