Viewpoint Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/aidcbc
Inhibitors of Influenza A Virus Polymerase Shuofeng Yuan, Lei Wen, and Jie Zhou* Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China ABSTRACT: The propensity of influenza virus to develop resistance to commonly prescribed drugs highlights the need for continuing development of new therapeutics. Biological and structural investigations of the enzymatic and interaction domains among influenza A virus polymerase subunits have broadened the target reservoir for drug screening. With the wealth of knowledge from these studies, identification of small-molecule and peptidic inhibitors that specifically abrogate polymerase activity or disrupt the polymerase assembly has emerged as an innovative and promising approach. Importantly, those domains are highly conserved among influenza subtypes and thus minimize the emergence of drug resistant mutants. An overview of the reported enzymatic inhibitors and protein−protein disruptors has been provided, in our effort to facilitate the development of next-generation anti-influenza therapeutics.
T
RNA genome, which is constituted of eight different viral RNA (vRNA) segments that encode for 11 identified viral proteins (Figure 1A).10 Each segment is associated with a viral RNAdependent RNA polymerase (RdRP) and multiple copies of viral nucleoprotein (NP), forming the viral ribonucleoprotein
he continuous zoonotic circulation and reassortment potential of influenza A viruses in nature have been posing an enormous public health threat to humans.1−3 Two classes of anti-influenza drugs have been approved for clinical treatment, the M2 channel inhibitors (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (oseltamivir, zanamivir, peramivir, and laninamivir). In addition, the first polymerase inhibitor, favipiravir (T-705), was recently approved in Japan for influenza treatment, highlighting the importance of a newgeneration of anti-influenza drugs targeting viral polymerase.4 As a broad-spectrum antiviral drug, however, the antiviral mode of action of favipiravir has not been fully elucidated. On the other hand, increasing usage of the licensed antivirals has resulted in the global emergence of amantadine- and/or oseltamivir-resistant strains of influenza virus and the occasional isolations of peramivir- or zanamivir-resistant influenza viruses, which are exemplified by the worldwide spread of adamantine resistant A(H3N2) viruses since 2003, oseltamivir-resistant seasonal A(H1N1) viruses since 2007, adamantane-resistant pandemic A(H1N1) viruses in 2009, and peramivir-resistant A(H7N9) viruses in 2013.5,6 Collectively, the ability of influenza virus to evade vaccines and become drug resistant calls for novel anti-influenza therapeutics using new targets and creative strategies. Recent advances in the molecular mechanisms of influenza A virus replication have hinted at new avenues for the development of antiviral agents that target viral proteins7 or host factors.8 Generally, virus-targeting antivirals functionally inhibit the biological process of viral proteins, mostly enzymatic activities. Alternatively, they may block viral protein−protein interactions (PPI) that are essential for the virus replication machinery.9 Host-targeting antivirals inspect the host−virus battleground and focus on the cellular factors that are involved in the viral life cycle or host immune response. Nevertheless, the virus-targeting antivirals, such as polymerase and protease inhibitors, will still be mainstream in the development of antiviral therapies, mainly due to our more comprehensive understanding of their underlying mechanisms.9 Influenza A virus belongs to the Orthomyxoviridae family. The infectious virion possesses a negative-sense single-stranded © XXXX American Chemical Society
Figure 1. Schematic representation of influenza A virus particle and viral ribonucleoprotein complex. (A) The influenza A virion is constituted by a lipid bilayer envelope that exposes three surface proteins, including hemagglutinin (HA), neuraminidase (NA), and the M2 ion channel. Viral matrix protein M1 coats the inside of the viral membrane. The viral genome lies within the M1 inner layer, consisting of eight viral RNA molecules that are encapsidated with nucleoprotein (NP) and associated with the viral polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA) to form the vRNP complex. Nonstructural protein 1 (NS1) is a multifunctional protein and a virulence factor, and nonstructural protein 2 (NS2) is a nuclear export protein (NEP). (B) Influenza vRNP complex. Each influenza vRNP consists of one single-stranded, negative-sense genomic RNA associated with multiple nucleoprotein (NP) monomers and a single trimeric polymerase complex (PB1, PB2, and PA). The 5′ and 3′ vRNA ends are complementary and base pair to form a double-stranded structure, which is bound by the polymerase complex at one end of the vRNP filament. The internal vRNA region is organized into an antiparallel double helix, the formation of which is driven by contacts between NP monomers. Received: December 15, 2017
A
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Viewpoint
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 in red, PB2 in green, and PB1 in cyan. Functional domains are labeled within a gay box. (B) Structural model of the PA endonuclease domain, with key catalytic residues highlighted. Other PDB codes are 4M5Q, 4NFZ, 4E5E, and 5CCY. (C) Structural model of the PB2 cap-binding domain, with key amino acid residues highlighted. Other PDB codes are 2VQ2, 4CB4, 4Q46, and 5EF9. (D) Structural model of the PA−PB1 interaction domain, with key residues in the hydrophobic pockets highlighted. Other PDB codes are 2ZNL and 3CM8. (E) Structural model of the PB1−PB2 interaction domain, with key interface highlighted. Other PDB codes are 2ZTT and 3A1G.
