Inhibitors of Influenza Virus Polymerase Acidic - American Chemical

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Inhibitors of Influenza Virus Polymerase Acidic (PA) Endonuclease: Contemporary Developments and Perspectives Han Ju,† Jian Zhang,† Boshi Huang,† Dongwei Kang,† Bing Huang,*,‡ Xinyong Liu,*,† and Peng Zhan*,† †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012, Jinan, Shandong, P. R. China ‡ Poultry Institute, Shandong Academy of Agricultural Sciences, 1, Jiaoxiao Road, 250023, Jinan, Shandong, P. R. China ABSTRACT: Influenza virus (IFV) causes periodic global influenza pandemics, resulting in substantial socioeconomic loss and burden on medical facilities. Yearly variation in the effectiveness of vaccines, slow responsiveness to vaccination in cases of pandemic IFV, and emerging resistance to available drugs highlight the need to develop additional small-molecular inhibitors that act on IFV proteins. One promising target is polymerase acidic (PA) endonuclease, which is a bridged dinuclear metalloenzyme that plays a crucial role in initiating IFV replication. During the past decade, intensive efforts have been made to develop small-molecular inhibitors of this endonuclease as candidate agents for treatment of IFV infection. Here, we review the current status of development of PA endonuclease inhibitors and we discuss the applicability of newer medicinal-chemistry strategies for the discovery more potent, selective, and safer inhibitors. (Figure 1).3,5,9−12 In addition, 7 (favipiravir, T-705), which inhibits the RNA-dependent RNA polymerase of multiple RNA viruses, was approved in Japan (in 2011) to treat IFV infection involving strains resistant to available antivirals.3,13 However, emergence of transmissible resistant variants and time-dependent effectiveness are major challenges for the currently approved antivirals3,14−18 (for example, almost 100% of IFV type A H3N2 and more than 95% of IFV type A H1N1 strains are resistant to adamantane). Besides, neuraminidase inhibitors must be administered within 1−2 days of infection to be effective. These therapeutics also have the potential to cause undesirable side effects, including unusual psychiatric or neurologic events such as hallucinations, confusion, delirium, and abnormal behavior, primarily in children, though these are rare occurrences with most drugs, including oseltamivir.19,20 Consequently, there is an urgent need to develop novel antiviral agents that act on targets different from those of the existing drugs used in clinic, in order to overcome drug resistance and to prevent and treat IFV infection effectively.21,22 To our knowledge, there has been no comprehensive and special review of IFV PA endonuclease inhibitors. The aim of this critical review is therefore to describe the current status of smallmolecular PA endonuclease inhibitors, highlighting structure− activity relationships (SARs), as well as the binding modes of the inhibitors in the enzyme active site as determined by X-ray crystallography. In the context of general approaches for lead

1. INTRODUCTION Influenza virus (IFV) is a respiratory pathogen that causes annual influenza epidemics as well as periodic global pandemics. The elderly and other high-risk patients are especially susceptible to influenza, which causes symptoms of fever, coughing, and headache and can lead to severe pneumonia and multiple organ failures.1−4 Adaptive mutations and genetic reassortment are the major reasons for high intraspecific variability, increase in virulence, and appearance of drug resistance in IFV. For example, the 2009 pandemic was caused by a novel H1N1 strain. Therefore, there is a continuing need for discovery of new antiviral agents and new targets to treat IFV infections. Currently, two main strategies are being used to control IFV infections: vaccination and antiviral treatment.5 Vaccination is the main prophylactic measure for healthy adults and people with chronic conditions, but it must be readministered annually and is markedly less effective for individuals with compromised immunity or similar high-risk medical conditions. In addition, the lack of efficacy was observed in very young children, and the live attenuated vaccines are not recommended for the very young. The efficacy of the vaccines is heavily dependent on correctly predicting the predominant infectious strains for each year, and incorrect predictions can render vaccination less than 25% effective.6,7 Hence, antiviral drugs may represent the first-line therapy.3−8 Two classes of virus protein-specific anti-IFV agents have been approved by the U.S. Food and Drug Administration (FDA), namely, M2 ion-channel blockers (1 (adamantine) and 2 (rimantadine)) and neuraminidase (NA) inhibitors (3 (oseltamivir), 4 (zanamivir), 5 (peramivir), and 6 (laninamivir octanoate)) © 2017 American Chemical Society

Received: August 13, 2016 Published: January 24, 2017 3533

DOI: 10.1021/acs.jmedchem.6b01227 J. Med. Chem. 2017, 60, 3533−3551

Journal of Medicinal Chemistry

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Figure 1. Approved antiviral agents for the treatment of IFV infection.

lack of a human counterpart of PA-Nter implies that the design of highly selective, nontoxic inhibitors should be feasible. The divalent metal ions (either two Mn2+ or two Mg2+ cations) present at the catalytic site (Figure 2) are critical for the endonuclease activity (Mn2+ is usually used in crystallography as a replacement for Mg2+ as the electron density of Mn2+ is higher than that of Mg2+, while they maintain similar coordination geometry). Thus, a metal-chelation strategy, i.e., discovering inhibitors that efficiently coordinate with the metal ions, could be effective for blocking the enzymatic activity and preventing the processing of the biological substrates. As a result of the rapid development of crystallographic and biochemical studies of PA-Nter (dozens of structures of complexes with various inhibitors are available in the Protein Data Bank), it is established that the central parts of inhibitors (such as diketo acids, exemplified by 4-dioxo-4-phenylbutanoic acid (8, DPBA)) bind to the active site of PA-Nter by chelating the divalent metal ions and also interacting with the surrounding amino acids (Figure 3).35 A detailed understanding of the mechanism of the metal-dependent catalysis should enable better mechanismbased design of PA-Nter inhibitors, and PA-Nter is considered a promising target for drug design.21,22,36,37

discovery, we then discuss currently used and new hit-to-lead and lead-to-candidate evolution strategies aimed at the discovery of more potent, selective, and safer anti-influenza drugs.

2. STRUCTURE AND FUNCTIONS OF PA ENDONUCLEASE AND BINDING MODE OF INHIBITORS IFV belongs to the orthomyxoviridae family, whose members contain eight negative-stranded RNA genomic segments. RNA-dependent RNA polymerase (RdRp), which catalyzes both the transcription and replication steps, is a heterotrimeric complex that comprises polymerase acidic (PA) protein, polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) subunits (Figure 2).23 The viral replication, namely, transcription of vRNA to mRNA, starts with the “cap-snatching” reaction. First, cellular capped RNAs are bound by an independent folding domain of PB2 and then are cleaved by PA at 10−15 nucleotides from the cap to yield 5′-capped RNA fragments. These fragments serve as primers for viral mRNA elongation catalyzed by the PB1 polymerase subunit. The cleavage is mediated by the endonuclease activity of the N-terminal region of PA, while the C-terminal region harbors a “mouth” into which the N-terminal domain (residues 1−16) of PB1 is inserted. Termination and polyadenylation occur at a stretch of five to seven U residues near the 5′ end of the vRNA.24−27 PA endonuclease function is crucial for IFV replication, and the endonuclease is therefore an attractive target for new antiviral therapies. The protein can be divided into N-terminal (PA-Nter) and C-terminal (PA-Cter) domains, which are linked by a long flexible peptide chain. PA-Nter consists of five β-strands surrounded by seven α-helices (Figure 2). The metal-chelating active site of PA-Nter is a negatively charged pocket, which consists of a histidine (His41), a conserved lysine (Lys134), and a cluster of three acidic residues (Glu80, Asp108, and Glu119), and binds two divalent metal ions (Mg2+ or Mn2+).25,28−34 The deep cleft at the endonuclease catalytic site of PA-Nter is a promising target for structure-based design of novel antiinfluenza agents for the following reasons:7 (i) the endonuclease activity is indispensable for the virus lifecycle; (ii) the PA-Nter domain is highly conserved across all strains and subtypes of the IFV family, namely, IFV types A, B, and C, and inhibitors may therefore have broad efficacy against multiple serotypes; (iii) the