and used as a primer for viral mRNA synthesis.10 Structurally, this heterotrimeric complex is assembled largely through the intersubunit interface between PA C-terminal domain (PAC) and PB2 N-terminal domain (PB1N)15,16 and that between PB1 C-terminal domain (PB1C) and PB2 N-terminal domain (PB2N).17
(vRNP) complex (Figure 1B). The vRNP constitutes the active transcription and replication component unit, in which the RdRP catalyzes the distinct processes of both transcription and replication using the same vRNA.11 Progresses on polymerase structures of bat influenza A virus,12 human influenza B virus,13 and influenza C virus14 have provided tremendous insights on the complex architecture of the RdRP. Functioning as a viral RNA synthesis machine, the RdRP can produce either cRNA or vRNA through de novo replication or viral mRNA transcription through “cap-snatching”. Functionally, PB1 houses the catalytic site for polymerase activity, while PB2 binds the cap structure of cellular pre-mRNA, which is cleaved by PA endonuclease
■
ENZYMATIC AND INTERACTION DOMAINS OF INFLUENZA A VIRUS POLYMERASE Two enzymatic domains of influenza A virus polymerase located in PB2 (Figure 2C) and PA subunit (Figure 2B), respectively, have been well characterized in the past decade. B
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Viewpoint
determined the PB1−PB2 interface are identified, containing residues Leu695, Phe699, Val715, Val719, Ala722, Ile746, and Ile750 that locate in PB1 C-terminus.17 Compared with the PA−PB1 interaction, the PB1−PB2 interface has been less investigated, a possible explanation might be the difficulty of obtaining “drug-like” inhibitors through disruption of PB1C and PB2N binding. As suggested by different groups, alternative binding/mediating site(s), other than the PB1 C-terminal and PB2 N-terminal interface, may contribute to the PB1−PB2 association.25,26
The conserved residues 318−483 in PB2 subunit (PB2cap) of influenza A polymerase is an independently folded cap-binding domain that exhibits a distinct binding mode from other host cap-binding proteins such as human eIF4E protein.18 Influenza viruses are obligate parasites that “steal” primers from the host to initiate the viral transcription. During this process, PB2cap binds the 5′-capped end of host pre-mRNA, which is cleaved by the PAN endonuclease. The “snatched” 5′-capped primer is then used for transcription of the viral genome by the viral PB1 polymerase. Thirteen amino acids are identified as the conserved residues responsible for cap-binding pocket formation of influenza A PB2 protein, including Phe323, Phe325, Lys339, Arg355, His357, Glu361, Phe363, Lys376, Phe404, Gln406, Asn429, Met431, and His432.19 Intriguingly, crystal structure of the PB2cap of influenza B virus reveals a different cap recognition mechanism.20 Unlike influenza A PB2cap that exhibits a preference for m7GDP (a cap mimic) rather than GDP, PB2cap of influenza B shows an analogous affinity for both substrates. In addition, influenza B PB2cap has a weaker affinity for m7GDP than that of influenza A, whereas replacement of influenza B PB2 Glu325 by Phe, the corresponding residue of influenza A PB2, increases the binding affinity significantly.20 Amino acid residues in the N-terminal (first 196 residues) of the PA subunit (PAN) play critical roles in endonuclease activity, protein stability, and vRNA promoter binding.21 The metal-chelating active site of PAN is a negatively charged pocket, which binds to two divalent metal ions (Mg2+ or Mn2+) and consists of a histidine (His41), a conserved lysine (Lys134), and a cluster of three acidic residues (Glu80, Asp108, and Glu119).22 A comprehensive study on (i) the modified PAN endonuclease activity and (ii) the enzyme− inhibitor cocrystal structure has been carried out by DuBois and colleagues,23 which highlights the important role of metal coordination in PAN inhibitor binding. Assembly of the individual subunits PB1, PA, and PB2 into the heterotrimeric polymerase complex form is a prerequisite for viral replication. Before entering the host nucleus to exert functions, the three subunits bind to each other noncovalently through a series of interactions that are essential for polymerase assembly. The cocrystallization of the PAC−PB1N complex has revealed that the PB1N (residues 1−25 of PB1 subunit) clamps into the open “jaws” of a dragon’s head, the hydrophobic grooves formed by PAC domain (residues 257−716 of PA subunit).24 These “jaws”, thus, represent a potential target for novel anti-influenza therapeutics (Figure 2D). The understanding of the structural basis of the PA−PB1 complex has been advanced by two papers published in 2008, in which X-ray crystal structures of the PA−PB1 complex were reported (Protein Data Bank (PDB) code: 3CM8) by He et al.16 and (2ZNL) by Obayashi et al.15 A few differences in the key interaction residues are noted despite significant similarities in the structures, whereas the consensus of opinions is that the contribution of the PAC residues W706, Q408, N412, L640, M595, V636, and Q670 are of primary importance. Later, the crystal structure of whole bat influenza A polymerase reveals that, aside from the interaction between the PB1N residues and the large PAC domain (residues 258−714), the PA linker (residues 196−257) that connects the PAN endonuclease and the PAC domains is wrapped around the external face of the PB1 fingers and palm domains.13 The interaction of PB1C and PB2N domain also modulates the assembly of PB1 and PB2 subunits (Figure 2E). In the pioneer work of Sugiyama and colleagues, key residues that