3. SMALL-MOLECULAR INHIBITORS OF IFV PA ENDONUCLEASE There are currently no FDA-approved endonuclease inhibitors, but several classes of small-molecular inhibitors have been reported by various research groups. These inhibitors can be chemically classified as diketo acid derivatives, flutimide derivatives, hydroxylated heterocycles (3-hydroxyquinolin2(1H)-ones, 5-hydroxypyrimidin-4(3H)-ones, 3-hydroxypyridin-2(1H)-ones), catechol derivatives, 2,3-dihydroxybenzoic acid and its bioisosteres, and other scaffolds. They will be systematically discussed in the following sections. 3.1. Diketo Acid Derivatives. 2,4-Dioxobutanoicacid derivatives are PA endonuclease inhibitors containing the diketo acid (DKA) motif. For example, 4-dioxo-4-phenylbutanoic acid (8, DPBA) is a prototypic inhibitor with an IC50 value of 21.3 μM in in vitro transcription assay. This value was unchanged upon methoxy substitution at the ortho or para position of the benzene ring. However, chlorine at the para position decreased the potency (Figure 4).38,39 The addition of an extra phenyl moiety to 8, as observed in compound 9, resulted in a 6-fold improvement in 3534



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Figure 3. PA endonuclease in complex with 8. (a) Binding mode of 8 (two molecules, in blue and pink, respectively) in the active site of avian IFA PA-Nter (PDB code 4E5G).32 Key active site residues are illustrated. (b) Surface presentation of 8 (one molecule) binding to the active site pocket of IFV strain (pH1N1 2009) PA endonuclease (PDB code 4AWF).35 The inhibitory activity of 8 has been attributed to the three contiguous oxygen atoms because at physiological pH, each oxygen atom has high negative charge and is well positioned to bind to and inhibit bridged dinuclear metalloenzymes.

Figure 2. Structurally characterized fragments of IFV type A RNA polymerase heterotrimeric complex. (a) The complete heterotrimeric RNA polymerase X-ray structure (PDB code 4WSB) is shown: PA is in green, PB1 in yellow, and PB2 in blue. (b) Structural model of the IFV RNA-dependent RNA polymerase PA subunit complexed with dTMP (green) (2009 H1N1 PA endonuclease active site, PDB code 5CCY). The PA endonuclease active site employs two divalent metal cations (shown as purple spheres) to facilitate hydrolytic cleavage of the phosphodiester backbone of nucleic acids.30 Other PDB codes are 4M5Q (apo influenza 2009 H1N1 endonuclease structure), 4NFZ (crystal structure of polymerase subunit PA-Nter endonuclease domain from bat-derived influenza virus H17N10),31 4E5E (crystal structure of apo avian influenza virus PA-Nter),32 3HW3 (crystal structure of avian influenza virus PA-Nter in complex with UMP).33

10 not only inhibited viral RNA synthesis but also had a secondary effect on virus entry. This series of diketo acid derivatives selectively inhibited the infectivity of IFV types A and B in both in vitro and in vivo assays with potency in the micromolar range but did not inhibit the initiation or elongation of IFV mRNA synthesis.38,39 Piperidinyl-diketo acid compounds 11−15 were found to selectively inhibit the endonuclease function of PA. The diketo acid moiety is linked to a piperidine group, which contains two cyclic substituents (“wings”). These molecules inhibited IFV replication in yield reduction assays with EC50 values ranging from 0.18 to 0.71 μM, which were in the same level with those for inhibition of in vitro transcription (0.32−0.54 μM).41 Notably, 11 (L-742,001, IC50 = 0.43 μM) is one of the most potent compounds, exhibiting dose-dependent inhibition of cell-based viral replication (EC50 = 0.35 μM).41 Compounds 12−15 also inhibited in vitro transcription and endonuclease activity with high potency and showed dose-dependent inhibition of viral replication in cell cultures. Interestingly, the activity was not changed when the phenyl moiety of 11 was replaced by a cyclohexyl group (12) or by chlorobenzene (13) and became

potency because the extra phenyl group probably had an additional interaction with Tyr24 (Figure 3). Derivatization of the carboxylic acids of 8 and 9 to methyl ester or amide abolished the potency, as did substitution of the α-carbonyl group of 8 with amine, methylene, or alcohol. These results indicated that the 2,4-dioxobutanoic side chain was indispensable for the inhibitory activity and that improvements might be achieved by optimization of substitution on the phenyl group.38,39 The pyrrole diketo acid derivative 10 was previously reported as a potent inhibitor of human immunodeficiency virus (HIV) integrase (IN), through a scaffold-refining approach.40 Stevaert et al. found that 10 possessed a favorable IC50 value (0.8 μM) in plasmid-based enzymatic assay.38 Further studies confirmed that 3535



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Figure 4. Two forms of diketo chelation of two divalent cations by 8 (top left; the purple spheres are manganese ions) and the structures of diketo acid based PA endonuclease inhibitors 8−15 and inhibitor candidates 16−19.

the newly designed compounds 16 and 17 with a carbonyl or sulfonyl moiety as a proton acceptor might potently inhibit PA endonuclease activity. By use of the above structural biology and structural bioinformatics as a starting point, 2,4-dioxobutanoic acid derivatives 18 and 19 were identified as novel PA endonuclease inhibitors by Chen and his colleagues through virtual screening of 450 000 compounds. These compounds displayed attractive endonuclease-inhibitory activity (IC50 = 0.6 and 0.74 μM) in a FRET-based assay. However, in spite of this, they exhibited neither antiviral activity in cell-based assay nor cytotoxicity, possibly due to poor cell permeability.45 3.2. Flutimide Derivatives. The 2,6-diketopiperazine derivative 20 (flutimide) is a fungal metabolite that was isolated from a new species of Delitschia confertaspora. It selectively inhibited the cap-dependent transcription of viral RNA of IFV types A and B to mRNA and blocked viral replication in cell cultures. In addition, 20 exhibited an IC50 value of 5.1 μM against the IFV endonuclease by chelating the two divalent metal ions in the PA-Nter active site, without affecting the activities of other polymerases. Further modification by replacing the left-side isopropyl moiety of 20 with a p-substituted phenyl group afforded compounds 21 and 22, which showed IC50 values of 0.8 and 0.9 μM, respectively (Figure 6). Substitution of both of the isopropyl moieties with (substituted) phenyl groups afforded compounds 23 and 24 with slightly improved or equivalent potency compared to 20. The SARs indicated that the N-hydroxyl and olefin groups are required for potency, and an increase in activity was observed by substituting the isopropyl side chains with aromatic groups. Unfortunately, these more active analogues demonstrated very high cytotoxicity in cell culture.46,47 Recently, through a structure-based scaffold-hopping approach, novel substituted indole derivatives of 20 (1,2-annulated indolediketopiperazines) were identified as PA endonuclease inhibitors. All the N-hydroxyimides showed potent inhibitory activity while displaying low cytotoxicity. The most active inhibitors were 25 and 26, with IC50 values of 12.7 μM and 17.3 μM, respectively.48 Docking calculations showed that the designed analogues retained the interaction with the two manganese ions, like diketo acid derivatives (compound 8), and the extended

only slightly lower when the piperidine nitrogen was moved to an adjacent position (14). Moreover, it is noteworthy that the introduction of a sulfonyl group on the side chain (15) significantly improved the potency. Crystal structures of PA-Nter complexed with various piperidinyl-diketo acid inhibitors have revealed a general chelating mode with the two catalytic site metal ions (M1 and M2), despite the involvement of different subpockets. As illustrated in Figure 5, the two “wings” of these molecules occupy different hydrophobic pockets.38 The chlorobenzyl group lies outside the active site, interacting with Phe105. In contrast, the aryl piperidine is located in a deeper position, having van der Waals contact with Ala20 and Tyr24; the piperidine nitrogen does not participate in hydrogen bonding. The complete diketobutanoic acid structure (i.e., chelating function with Mn2+ ions) is required for antiviral activity. Specifically, the carboxylate of the DKA moiety chelates the first Mn2+ ion, while the second Mn2+ ion of PA-Nter interacts with the oxygen atom of its carboxylate moiety and the α-hydroxyl group, or the two Mn2+ ions simply form a chelate with the β-diketone group (Figure 5).42,43 These crystal structures provide important insights into the binding mode of piperidinyl-diketo acids in the PA endonuclease catalytic center. Besides, some potential resistance sites, such as H41A, G81F/V/T, and I120T mutants, should be taken into account in the optimization process of PA endonuclease inhibitors, from the viewpoint of tight binding in any of the subpockets (in general, hydrophobic) surrounding the active site of the enzyme.30,43 Moreover, monodentate binding of two ligand molecules rather than bidentate complexation of the metal ion(s) was observed. Monodendate ligands usually have flexible conformation and can bind with multiple binding sites.44 The availability of crystal structures of endonuclease bound to various inhibitors has enabled researchers to use the structurebased drug design approach to develop improved endonuclease inhibitors by identifying chemical moieties capable of interacting with additional sites of the endonuclease. Ishikawa and co-workers designed potential inhibitors 16 and 17 by optimization of 11 using a thermodynamic method with structureguided and computer-assisted modeling, targeting the proton donor Arg84.42 The molecular simulation studies suggested that 3536



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indole system of the molecules facilitated hydrophobic and stacking interactions with the residues, especially with the side chains of Tyr24, Lys34, and Ile38. Notably, the 8-position of the indole ring extended toward unstable water molecules and the N-terminal cavity. Consequently, this characteristic should be helpful for modulation of the physicochemical properties of N-hydroxyimides.48 As early as in 2003, 2-hydroxyisoquinoline-1,3(2H,4H)-dione (27) was reported as a PA endonuclease inhibitor with an IC50 of 15 μM via screening (in cap-dependent RNA polymerase assay) of the focused Roche compound collections selected to include molecules with a range of potential metal-binding groups (MBGs).49 The core part is clearly similar to 20. In preliminary structure−activity relationship studies, it was found that the N-hydroxyl group and the benzoyl carbonyl group are functionally necessary. More importantly, the two benzoyl carbonyl groups functioned together: removing either of them impaired the activity. In addition, replacing the benzene ring with heterocyclic analogues was unsuccessful.49 In 2012, the crystal structure of avian IFV PA-Nter bound to 27 was reported (Figure 7).32 Compound 27 is a compact fragment structure, and this should minimize steric hindrance during binding with the protein but may also favor interactions with binding pockets in unrelated targets, resulting in unacceptable selectivity (this will be discussed below). Careful analysis of structural biology information on the endonuclease complexed with loose binders could offer important inspiration to a suitable decoration pattern for this fragment. 3.3. Hydroxylated Heterocycles. LaVoie and co-workers have recently reported a series of hydroxylated heterocycles, including 3-hydroxyquinolin-2(1H)-ones, 5-hydroxypyrimidin4(3H)-ones, and 3-hydroxypyridin-2(1H)-ones, that act as potent PA endonuclease inhibitors by chelating the two metal ions.50,51 In the 3-hydroxyquinolin-2(1H)-one subseries, 28 and 29 exhibited the best inhibitory activities, each with an IC50 value of 0.5 μM. The presence of a p-fluorophenyl substituent at either the 6- or 7-position of 3-hydroxyquinolin-2(1H)-one significantly enhanced the inhibitory activity relative any other halogen, but substitution at the 4- and 8-positions reduced the activity, as exemplified by compound 30 (Figure 8).50 In the 5-hydroxypyrimidin-4(3H)-one subseries, the 4-(tetrazolyl)phenyl derivative 31 was the most potent compound (IC50 = 0.15 μM), while the 3-(tetrazolyl)phenyl derivative 32 showed 3 times lower potency. The 2-(cyanophenyl) precursors of compounds 31 and 32 also possessed surprising inhibitory activities. For example, the 4-cyanophenyl compound 33 showed activity equal to that of 32, while the 3-cyanophenyl isomer 34 is approximately twice as potent. Another potent inhibitor in this series is 35 (IC50 = 0.40 μM), which seems to be a promising lead compound for further modification, especially on the phenyl rings.51 Crystallographic fragment screening is a new technique for initiating drug discovery in which crystals are soaked with high concentrations of small fragment-like molecules.52−54 Fragment screening identified 5-bromo-3-hydroxypyridin-2(1H)-one (36) as an initial active site chelating hit. Refinement of the structure displayed that three molecules of 36 bound to PA-Nter. Two coordinated with M3 and were bound to subpockets 2 and 3 (analogous to the binding of 11), while the third chelated to M1 and M2 at the endonuclease catalytic center (Figure 9a). 36 exhibited potent activity in the enzymatic assay (IC50 = 16 μM). Further hit-to-lead modification was performed, and in the 3-hydroxypyridin-2(1H)-one series, compounds 37 and 38

Figure 5. 2009 H1N1 PA endonuclease in complex with piperidinyldiketo acids: (a) 11 (pink) (PDB code 5CGV);30 (b) 12 (blue) (PDB code 4AVG);35 (c) crystallographic overlays of 14 (yellow) (PDB code 4AWG) and 15 (orange) (PDB code 4AWK) in the catalytic site of PA endonuclease.35 Key hydrogen bond interactions are shown as yellow dashed lines. In addition, crystal structures of 11 in complexes with four additional types of endonucleases, namely, 2009 H1N1 PA endonuclease mutant E119D (PDB code 5D8U), mutant F105S (PDB code 5D9J),30 IFV strain H1N1 polymerase acidic subunit N-terminal region (PDB code 5FDG),44 and avian IFV PA (PDB code 4E5H),32 have also been disclosed. 3537



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Figure 6. Structures of derivatives derived from 20 and its heterocyclic counterparts.

showed IC50 values of 11 and 23 nM, respectively, in the enzymatic assay. Indeed, all of the 5,6-diphenyl-substituted 3-hydroxypyridin-2(1H)-ones exhibited significant activities as IFV type A endonuclease inhibitors. For example, compounds 39, 40 exhibited IC50 values of 47 and 41 nM, respectively, which are slightly lower than those of the tetrazolyl group-containing derivatives and equal to those of most compounds substituted only at the 5 or 6-position (such as 41−43). These results showed that the placement of phenyl groups at both the 5- and 6-positions could significantly enhance the inhibitory activities. In addition, different substituents on these phenyl moieties affected the potency in different ways.55,56 Crystal structures of endonuclease-hydroxylated heterocycle complexes have been determined and represent the most accurate reference points for understanding how these compounds chelate the enzyme active-site metals, as well as the basis of the structure−affinity relationships. For example, the quinoline scaffold of 29 forms a hydrogen bond between the hydroxyl group at the 3-position and Lys134 (Figure 9b).50 The protonated nitrogen of the quinoline also interacts with a water molecule chelating to Mn2+. Importantly, the ring system shows a 50° tilt

Figure 7. Crystal structure of avian IFV PA-Nter bound to 27 (PDB code 4E5F).32 Potential decoration sites are highlighted.