■
INHIBITORS OF INFLUENZA A POLYMERASE Inhibition of PA endonuclease27 and PB2 cap-binding28 are valid means for developing the virus-targeting antivirals. Another appealing approach to inhibit the RdRP function is to interfere with its proper assembly using PPIs. Both strategies have been actively pursued in the past decades.29−31 VX-787 (pimodivir) is a potent influenza A PB2cap inhibitor that is now under preclinical characterization.32 This orally bioavailable azaindole inhibitor binds the purified central domain of PB2, occupies the cap-binding site as defined by X-ray crystallography, and blocks cap-dependent production of positive-strand viral RNA in time course experiments. Importantly, VX-787 is highly effective in both prophylaxis and treatment mouse models of influenza A and is superior to the neuraminidase inhibitor oseltamivir, even in delayed-startto-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in the mice when the initiation of therapy was delayed up to 120 h postinfection. In mouse lungs, VX-787 administration shows a 5-log reduction in viral load (relative to vehicle controls) with the treatment dosage of 10 mg/kg.33 Using reverse chemical genetic screening, we established an innovative fluorescence polarization assay for the identification of anti-influenza PB2cap inhibitors.34 A panel of PB2 cap-binding inhibitors is selected from a chemical library, followed by the evaluation of their antiviral efficacies. A novel small-molecule compound PB2−39 (Figure 3) is identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, in vitro and in vivo.28 Collectively, these studies highlight PB2cap as a useful anti-influenza therapeutic target. Determination of the PAN crystal structure22,24 has also paved the path for the development of endonuclease inhibitors. These inhibitors can be chemically classified into different groups, including 2,4-dioxobutanoic acid derivatives,23,35−37 flutimide derivatives,23,35,38 3-hydroxyquinolin-2(1H)-ones, and 3-hydroxypyridin-2(1H)-ones,39,40 as well as tetramic acid derivatives.41 Strategically, these endonuclease inhibitors are screened by computational modeling42−44 or identified through the measurement of nucleic acid hydrolysis45,46 or capsnatching activity.47 The great success in the development of PAN inhibitors is partially due to the spacious active site of PA endonuclease. For example, even a short DNA aptamer can fit into the PAN endonuclease cavity.48 Among the reported PAN endonuclease inhibitors with in vivo efficacy, RO-7 (Figure 3) exhibits impressive potential for clinical usage. Prophylactic administration completely protects mice from lethal infection by influenza A or B virus. The level of therapeutic protection varies from 60% to 100% and 80% to 100% survival with influenza A and B viruses, respectively. RO-7 treatment significantly decreases virus titers in the lung and lessens the extent and severity of lung damage.49 C
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Viewpoint
ELISA-based screening, we identified a novel small-molecule compound PP7 (Figure 3) with antiviral activities against influenza A virus, including A(H1N1)pdm09, A(H7N9), and A(H9N2) subtypes, but noneffective against A(H7N7) and A(H5N1) strains.26 The subtype specific antiviral property of PP7 suggests that alternative binding/mediating site(s), other than the PB1C and PB2N interface, may contribute to the PB1− PB2 association. Failure of cross-subtype protection of PP7 might also be restricted by the unique features of PPI. Protein− protein interaction often involves a large, flat surface area and multiple contacts; therefore, they cannot be easily disrupted through the binding and competition of a small molecule. Accordingly, there are a few examples of small “drug-like” molecules that selectively disrupt these interactions,52 in contrast to the numerous reports of using dominant negative proteins, antibodies, or peptides to inhibit protein−protein interactions. Our experiences in the discovery of PA−PB1 or PB1−PB2 inhibitors suggest that the PA−PB1 interface shall be prioritized as a druggable antiviral target for searching influenza A polymerase assembly inhibitors.
■
CONCLUDING REMARKS The limited ability of influenza viruses to compensate for mutations deliberately introduced into the enzymatic or interactive domains is observed, suggesting that escape mutations in these domains are a rare occurrence.53 Usually, mutations in these individual domains, alone or in combination, impair polymerase activity and viral growth. By searching the PubMed database, we analyzed the publication activity during the past decade in terms of the drug development upon influenza A polymerase inhibitors. PA endonuclease inhibitors have been intensively screened and represent a promising class of drugs for the next-generation anti-influenza therapeutics. Despite the extensive studies, apart from favipiravir (T-705) and pimodivir (VX-787), very few influenza A polymerase inhibitors have shown promising clinical potential. Under this circumstance, modifications of the pharmacophore and substituent groups are essential. Structural optimizations of lead compounds have been mostly affinity-driven; however, improvement on the physicochemical properties of the compounds bears the same importance. Toxicity, metabolic stability, solubility, and cellular compartment tropism are all critical factors that should be considered. Therefore, apart from the focused structure−activity exploration of the initial hits, multiobjective optimization, particularly the assessments of absorption, distribution, metabolism, excretion, and toxicity (ADMET), of the identified lead compounds is needed to complement the classical approaches.54 Segmented negative strand RNA viruses of the arena-, bunya-, and orthomyxovirus families uniquely carry out viral mRNA transcription by the “cap-snatching” mechanism.55 Bunyaviruses, the emerging zoonotic pathogens of publichealth concern, caused endemics of Severe Fever with Thrombocytopenia Syndrome (SFTS) in Asia56 and human infections of Heartland virus (HRTV) cases in the USA.57 With the estimated case fatality rate of around 30%, there is no approved antiviral or vaccine for SFTS. One of the common features of SFTSV, HRTV, and influenza A viruses is that viral mRNA transcription requires the action of a metal iondependent endonuclease. In this regard, drug repurposing of influenza PAN endonuclease inhibitors, in particular the divalent metal ion chelators such as N-hydroxyimides, diketo acids, and pyrimidinol carboxylic acids, could be considered as a
Figure 3. Structures of highlighted inhibitors in each enzymatic/ interaction domain. Shown are chemical structures of the compounds and their corresponding drug targets.