Figure 8. Hydroxylated heterocycles as PA endonuclease inhibitors. Reported IC50 values for each compound are shown. The MBG portion of each molecule is shown in red. 3538



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Figure 9. Crystal structures of IFV 2009 H1N1 PA endonuclease bound to a set of hydroxylated heterocycles: (a) 36 (three molecules) (PDB code 4MK1);56 (b) 29 (pink) (PDB code 4KIL);50 (c) 31 (white) (PDB code 4W9S);51 (d) 38 (green) (PDB code 4M5U);55 (e) 37 (yellow) (PDB code 4M4Q);56 (f) crystallographic overlays of 37 (yellow) and 38 (green) in the catalytic site of PA endonuclease. Metal ions are depicted as purple spheres, and hydrogen bonds are depicted as yellow dashed lines. Crystal structures of IFV 2009 pH1N1 endonuclease bound to 40 (PDB code 4LN7), 41 (PDB code 4M5O), 42 (PDB code 4MK5), 43 (PDB code 4MK2) have also been disclosed.56

toward His41, which is different from other inhibitors. The binding angle favors π−π stacking interactions with His41, which

facilitates additional interactions with adjacent pockets. Thus, the 7-p-fluorophenyl moiety of the molecule extends into a subpocket 3539



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formed by Ala20, Met21, Tyr24, Glu26, Lys34, and Ile38 (highlighted in Figure 9b). There also appears to be a hydrophobic interaction between the 7-p-fluorophenyl moiety and the pocket.50 The N-1 atom of the pyrimidine ring in 31 forms a hydrogen bond with a water molecule (small red ball, in Figure 9c), while N-3 interacts with Tyr130 through two bridging waters (small red balls, in Figure 9c). Moreover, the 2-[4-(tetrazolyl)phenyl] structure has potential bidentate hydrogen-bonding interactions with Arg124 (not shown in Figure 9c), which could significantly increase the affinity.51 The pyridinone ring of 38 could chelate the two metal ions in the catalytic center and undergo π-stacking interaction with His41. The pyridinone nitrogen forms hydrogen bonds to a coordinating water of one metal ion and to the Glu80 residue. When apo crystals of PA-Nter were soaked in a solution of 38, the p-fluorophenyl group formed a cation−π interaction with one metal ion and exhibited hydrophobic interactions with the side chains of Ala20, Tyr24, and Ile38, while the 4-(tetrazol-5yl)phenyl group showed hydrophobic interactions with the side chains of Lys34, Ala37, and Ile38 (Figure 9d). Compounds 37 and 38 displayed very similar binding conformations (Figure 9e and Figure 9f), but 37 was more potent than 38, probably because of the optimal position of the pyridinone nitrogen and its favorable interactions with metal-chelating atoms.55,56 Thus, the X-ray crystal structures suggest potential new avenues for modifications that could further modulate the biological activity. Fragment-based drug discovery (FBDD) has become an increasingly common method for hit identification and for exploration of chemical diversity space in drug discovery as an alternative to HTS. It has the advantage of being very flexible, as it can exploit diverse methodologies, such as virtual, biophysical, and biochemical approaches. Moreover, it can afford optimal chemical platforms that bind more efficiently to alternative regions, affording molecules with higher ligand efficiency (LE) and favorable druglike properties than those originating from classical high-throughput screening (HTS) approaches and thus delivering higher hit rates.57

In an effort to efficiently discover novel, potent inhibitors of IFV PA endonuclease, a FBDD campaign was launched, using a carefully designed metal-binding pharmacophore (metalloenzyme-focused) library consisting of ∼300 fragment molecules designed to interact with the metal ions in the metalloenzyme active sites. This resulted in the discovery of the pyromeconic acid scaffold (exemplified by 44, in Figure 10) as a subject for further elaboration. The findings on the presence of multiple binding subpockets located around the PAN substrate-binding site will provide useful clues for enhancing the binding affinity of compounds.44 Subsequently, guided by modeling of this hit, as well as SAR identified in the initial library screen and previously reported structural data, a modest sublibrary of molecules (containing 45 and 46) was constructed to establish precise structure−activity relationships, using fragment growth and fragment merging strategies. This ultimately afforded a lead compound 47, which showed an IC50 value of 14 nM in enzymatic assays, an EC50 value of 2.1 μM against an IFV H1N1 strain in MDCK cells, and a CC50 value of 280 μM (Figure 10).58 Docking analysis of 47 predicts a very similar binding mode to that observed crystallographically for phenyltetrazole-containing inhibitors (exemplified by 37). This approach could be applied to facilitate hit-to-lead optimization of a range of biological metalloenzyme targets. Very recently, a new series of 5-hydroxy-4-pyridone-3-carboxy acid derivatives was reported as influenza endonuclease inhibitors from an in-house chelate library. The lipophilic region proved to be very important to endonuclease inhibition. Particularly, the distance between the lipophilic region and the chelate was a critical factor for the activity. An SAR study of hit molecule 48 based on virtual modeling resulted in the discovery of 49 as a potent endonuclease inhibitor with an IC50 of 5.12 nM (Figure 11). However, 49 had low antiviral potency (EC50 = 201 μM) compared to its enzyme−inhibitory potency, likely due to poor membrane permeability.59 3.4. Catechol Derivatives. Natural products are always a good source for the discovery of novel lead compounds. Natural products containing a catechol fragment form an important class

Figure 10. Fragment-based identification of PA endonuclease inhibitor 47 from a metalloenzyme-focused library. The MBG portion of each molecule is shown in red. 3540



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screening and bioactivity assay, two catechol derivatives 50 (IC50 = 0.94 μM) and 51 (IC50 = 7.0 μM) were identified as PA endonuclease inhibitors.60 In a virus yield assay using IFV-infected MDCK cells, compound 50 achieved EC90 and EC99 values of 3.2 and 5.7 μM, compared with 6.3 and 12 μM for compound 51 (the reference compound 11 had EC90 and EC99 values of 5.4 and 8.4 μM, respectively) (Figure 12). Docking study indicated that the dihydroxyindole moiety was directed toward the two catalytic metal ions, and both hydroxyl groups chelated metal ions. The catechol functional group of 50 and the second dihydroxyindole ring of 51 were engaged in the Val122-Arg124Tyr130 cavity, which is of critical importance, based on PA-Nter crystallization experiments. The structures of these novel classes of catechols have not yet been thoroughly investigated for medicinalchemistry purposes, and these compounds could constitute important starting points for detailed structural optimization.

Figure 11. Identification of newer 5-hydroxy-4-pyridone-3-carboxy acids as potent endonuclease inhibitors. The MBG portion of each molecule is shown in red.

of bioactive medicinal compounds and have recently drawn more and more attention for their diverse and complicated bioactivities. Through a combination of pharmacophore-docking virtual

Figure 12. Structures of catechol-containing PA endonuclease inhibitors. The MBG portion of each molecule is shown in red. The toxicity of the bisbibenzyl derivatives 62 and 63 was not reported in the original paper.66 3541