Alternatively, an appealing strategy to inhibit functions of RdRP is to interfere with its proper assembly using PPI inhibitors. Among the three interactions of polymerase subunit, PB1−PB2, PA−PB2, and PA−PB1, only the PA−PB1 interaction has been reported to be inhibited by both peptides and small molecules. Wunderlich et al. pioneers the inhibition of the PA−PB1 complex formation and provides preliminary evidence that vRNA synthesis can be blocked by the specific inhibition of the polymerase PA−PB1 subunit interaction using small peptides.50 Later, studies by Schwemmle and co-workers solve the PA−PB1 crystal structures and furnish significant insights into the key residues involved in the interaction. A 25amino acid peptide, derived from the N-terminus of influenza A PB1, can compete with the corresponding PAC binding interface. This short peptide, when fused the HIV Tat cellpenetrating domain, inhibits the growth of influenza A virus by interfering with viral polymerase activity.51 However, the peptide is noneffective in blocking the polymerase activity of influenza B. On the basis of PA−PB1 crystallographic structures, Loregian and colleagues perform a virtual screening and identify small-molecule inhibitors of PA−PB1 interaction.30 Hit compounds are validated by an ELISA-based assay for their ability to disrupt His-PAC and GST-PB1N protein−protein binding. Notably, compound 1 (Figure 3) is found to potently inhibit the replication of a variety of influenza A virus strains, including seasonal H3N2 and H1N1 2009 pandemic strains, an oseltamivir-resistant isolate, and influenza B viruses. Making use of the ELISA-based assay, we performed a systematic screening of a chemical library and identified a small-molecule inhibitor ANA-1 with broad-spectrum antiviral activity against different subtypes of influenza A virus.31 In our effort to demonstrate the possibility of suppressing viral replication by abrogating the PB1-Cter and PB2-Nter binding, we verified the antiviral activity of a PB2N derived peptide reported by Reuther et al.25 Similar to the PA−PB1 D
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Viewpoint
for an essential subunit interaction in influenza virus RNA polymerase. Nature 454 (7208), 1127−1131. (16) He, X., Zhou, J., Bartlam, M., Zhang, R., Ma, J., Lou, Z., Li, X., Li, J., Joachimiak, A., Zeng, Z., Ge, R., Rao, Z., and Liu, Y. (2008) Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus. Nature 454 (7208), 1123−1126. (17) Sugiyama, K., Obayashi, E., Kawaguchi, A., Suzuki, Y., Tame, J. R., Nagata, K., and Park, S. Y. (2009) Structural insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase. EMBO J. 28 (12), 1803−1811. (18) Yanguez, E., Rodriguez, P., Goodfellow, I., and Nieto, A. (2012) Influenza virus polymerase confers independence of the cellular capbinding factor eIF4E for viral mRNA translation. Virology 422 (2), 297−307. (19) Liu, Y., Qin, K., Meng, G., Zhang, J., Zhou, J., Zhao, G., Luo, M., and Zheng, X. (2013) Structural and functional characterization of K339T substitution identified in the PB2 subunit cap-binding pocket of influenza A virus. J. Biol. Chem. 288 (16), 11013−11023. (20) Liu, Y., Yang, Y., Fan, J., He, R., Luo, M., and Zheng, X. (2015) The crystal structure of the PB2 cap-binding domain of influenza B virus reveals a novel cap recognition mechanism. J. Biol. Chem. 290 (14), 9141−9149. (21) Hara, K., Schmidt, F. I., Crow, M., and Brownlee, G. G. (2006) Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. J. Virol 80 (16), 7789−7798. (22) Dias, A., Bouvier, D., Crepin, T., McCarthy, A. A., Hart, D. J., Baudin, F., Cusack, S., and Ruigrok, R. W. (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458 (7240), 914−918. (23) DuBois, R. M., Slavish, P. J., Baughman, B. M., Yun, M. K., Bao, J., Webby, R. J., Webb, T. R., and White, S. W. (2012) Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease. PLoS Pathog. 8 (8), e1002830. (24) Yuan, P. W., Bartlam, M., Lou, Z. Y., Chen, S. D., Zhou, J., He, X. J., Lv, Z. Y., Ge, R. W., Li, X. M., Deng, T., Fodor, E., Rao, Z. H., and Liu, Y. F. (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458 (7240), 909−912. (25) Reuther, P., Manz, B., Brunotte, L., Schwemmle, M., and Wunderlich, K. (2011) Targeting of the influenza A virus polymerase PB1-PB2 interface indicates strain-specific assembly differences. J. Virol 85 (24), 13298−13309. (26) Yuan, S., Chu, H., Ye, J., Singh, K., Ye, Z., Zhao, H., Kao, R. Y., Chow, B. K., Zhou, J., and Zheng, B. J. (2017) Identification of a novel small-molecule compound targeting the influenza A virus polymerase PB1-PB2 interface. Antiviral Res. 137, 58−66. (27) Yuan, S., Chu, H., Singh, K., Zhao, H., Zhang, K., Kao, R. Y., Chow, B. K., Zhou, J., and Zheng, B. J. (2016) A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase. Sci. Rep. 6, 22880. (28) Yuan, S., Chu, H., Zhang, K., Ye, J., Singh, K., Kao, R. Y., Chow, B. K., Zhou, J., and Zheng, B. J. (2016) A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication. J. Antimicrob. Chemother. 71, 2489. (29) Massari, S., Goracci, L., Desantis, J., and Tabarrini, O. (2016) Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics. J. Med. Chem. 59, 7699. (30) Muratore, G., Goracci, L., Mercorelli, B., Foeglein, A., Digard, P., Cruciani, G., Palu, G., and Loregian, A. (2012) Small molecule inhibitors of influenza A and B viruses that act by disrupting subunit interactions of the viral polymerase. Proc. Natl. Acad. Sci. U. S. A. 109 (16), 6247−6252. (31) Yuan, S., Chu, H., Zhao, H., Zhang, K., Singh, K., Chow, B. K., Kao, R. Y., Zhou, J., and Zheng, B. J. (2016) Identification of a smallmolecule inhibitor of influenza virus via disrupting the subunits interaction of the viral polymerase. Antiviral Res. 125, 34−42.
therapeutic option for SFTSV and HRTV. The strategy is also applicable to Lassa or Hantaan viruses that are responsible for a large number of severe human infectious diseases and retain the “cap-snatching” mechanism in their life cycle as well.55
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +852 22554818. Fax: +852 28551241. E-mail: jiezhou@ hku.hk. Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Novel Swine-Origin Influenza Virus Investigation Team, Dawood, F. S., Jain, S., Finelli, L., Shaw, M. W., Lindstrom, S., Garten, R. J., Gubareva, L. V., Xu, X., Bridges, C. B., and Uyeki, T. M. (2009) Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 360 (25), 2605−2615. (2) Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., Chen, J., Jie, Z., Qiu, H., Xu, K., Xu, X., Lu, H., Zhu, W., Gao, Z., Xiang, N., Shen, Y., He, Z., Gu, Y., Zhang, Z., Yang, Y., Zhao, X., Zhou, L., Li, X., Zou, S., Zhang, Y., Li, X., Yang, L., Guo, J., Dong, J., Li, Q., Dong, L., Zhu, Y., Bai, T., Wang, S., Hao, P., Yang, W., Zhang, Y., Han, J., Yu, H., Li, D., Gao, G. F., Wu, G., Wang, Y., Yuan, Z., and Shu, Y. (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368 (20), 1888−1897. (3) Peiris, J. S., de Jong, M. D., and Guan, Y. (2007) Avian influenza virus (H5N1): a threat to human health. Clin Microbiol Rev. 20 (2), 243−267. (4) Furuta, Y., Gowen, B. B., Takahashi, K., Shiraki, K., Smee, D. F., and Barnard, D. L. (2013) Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 100 (2), 446−454. (5) Hayden, F. G., and de Jong, M. D. (2011) Emerging influenza antiviral resistance threats. J. Infect. Dis. 203 (1), 6−10. (6) Hai, R., Schmolke, M., Leyva-Grado, V. H., Thangavel, R. R., Margine, I., Jaffe, E. L., Krammer, F., Solorzano, A., Garcia-Sastre, A., Palese, P., and Bouvier, N. M. (2013) Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat. Commun. 4, 2854. (7) Das, K., Aramini, J. M., Ma, L. C., Krug, R. M., and Arnold, E. (2010) Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 17 (5), 530−538. (8) Muller, K. H., Kakkola, L., Nagaraj, A. S., Cheltsov, A. V., Anastasina, M., and Kainov, D. E. (2012) Emerging cellular targets for influenza antiviral agents. Trends Pharmacol. Sci. 33 (2), 89−99. (9) Lou, Z., Sun, Y., and Rao, Z. (2014) Current progress in antiviral strategies. Trends Pharmacol. Sci. 35 (2), 86−102. (10) Eisfeld, A. J., Neumann, G., and Kawaoka, Y. (2015) At the centre: influenza A virus ribonucleoproteins. Nat. Rev. Microbiol. 13 (1), 28−41. (11) Resa-Infante, P., Jorba, N., Coloma, R., and Ortin, J. (2011) The influenza virus RNA synthesis machine: advances in its structure and function. RNA Biol. 8 (2), 207−215. (12) Pflug, A., Guilligay, D., Reich, S., and Cusack, S. (2014) Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516 (7531), 355−360. (13) Reich, S., Guilligay, D., Pflug, A., Malet, H., Berger, I., Crepin, T., Hart, D., Lunardi, T., Nanao, M., Ruigrok, R. W., and Cusack, S. (2014) Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516 (7531), 361−366. (14) Hengrung, N., El Omari, K., Serna Martin, I., Vreede, F. T., Cusack, S., Rambo, R. P., Vonrhein, C., Bricogne, G., Stuart, D. I., Grimes, J. M., and Fodor, E. (2015) Crystal structure of the RNAdependent RNA polymerase from influenza C virus. Nature 527 (7576), 114−117. (15) Obayashi, E., Yoshida, H., Kawai, F., Shibayama, N., Kawaguchi, A., Nagata, K., Tame, J. R., and Park, S. Y. (2008) The structural basis E