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Compound 52 (thalidomide), a hypnotic/sedative drug with anticachexia, anti-inflammatory, and antiangiogenic activities, was utilized as a platform to develop a class of phenethylphenylphthalimide analogues as novel PA endonuclease inhibitors. Compounds 53−55 bearing a 3,4-dihydroxyphenethyl group displayed remarkable potency against replication of IFV type A, with EC50 values of 24, 48, and 26 μM, respectively, and CC50 values above 80 μM. Only 53 and 54 are inhibitors of PA endonuclease. The loss of inhibition with conversion of the 3,4-dihydroxyphenethyl moiety to a 3,4-dihydroxycinnamyl moiety indicated that the 3,4-dihydroxyphenethyl group plays an indispensable role in the inhibitory activity.61 Recently, novel chemotypes of natural origin have been found to modulate PA endonuclease function. (−)-Epigallocatechin gallate (56, EGCG) and epicatechin gallate (57, ECG) are major polyphenol components of green tea. EGCG inhibited the endonuclease activity of PA-Nter at a dose of 10 μM (EC50 = 28.4 μM in plaque inhibition assay using MDCK cells). ECG also showed inhibitory activity, though similar compounds without the galloyl motif gave disappointing results. The galloyl group appears to be crucial for inhibitory activity, and in the case of EGCG, its binding location in the endonuclease domain was predicted.62,63 In 2012, the high-resolution X-ray cocrystal structure of the 2009 pandemic H1N1 PA endonuclease region with (−)-epigallocatechin gallate (PDB code 4AWM) was described. The conformation, details of the interactions, and the placement of the EGCG in the catalytic center are illustrated in Figure 13a,b. Two manganese ions are cochelated with two of the hydroxyl groups of the gallo group, while the galloyl moiety is orientated to helix α3, forming hydrogen bond with the carbonyl oxygen of Val122, and stacking on Ile38. The planes of the gallo- and galloylphenyls are parallel but not entirely overlapped. The double ring of EGCG is orientated to the preceding loop, notably with the resorcinol group stacking on Tyr24 and forming a hydrogen bond with Glu26. However, three of the eight hydroxyls of EGCG do not form direct contacts with endonuclease. These finding will be important for designing more active compounds targeting the cap-snatching activity of PA endonuclease.35 On the other hand, to improve the low lipid membrane permeability, poor chemical stability, and metabolic stability of EGCG, a set of EGCG fatty acid monoester derivatives was obtained through lipase-catalyzed transesterification. EGCG monoesters (a mixture of 58a−d) demonstrated the best potency with an EC50 value of 4 μM, and the inhibitory effect was 24-fold greater than that of EGCG. Although 58a−d showed relatively high cytotoxicity, the antiviral activity was significantly enhanced and consequently the selectivity index was increased compared to other derivatives.64 The biological potencies of thiosemicarbazones (TSCs) are often involved in the coordination of metal ions in an N,S-bidentate mode. Thus, in principle, they could be potential chelating inhibitors of PA endonuclease. On the basis of this idea, Rogolino et al. synthesized several salicylaldehyde thiosemicarbazone derivatives and evaluated their inhibitory activity in enzymatic PA-Nter inhibitory assays and MDCK-cells-based virus yield assays. Notably, compounds 59 and 60 displayed weak activities in enzymatic assay with PA-Nter; the catechol 59 gave the best result (IC50 = 37 μM), which is consistent with indispensability of the catechol pharmacophore for inhibition of PA-Nter mentioned above. To probe the role of the thiosemicarbazone motif in the activity, compound 61, a hydrazonic analogue of 59, was synthesized and tested. It is noteworthy that

Figure 13. IFV strain (H1N1) 2009 polymerase subunit PA endonuclease complexed with 56 (PDB code 4AWM).35 56 is shown as a stick model with gray carbons. (a) The divalent cations (two manganese ions, purple spheres) and key catalytic center residues that interact with 56 or are close to it are illustrated. Possible hydrogen bonds are illustrated as yellow dotted lines. (b) Surface presentation of 56 bound in the H1N1 PA endonuclease active site. Manganese ions are shown as pink spheres.

replacement of the sulfur atom with an oxygen afforded a slight improvement in the inhibitory potency toward PA-Nter, with the IC50 changing from 37 μM to 24 μM, but the inhibition of 61 in the cellular vRNP assay was lower, which indicates that for the thiosemicarbazone derivatives, inhibition of PA-Nter endonuclease activity is not the only mode of action. Further, in the vRNP reconstitution assay, 60 showed antiviral activity at the concentration of 22 μM and 59 was effective at 63 μM, while in the PA-Nter inhibitory assay the IC50 value was 341 μM. This suggested that the inhibitory effect of 60 on IFV replication may not be solely due to direct inhibition of PA endonuclease.65 Marchantin is a phytochemical found in several liverworts and has a unique macrocyclic bisbibenzyl structure. By screening 33 different series of phytochemicals using an in vitro enzymatic assay, 62 (marchantin A) and 63 (marchantin E), both bearing a 3,4-dihydroxyphenethyl group, were identified as PA endonuclease inhibitors, indicating the importance of the 3,4-dihydroxyphenethyl group for the inhibition of the endonuclease. In silico docking simulation analysis indicated that the dihydroxyphenethyl group of 63 could chelate the Mn2+ ions 3542



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within PA endonuclease. Moreover, the aromatic groups and the methoxy moiety bind to the active pocket via hydrophobic interaction.66 Compounds 64−66 are catechol derivatives that were identified by virtual screening, and they showed IC50 values of around 2 μM in a fluorescence resonance energy transfer (FRET)-based assay using a recombinant H1N1 2009 PA-Nter construct. However, 64 was cytotoxic, while 65 and 66 lacked both cytotoxicity and antiviral activity (the cytotoxicity was determined in MDCK cells). On the other hand, compound 67, an oxidized analogue of 64−66, had negligible cytotoxicity, promoted infected cell survival with an EC50 of 18 μM, and inhibited endonuclease with an IC50 of 14 μM.45 An in vitro chemical screening of approximately 410 compounds by Fudo et al. resulted in the discovery of several PA-Nter endonuclease inhibitors, including 68, 69, and 70.44,67 In FRET-based endonuclease assay, 68 and 70 had IC50 values of the order of 10−6 M, while 69 had an IC50 value of the order of 10−5 M. All the inhibitors were found to have similar antiviral potencies in an IFV cell culture assay (EC50 values of 10−15 μM), although 69 was cytotoxic (CC50 = 74 μM). Compounds 68 and 70 displayed no apparent cytotoxicity (CC50 > 200 μM). As the molecular weights of these compounds are comparatively low, there is considerable potential for structural optimizations to improve the activity. To clarify the binding poses of these molecules to PA-Nter, a crystallographic investigation on the PA-Nter complexed with each inhibitor was performed (68, PDB code 4YYL; 69, PDB codes 5FDD and 4ZHZ; 70, PDB codes 5I13 and 4ZI0).44,67 The information from this study will be useful in designing and seeking more active agents. Very recently, the activities of a class of N-acylhydrazones in PA-Nter endonuclease inhibition assay and in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution (in HEK293T cells) and virus yield (in influenza virus-infected MDCK cells) assays were reported.68 Some N-acylhydrazones (exemplified by 71) exhibited remarkable anti-influenza potency in the low micromolar concentration level, together with high selectivity. Furthermore, the crystal structure of PA-Nter complexed with 71 was disclosed, revealing its interactions in and around the catalytic center of the enzyme (PDB code 5EGA). It is interesting to note that the galloyl moiety efficiently coordinates the manganese ions within the protein’s active site, while the trihydroxyphenyl moiety stacks against the side chain of Tyr24. Two of these hydroxyl moieties are in suitable positions to generate hydrogen bonds with the Glu26 and Lys34 side chains (Figure 14), suggesting that, in further optimization of these compounds, other favorable functional moieties could be introduced to replace the hydroxyls in order to obtain stronger interactions with the side chains of Glu26 and Lys34 and possibly higher inhibitory potency. 3.5. 2,3-Dihydroxybenzoic Acid and Its Bioisosteres. Bioisosterism is considered to be a useful strategy for molecular optimization and drug design.69 Through an in vitro endonuclease inhibition assay, it was demonstrated that the bioisosteres of 2,3-dihydroxybenzoic acid (72) (exemplified by 5-hydroxy-6-oxopyrimidine-4-carboxylic acids 73 and 74) inhibit the enzymatic activity of the isolated PA-Nter endonuclease domain (Figure 15).32 Furthermore, the way in which these compounds coordinate the two metal ions in the endonuclease catalytic center and engage with the catalytic center residues was visualized by means of X-ray crystallography (Figure 16a−c). Interestingly, an induced-fit mode of compound binding was observed in the cocrystal structures of PA-Nter and the inhibitors.32

Figure 14. Structure of 71 and crystal structure of 2009 H1N1 PA-Nter endonuclease complexed with 71 (PDB code 5EGA).68 Compound 71 is shown as a stick model with pink carbons. The catalytic residues are shown as stick models with yellow carbons, and Mn2+ and water molecules are shown as purple and blue spheres, respectively. Hydrogen bonds are shown as yellow dotted lines.

For instance, the chlorobenzyl group of 11 causes a movement of Tyr24, highlighting the point that conformation changes can occur when inhibitors bind to the catalytic center. These results should be helpful to guide further hit-to-lead optimization. On the basis of the functional resemblance between the catalytic centers of PA-Nter and HIV integrase (IN), another bridged dinuclear metalloenzyme,70 it was speculated that HIV IN inhibitors might also inhibit PA endonuclease. Accordingly, Carcelli and his co-workers repositioned a HIV IN inhibitor 2,3dihydroxybenzamide 75 as a novel endonuclease inhibitor with IC50 = 33 μM. Interestingly, its magnesium complex displayed a 2-fold higher activity (IC50 = 18 μM) than the free ligand 75, which suggested that the effective inhibitor of PA activity could be a metal complex.71 This was the first report to suggest that a metal complex might serve as an inhibitor of PA-Nter endonuclease. Molecular simulation showed that 75 binds to 3543



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Figure 15. Structures of 2,3-dihydroxybenzoic acid (72) and its bioisosteres 73−76.

Figure 16. Crystal structure of avian IFV PA-Nter bound to 2,3-dihydroxybenzoic acid and several bioisosteres: (a) 72 (pink) (PDB code 4E5L);32 (b) 73 (yellow) (PDB code 4E5I);32 (c) 74 (green) (PDB code 4E5J);32 (d) binding model of 72 (yellow) compared with 11 (white) in the PA-Nter complex (PDB code 4E5H). The two metal ions are shown in orange.71

endonuclease inhibitor with broad-spectrum potency in multiple in vitro assays, including IFV type A (seasonal and 2009 pandemic H1N1, seasonal H3N2) and type B (Yamagata and Victoria lineages), zoonotic viruses (H5N1, H9N2, and H7N9), and NAI-resistant mutations (with nanomolar to submicromolar EC50 values).72 76 could specifically block the ability of the PA endonuclease domain to split a nucleic acid substrate, displaying low toxicity and a favorable selectivity index. In cell culture

one metal ion via the catechol group and coordinates with the second cation through the amide-linked carbonyl and the α-hydroxyl groups. The chelating motif of 75 strictly overlapped with the three coplanar oxygen atoms of 11 (Figure 16d), and this result could offer hints for structure-based rational design and modification of PA endonuclease inhibitors. Very recently, a novel 5-hydroxypyrido[2,1-f ][1,2,4]triazine4,6-dione derivative 76 (RO-7) was identified as a novel PA 3544



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Figure 17. Other scaffolds with PA endonuclease-inhibitory activity.

scaffolds or novel mechanisms of action are attracting great interest.

(in primary, normal human bronchial epithelial cells), 76 also blocked IFV replication.72 These results therefore suggest that 76 has great potential to be developed as a promising anti-influenza agent. 3.6. Other Scaffolds with Alternative Mechanisms of Action. As described above, the vast majority of potent PA endonuclease inhibitors are metal-chelating agents. The intrinsic conformational flexibility of the endonuclease can give additional allosteric sites and/or alternative mechanisms that can be exploited to seek new antiviral agents. In recent years, several endonuclease inhibitors bearing innovative scaffolds or acting via other mechanisms have been disclosed (Figure 17). In 2012, THC19 (77) was identified as a PA inhibitor via phenotypic screening (experimental and computational screening of compound collection) and followed by target identification based on time-of-addition experiments and minigenome assays.73 It showed no cytotoxicity and did not interfere with cell-cycle progression or induce apoptosis in MDCK cells. Preliminary SAR investigation revealed that substituting the 1,2,3,4-tetrahydrocarbazole ring and replacing this ring with a carbazole or indole ring resulted in reduced potency. The authors concluded that the 1,2,3,4-tetrahydrocarbazole moiety, the propanol linker, and the piperidine ring were all essential for antiviral activity and low cytotoxicity. Although the binding mode of 77 has not been clarified, this compound should be a useful tool to probe the mechanisms of PA endonuclease activity.73 In 2016, 78 (PA-30) was identified as a potent PA endonuclease inhibitor with high activity against IFV type A through systematic screening of a chemical library containing 950 compounds.74 The binding affinity of 79 (ANA-0) (Kd = 1.1 μM), one of a series of analogues of 78, was greater than that of the known inhibitor 8 (Kd = 4.5 μM). Subsequently, it was found that 79 potently inhibited replication of multiple subtypes of IFV type A, including H1N1, H3N2, H5N1, H7N7, H7N9, and H9N2, in cell cultures. Interestingly, a synergistic antiviral effect was observed in combinational treatment with zanamivir and 79.74 Thus, 79 is considered a promising anti-influenza candidate for clinical applications. Often resistance can occur by mutation of the drug target to reduce or prevent binding. From the clinical point of view, the combination of a classical inhibitor with an inhibitor acting via a different mechanism is expected to be less susceptible to development of resistance.75 Multidrug combinations, as exemplified by the tritherapies developed to treat HIV infection, may also be useful in severe influenza cases.29 Thus, efforts to develop additional endonuclease inhibitors with alternative

4. CONCLUSIONS AND PROSPECTS Yearly variance in the effectiveness of vaccination, the slow response to vaccines in dealing with epidemic outbreaks, and the frequent emergence of drug resistance necessitate the development of new drugs to treat influenza infections, especially drugs that are not susceptible to escape mutation by the virus. The essential function of the PA endonuclease domain in the synthesis of viral mRNAs and in the virus life cycle, as well as the presence of the deep cleft at the endonuclease catalytic center of PA-Nter, makes this enzyme a promising target for developing effective antivirals. During the past decade, many different structural classes of inhibitors targeting PA endonuclease have been designed and developed. Indeed, two PA endonuclease inhibitors (AL-794 and S-033188) have recently entered clinical trials (the safety and pharmacokinetics profile of S-033188 had been confirmed in a phase I study). This is a significant milestone in the development of potent and specific inhibitors, but the structures of these two compounds have not been disclosed for reasons of commercial confidentiality.76,77 This review has covered the progress made to date in inhibiting the activity of PA endonuclease with small molecules, focusing on the discovery, structural modifications, SARs, and mechanisms of action of inhibitors. Although many different chemical series of PA endonuclease inhibitors have been described in this review, it is important to note that many of the scaffolds have never been further investigated after the initial report. There may be many reasons for this, including unconfirmed activity, poor oral bioavailability, cytotoxicity, and/or poor specificity (due to drug promiscuity, namely, drug binding to more than one target). Thus, potent, selective, orally active anti-endonuclease drugs to treat influenza have remained elusive. Regarding the lead discovery phase, most of these endonuclease inhibitors with potential for further optimization were identified by experimental HTS screening of large libraries obtained via highly resource-intensive and time-consuming synthetic work. On the other hand, fragment screening by X-ray crystallography has been employed to seek novel endonuclease binders by using previously assembled diverse compound libraries and soaking preformed crystals in solutions of the test compounds.56 This approach has been extensively discussed in dozens of papers.52−54 Recent work has demonstrated that a metal-centric FBDD approach (also known as the targeted library screening method) can enable rapid and efficient 3545