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
ACS Infectious Diseases
Viewpoint
(32) Clark, M. P., Ledeboer, M. W., Davies, I., Byrn, R. A., Jones, S. M., Perola, E., Tsai, A., Jacobs, M., Nti-Addae, K., Bandarage, U. K., Boyd, M. J., Bethiel, R. S., Court, J. J., Deng, H., Duffy, J. P., Dorsch, W. A., Farmer, L. J., Gao, H., Gu, W., Jackson, K., Jacobs, D. H., Kennedy, J. M., Ledford, B., Liang, J., Maltais, F., Murcko, M., Wang, T., Wannamaker, M. W., Bennett, H. B., Leeman, J. R., McNeil, C., Taylor, W. P., Memmott, C., Jiang, M., Rijnbrand, R., Bral, C., Germann, U., Nezami, A., Zhang, Y., Salituro, F. G., Bennani, Y. L., and Charifson, P. S. (2014) Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2. J. Med. Chem. 57 (15), 6668−6678. (33) Byrn, R. A., Jones, S. M., Bennett, H. B., Bral, C., Clark, M. P., Jacobs, M. D., Kwong, A. D., Ledeboer, M. W., Leeman, J. R., McNeil, C. F., Murcko, M. A., Nezami, A., Perola, E., Rijnbrand, R., Saxena, K., Tsai, A. W., Zhou, Y., and Charifson, P. S. (2015) Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit. Antimicrob. Agents Chemother. 59 (3), 1569−1582. (34) Yuan, S., Chu, H., Zhang, K., Ye, J., Singh, K., Kao, R. Y., Chow, B. K., Zhou, J., and Zheng, B. J. (2016) A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication. J. Antimicrob. Chemother. 71 (9), 2489−2497. (35) Kowalinski, E., Zubieta, C., Wolkerstorfer, A., Szolar, O. H., Ruigrok, R. W., and Cusack, S. (2012) Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase. PLoS Pathog. 8 (8), e1002831. (36) Hastings, J. C., Selnick, H., Wolanski, B., and Tomassini, J. E. (1996) Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors. Antimicrob. Agents Chemother. 40 (5), 1304−1307. (37) Tomassini, J., Selnick, H., Davies, M. E., Armstrong, M. E., Baldwin, J., Bourgeois, M., Hastings, J., Hazuda, D., Lewis, J., McClements, W., et al. (1994) Inhibition of cap (m7GpppXm)dependent endonuclease of influenza virus by 4-substituted 2,4dioxobutanoic acid compounds. Antimicrob. Agents Chemother. 38 (12), 2827−2837. (38) Singh, S. B. (1995) Total synthesis of flutimide, a novel endonuclease inhibitor of influenza virus. Tetrahedron Lett. 36 (12), 2009−2012. (39) Sagong, H. Y., Parhi, A., Bauman, J. D., Patel, D., Vijayan, R. S., Das, K., Arnold, E., and LaVoie, E. J. (2013) 3-Hydroxyquinolin2(1H)-ones As Inhibitors of Influenza A Endonuclease. ACS Med. Chem. Lett. 4 (6), 547−550. (40) Sagong, H. Y., Bauman, J. D., Patel, D., Das, K., Arnold, E., and LaVoie, E. J. (2014) Phenyl substituted 4-hydroxypyridazin-3(2H)ones and 5-hydroxypyrimidin-4(3H)-ones: inhibitors of influenza A endonuclease. J. Med. Chem. 57 (19), 8086−8098. (41) Parkes, K. E., Ermert, P., Fassler, J., Ives, J., Martin, J. A., Merrett, J. H., Obrecht, D., Williams, G., and Klumpp, K. (2003) Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors. J. Med. Chem. 46 (7), 1153−1164. (42) Carcelli, M., Rogolino, D., Bacchi, A., Rispoli, G., Fisicaro, E., Compari, C., Sechi, M., Stevaert, A., and Naesens, L. (2014) Metalchelating 