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design of highly specific endonuclease inhibitors to fight influenza viral infections.85,86 Consequently, screening for special inhibitors using endonuclease-containing complexes rather than isolated endonuclease or the addition of requisite multiprotein interaction assays is advocated. For example, it was reported that both PA(1−27) and NP domains involved in the binding are highly conserved among influenza A strains, so newly identified protein−protein complexes could offer novel targets for antiviral drug design.86 As described above, the vast majority of PA endonuclease inhibitors act by coordinating to the catalytic center metal ions. In general, inhibitors can be divided into two parts: a MBG core and a backbone substituent (as discussed below). As an alternative approach to structure-based methods, ligand-based virtual screening methods that would enable us to select molecular shape and pharmacophore analogues of previously known endonuclease inhibitors based on similarity to reference compounds, could enable faster and more efficient drug discovery.87 In particular, we can learn from the ligand-based virtual screening methodologies (3D pharmacophore-guided database search) used in the discovery of novel strand-transfer inhibitors targeting HIV-1 IN, another metalloenzyme.88−90 The endonuclease and other endoribonucleases usually use the phosphodiester group 3′ of the scissile bond to favor catalysis, and thus their catalytic sites often share high sequence homology. It is generally agreed that compounds targeting only the catalytic centers may lack selectivity. For example, the divalent metal ion chelator moieties of representative PA endonuclease inhibitors, such as N-hydroxyimides, diketo acids, pyrimidinol carboxylic acids, etc., are also potent active-site inhibitors of other metalloenzymes via the same mechanism of chelation of activesite divalent metal ions (Figures 18 and 19). Examples include HIV-1 IN inhibitors 27 and 80 (an analogue of 75),91,92 HIV RNase H inhibitor 81 (an analogue of 74),93 La Crosse virus Bunyaviridae RNA polymerase,94 and Mycobacterium tuberculosis malate synthase binder 8.95 These facts may suggest the existence of some so-far-uncharacterized divalent metal ion-chelating “privileged structure” that is shared by these inhibitors. Thus, it might be possible to modify inhibitors of other bridged dinuclear metalloenzymes with similar active sites, including the abovementioned metalloenzymes and hepatitis C virus (HCV) NS5B polymerase, to obtain novel inhibitors of PA endonuclease via structure-based scaffold re-evolution/refining.96 In fact, “privileged structure”-guided scaffold re-evolution has been extensively used by our group97 and other groups98−101 as a primary strategy to develop novel bioactivities by making use of readily derivatizable intermediates with well-established preparation methods (we have previously reviewed this topic40). However, privileged motifs may interact with multiple targets,

identification of leadlike molecules with good physiochemical properties via screening a vast chemical space using a small library of fragments, especially when compared to traditional FBDD using unbiased libraries or HTS.58,78 Of course, because each fragment has relatively few interactions, the binding affinities are probably lower than those of larger compounds, requiring higher assay sensitivity. In addition, physicochemical or other empirical filters based on key characteristics should be adopted to exclude reactive, metabolically active groups, known aggregators, and pan-assay interference compounds when selecting fragments to construct a library for lead discovery. We also envisage that combining new technologies (such as click-chemistry-based combinatorial libraries and DNA encoded compound libraries) with orthogonal HTS methods will emerge as a faster and more efficient approach for screening of large chemical libraries.79,80 Large amounts of X-ray structural biology data have been accumulated in recent years for complexes of PA endonuclease with the major classes of inhibitors. Therefore, besides classical and modified HTS campaigns, structure-based computational screening of virtual libraries and computer-aided de novo drug design utilizing these data will undoubtedly be beneficial to discover novel effective chemical entities. Indeed, some progress has already been achieved in the identification of novel endonuclease inhibitors via computer-aided technologies.60 To emphasize an important point, molecular simulation may not reflect the real binding process and in some cases (despite a high score) may afford questionable results.81 Therefore, in docking simulation, the following issues should be paid sound attention, including conformational flexibility of endonuclease and conformational changes upon ligand binding (induced fit).81 More specifically, for metalloenzymes, the affinity of MBGs is related to many factors including donor atom identity, orientation, electrostatic interactions, and van der Waals interactions.82 Molecular modeling with the existing docking programs to illustrate metal coordination process (coordination geometry, atomic charge variability) and proton transfers during ligand binding to metalloenzymes (from small molecules to a neighboring basic residue) still has a lot of limitations.83 In this context, it is important to develop specific functions and parameters (such as a noncovalent scoring function) to describe metal−ligand coordination.83,84 It is important to note that endonuclease is just one component of sophisticated biomacromolecular complexes (incorporating host mRNA and nucleoprotein (NP)), and the observed selectivity of inhibitors toward purified endonuclease may be different from that toward biomacromolecular complexes. Therefore, a detailed understanding of the relevant protein (or RNA)−protein interactions will be important in the

Figure 18. Putative divalent metal ion-chelating “privileged structure” with potential activity against multiple metalloenzymes. 3546



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and the different amino acid residues chelated wih the two metal ions, especially the additional His residue in PA compared to a water molecule in RNase H.85 Taking into account the current understanding of endonuclease structure, the combination of diversity-oriented synthesis (DOS) around privileged structures and focused library preparation (target-oriented synthesis, TOS) should prove to be efficient methodology for exploring a large chemical space to discover innovative and selective endonuclease inhibitors. This approach may be more reliable than computational virtual screening alone and less costly than conventional approaches such as serendipitous discovery or HTS of compound libraries.104 Most endonuclease inhibitors so far reported contain two functional motifs. One is a metal-binding group (MBG) that coordinates the divalent metal ion(s) and interacts with residues at the endonuclease active site; examples are a diketo acid or catechol group. The other is a backbone motif, namely, an aromatic or hydrophobic moiety, interacting with subpockets distant from the metal ion(s) center. Both moieties can be optimized in various ways. For example, the backbone can be extended to explore additional regions around the endonuclease active site. Thus, in the lead optimization process, an initial weakly binding hit fragment can be developed into potent endonuclease inhibitors via structure-based core-refining, substituents-decorating (bioisosteric replacement and scaffoldhopping), and crystallographic overlay-based molecular hybridization. In other words, previous empirical trial-and-error approaches are being replaced by more sophisticated strategies that rely heavily on detailed structural information about endonuclease-ligand interactions and medicinal-chemistry strategies to improve drug resistance profiles,105 physicochemical profiles, and synthetic accessibility.106 Notably, recent structure optimizations of endonuclease inhibitors have not been purely affinity-driven, but instead the LE value, an important metric of “druglikeness” that reflects the activity and physicochemical properties of compounds,107 has been employed to guide modification of the scaffold and substituent groups.56 Of course, overcoming toxicity and improving metabolic stability are improving the solubility, and targeting to the proper cellular compartment may decrease side effects. Therefore, apart from SAR exploration around initial hits, multiobjective optimization,108 including early absorption, distribution, metabolism, excretion, and toxicity (ADMET) assessments, of endonuclease inhibitors is needed to complement the classical approaches. Already, some new agents have yielded promising results in clinical trials, and it seems clear that there are excellent prospects for eventual introduction of PA endonuclease inhibitors as anti-influenza therapeutics.

Figure 19. Crystal structures of 8 in complexes with (a) N-terminal endonuclease domain of La Crosse virus Bunyaviridae RNA polymerase (L-protein) (PDB code 2XI7)94 and (b) Mycobacterium tuberculosis malate synthase (PDB code 3S9I).95

which can lead to wide-ranging, even unfavorable, bioactivities. Crystal structures of metalloenzymes indicate that protein surface topology proximal to the catalytic centers affords features that are unique to each metalloenzyme and that amino acids in these peripheral sites may be very important for the unique substrate recognition properties of each metalloenzyme. Thus, multisite-binding ligands derived from a divalent metal ionchelating “privileged structure”, which can simultaneously target the catalytic pocket as well as peripheral sites outside the active site, may show enhanced potency and selectivity. Meanwhile, computer-aided drug design and dynamics simulation techniques can be utilized to understand the metal−ligand interactions and the effects of structural differences (preferential interactions) in the active sites of metalloenzymes and to help overcome the selectivity issue.102,103 For example, in both PA endonuclease and HIV RNase H, a hydrogen bond is formed between the 3′ phosphodiester group and the adjacent nucleophilic water, which may help to properly position the attacking water. The subtle differences between these two ribonucleases probably result from the different nucleic acid substrates (double-stranded DNA or an RNA:DNA hybrid for RNase H but single-stranded RNA for PA)

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AUTHOR INFORMATION

Corresponding Authors

*B.H.: e-mail, [email protected]; phone, 086-531-85999436. *X.L.: e-mail, [email protected]; phone, 086-531-88380270. *P.Z.: e-mail, [email protected]; phone, 086-53188382005. ORCID

Peng Zhan: 0000-0002-9675-6026 Notes

The authors declare no competing financial interest. All binding mode illustrations were produced with PyMol (www.pymol.org). 3547



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Biographies

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC Grant 81573347), Young Scholars Program of Shandong University (YSPSDU Grant 2016WLJH32 to P.Z.), Key Project of NSFC for International Cooperation (Grant 81420108027), Shandong Provincial Key Research and Development Program (Grant 2015GNC110009 to B.H.), the Science and Technology Development Project of Shandong Province (Grant 2014GSF118175), and Major Project of Science and Technology of Shandong Province (Grant 2015ZDJS04001) is gratefully acknowledged.