2-hydroxyphenyl amide pharmacophore for inhibition of influenza virus endonuclease. Mol. Pharmaceutics 11 (1), 304−316. (43) Yan, Z., Zhang, L., Fu, H., Wang, Z., and Lin, J. (2014) Design of the influenza virus inhibitors targeting the PA endonuclease using 3D-QSAR modeling, side-chain hopping, and docking. Bioorg. Med. Chem. Lett. 24 (2), 539−547. (44) Kim, J., Lee, C., and Chong, Y. (2009) Identification of potential influenza virus endonuclease inhibitors through virtual screening based on the 3D-QSAR model. SAR QSAR Environ. Res. 20 (1−2), 103−118. (45) Shoji, M., Takahashi, E., Hatakeyama, D., Iwai, Y., Morita, Y., Shirayama, R., Echigo, N., Kido, H., Nakamura, S., Mashino, T., Okutani, T., and Kuzuhara, T. (2013) Anti-influenza activity of c60 fullerene derivatives. PLoS One 8 (6), e66337. (46) Noble, E., Cox, A., Deval, J., and Kim, B. (2012) Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase. Virology 433 (1), 27−34.
(47) Shibagaki, Y., Ikuta, N., Iguchi, S., Takaki, K., Watanabe, S., Kaihotsu, M., Masuda, C., Maeyama, K., Mizumoto, K., and Hattori, S. (2014) An efficient screening system for influenza virus cap-dependent endonuclease inhibitors. J. Virol. Methods 202, 8−14. (48) Yuan, S., Zhang, N., Singh, K., Shuai, H., Chu, H., Zhou, J., Chow, B. K., and Zheng, B. J. (2015) Cross-protection of influenza A virus infection by a DNA aptamer targeting the PA endonuclease domain. Antimicrob. Agents Chemother. 59 (7), 4082−4093. (49) Jones, J. C., Marathe, B. M., Vogel, P., Gasser, R., Najera, I., and Govorkova, E. A. (2017) The PA Endonuclease Inhibitor RO-7 Protects Mice from Lethal Challenge with Influenza A or B Viruses. Antimicrob. Agents Chemother. 61 (5), e02460-16. (50) Wunderlich, K., Juozapaitis, M., Manz, B., Mayer, D., Gotz, V., Zohner, A., Wolff, T., Schwemmle, M., and Martin, A. (2010) Limited compatibility of polymerase subunit interactions in influenza A and B viruses. J. Biol. Chem. 285 (22), 16704−16712. (51) Ghanem, A., Mayer, D., Chase, G., Tegge, W., Frank, R., Kochs, G., Garcia-Sastre, A., and Schwemmle, M. (2007) Peptide-mediated interference with influenza A virus polymerase. J. Virol 81 (14), 7801− 7804. (52) Loregian, A., and Palu, G. (2005) Disruption of protein-protein interactions: towards new targets for chemotherapy. J. Cell. Physiol. 204 (3), 750−762. (53) Nobusawa, E., and Sato, K. (2006) Comparison of the mutation rates of human influenza A and B viruses. J. Virol 80 (7), 3675−3678. (54) Lin, J., Sahakian, D. C., de Morais, S. M., Xu, J. J., Polzer, R. J., and Winter, S. M. (2003) The role of absorption, distribution, metabolism, excretion and toxicity in drug discovery. Curr. Top. Med. Chem. 3 (10), 1125−1154. (55) Reguera, J., Gerlach, P., Rosenthal, M., Gaudon, S., Coscia, F., Gunther, S., and Cusack, S. (2016) Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases. PLoS Pathog. 12 (6), e1005636. (56) Liu, Q., He, B., Huang, S. Y., Wei, F., and Zhu, X. Q. (2014) Severe fever with thrombocytopenia syndrome, an emerging tickborne zoonosis. Lancet Infect. Dis. 14 (8), 763−772. (57) Stubbs, A. M., and Steele, M. T. (2014) Commentary. Ann. Emerg Med. 64 (3), 314−315.
F
DOI: 10.1021/acsinfecdis.7b00265 ACS Infect. Dis. XXXX, XXX, XXX−XXX