Han Ju completed his B.S. degree at Shenyang Pharmaceutical University in 2015 and then started work with Professor Xinyong Liu as a graduate student in the School of Pharmaceutical Science, Shandong University. His current research focuses on the discovery of influenza virus neuraminidase inhibitors and PA endonuclease inhibitors. Jian Zhang received his Master’s degree from Shandong Normal University in 2010 and then worked in a pharmaceutical company from 2010 to 2014. Since September 2014 he has been working in the School of Pharmaceutical Sciences of Shandong University as a Ph.D. candidate, supervised by Professor Xinyong Liu. His research interests focus on rational design, synthesis, and biological evaluation of novel potent inhibitors of influenza virus neuraminidase.

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ABBREVIATIONS USED DKA, diketo acid; DOS, diversity-oriented synthesis; DPBA, 4-dioxo-4-phenylbutanoic acid; EGCG, epigallocatechin gallate; FBDD, fragment-based drug discovery; FRET, fluorescence resonance energy transfer; HIV, human immunodeficiency virus; HTS, high-throughput screening; IFV, influenza virus; IN, integrase; LE, ligand efficiency; MBG, metal-binding group; PA, polymerase acidic protein; PB1, polymerase basic protein 1; PB2, polymerase basic protein 2; PA-Nter, N-terminal of polymerase acidic protein; PA-Cter, C-terminal of polymerase acidic protein; RdRp, RNA-dependent RNA polymerase; SAR, structure−activity relationship; TOS, target-oriented synthesis; TSC, thiosemicarbazone

Boshi Huang completed his B.S. degree at Shandong University in 2012. He is currently studying for his Ph.D. degree in the School of Pharmaceutical Science in Shandong University. His current research focuses on the discovery of antiviral agents based on rational drug design. Dongwei Kang graduated from the School of Hebei University of Technology in 2012 and earned his M.S. degree in the School of Pharmaceutical Sciences in Shandong University in 2015. He is studying for his Ph.D. degree in Shandong University. His current research focuses on the discovery of antiviral agents based on rational drug design.

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Bing Huang received his B.S. degree from Guizhou University in 1995 and his Master’s degree from Nanjing Agricultural University in 1998. Then, he worked at Guizhou University. In 2001 he joined Professor Xiumei Zhang’s group at the Institute of Poultry Science, Shandong Academy of Agricultural Science as an Assistant Scientist. He completed his Ph.D. under the supervision of Professor Puyan Chen at Nanjing Agricultural University in 2005. He spent a year and a half as a Visiting Scholar working with Professor Feng Li (South Dakota State University, U.S.) on the phylodynamics of bovine influenza D virus. In March 2014, he was appointed a Professor at the Institute of Poultry Science of SAAS. His research interests include molecular biology, pathogenesis, and control of animal virus.

REFERENCES

(1) Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases (NCIRD) Website; October 31, 2016. https://www.cdc.gov/flu/about/disease/. (2) Chowell, G.; Bertozzi, S. M.; Colchero, M. A.; Lopez-Gatell, H.; Alpuche-Aranda, C.; Hernandez, M.; Miller, M. A. Severe respiratory disease concurrent with the circulation of H1N1 influenza. N. Engl. J. Med. 2009, 361, 674−679. (3) Li, T. C.; Chan, M. C.; Lee, N. Clinical implications of antiviral resistance in influenza. Viruses 2015, 7, 4929−4944. (4) Yen, H. L. Current and novel antiviral strategies for influenza infection. Curr. Opin. Virol. 2016, 18, 126−134. (5) Du, J.; Cross, T. A.; Zhou, H. X. Recent progress in structure-based anti-influenza drug design. Drug Discovery Today 2012, 17, 1111−1120. (6) Nichol, K. L.; Treanor, J. J. Vaccines for seasonal and pandemic influenza. J. Infect. Dis. 2006, 194 (Suppl. 2), S111−S118. (7) Subbarao, K.; Joseph, T. Scientific barriers to developing vaccines against avian influenza viruses. Nat. Rev. Immunol. 2007, 7, 267−278. (8) Das, K.; Aramini, J. M.; Ma, L. C.; Krug, R. M.; Arnold, E. Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 2010, 17, 530−538. (9) Davies, W. L.; Grunert, R. R.; Haff, R. F.; Mcgahen, J. W.; Neumayer, E. M.; Paulshock, M.; Watts, J. C.; Wood, T. R.; Hermann, E. C.; Hoffmann, C. E. Antiviral activity of 1-adamantanamine (amantadine). Science 1964, 144, 862−863. (10) Dunning, J.; Baillie, J. K.; Cao, B.; Hayden, F. G. International severe acute respiratory and emerging infection consortium (ISARIC). antiviral combinations for severe influenza. Lancet Infect. Dis. 2014, 14, 1259−1270. (11) Smee, D. F.; Sidwell, R. W. Peramivir (BCX-1812, RWJ-270201): potential new therapy for influenza. Expert Opin. Invest. Drugs 2002, 11, 859−869. (12) Cheng, C. K.; Tsai, C. H.; Shie, J. J.; Fang, J. M. From neuraminidase inhibitors to conjugates: a step towards better antiinfluenza drugs? Future Med. Chem. 2014, 6, 757−774. (13) Furuta, Y.; Gowen, B. B.; Takahashi, K.; Shiraki, K.; Smee, D. F.; Barnard, D. L. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 2013, 100, 446−454. (14) Bright, R. A.; Shay, D. K.; Shu, B.; Cox, N. J.; Klimov, A. I. Adamantane resistance among influenza A viruses isolated early during

Xinyong Liu received his B.S. and M.S. degrees from School of Pharmaceutical Sciences, Shandong University, China, in 1984 and in 1991, respectively. From 1997 to 1999 he worked at Instituto de Quimica Medica (CSIC) in Spain as a Senior Visiting Scholar. He obtained his Ph.D. from Shandong University in 2004. He is currently a Full Professor of the Institute of Medicinal Chemistry, Shandong University. His research interests include rational drug design and synthesis and antiviral activity evaluation of a variety of molecules that interact with specific enzymes and receptors in the viral life cycle (HIV, HBV, HCV, and FluV). Other ongoing programs include molecular modification and structure− activity relationships study of natural products used to treat cardiovascular diseases, and drug delivery research using PEGylated small-molecular agents. Peng Zhan obtained his B.S. degree at Shandong University, China, in 2005 and earned his M.S. and Ph.D. degrees at Shandong University in 2008 and 2010, respectively. He subsequently joined the research group of Professor Xinyong Liu as a Lecturer (2010−2012). From 2012 to 2014, he worked as a Postdoctoral Fellow funded by JSPS (Japan Society for the Promotion of Science) in the Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan. He is currently an Associate Professor in the laboratory of Professor Xinyong Liu. His research interests include the discovery of novel antiviral, anticancer, and neurodegenerative disease-related agents based on rational drug design and combinatorial chemistry approaches. 3548



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