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ScienceDirect Capsid proteins of enveloped viruses as antiviral drug targets Klaus Klumpp1 and Thibaut Cre´pin2 Viral proteins have enabled the design of selective and efficacious treatments for viral diseases. While focus in this area has been on viral enzymes, it appears that multifunctional viral proteins may be even more susceptible to small molecule interference. As exemplified by HIV capsid, small molecule inhibitors can bind to multiple binding sites on the capsid protein and induce or prevent protein interactions and conformational changes. Resistance selection is complicated by the fact that the capsid proteins have to engage in different protein interactions at different times of the life cycle. Viral capsid assembly and disassembly have therefore emerged as highly sensitive processes that could deliver a new generation of antiviral agents across viral diseases. Addresses 1 Novira Therapeutics, Inc., 3805 Old Easton Road, Doylestown, PA 18902, United States 2 University of Grenoble Alpes-EMBL-CNRS, Unit for Virus Host-Cell Interactions, 6 rue Jules Horowitz, 38042, France Corresponding author: Klumpp, Klaus ([email protected], [email protected]) and
Current Opinion in Virology 2014, 5:63–71 This review comes from a themed issue on Virus structure and function Edited by Wah Chiu and Thibaut Crepin
1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2014.02.002
Introduction Viral diseases are the source of significant morbidity and mortality worldwide [1]. More than 20 virus families contain known human pathogens. The development of prophylactic vaccines and efficacious antiviral treatments has been very successful against a few of these pathogens, but large medical needs remain un-addressed and major challenges remain even in those diseases for which treatments are available. For example, the current treatment options for HIV or Hepatitis B infections have had a major impact on survival of chronically infected patients. Optimal treatment can significantly delay the onset of severe disease, but ongoing low level virus replication, safety and tolerability issues and the difficulty to maintain adherent to life-long daily drug administration are main causes of treatment failure. Chronic inflammation, long term drug administration and complexities associated with polypharmacy and drug–drug interactions in many www.sciencedirect.com
patients present additional health issues that need to be addressed ([2–9]; http://aidsinfo.nih.gov/guidelines). It is also of concern that only very few options are available to manage severe acute infections that are caused by a number of different viruses. Treatments are needed that provide very fast onset of antiviral activity and highly efficient shut-down of viral replication in these cases [9– 11]. There is therefore an urgent need for additional classes of antiviral agents to address serious unmet medical need across a range of viral diseases. Viral core or capsid proteins are emerging as interesting targets for the development of new potent antiviral agents. The process of encapsidation of viral nucleic acid to enable the formation of virions and the infection of new cells is complicated by the fact that the capsid forming protein subunits have to interact with each other, and with other viral and host proteins to form the capsid at the right time during the viral life cycle and at the right location in the infected cell. The capsid has to be stable to protect the genome in extracellular environments, but not too stable as to prevent efficient genome release after entry into new host cells. The typical icosahedral or conical capsid structures are formed from hexameric and pentameric building blocks that emerge from slightly different interaction of the same capsid protein subunits. The capsid proteins have therefore evolved to be conformationally flexible to allow different functional interactions with themselves (to form hexamers or pentamers) and with other proteins. Even small interferences with the ability of the capsid forming proteins to undergo required conformational changes or changes in the stability of protein interactions can disrupt critical steps in the process between genome encapsidation and release. In addition, many capsid forming proteins are performing additional regulatory functions in infected cells, such as modulation of host gene expression and interference with immune recognition. Not surprisingly therefore, the sequences of capsid proteins show high sequence conservation levels and many single point mutations are associated with replication deficiency. Recent studies have described the identification of structurally diverse small molecule inhibitors of viral replication that target these sensitive processes of genome encapsidation and release. This short review will focus on examples of inhibitors of HIV replication, which exemplify the principle that targeting capsid forming proteins with small molecules is feasible and can result in different biological phenotypes, depending which step in the encapsidation–release–host factor interaction network is primarily inhibited. The structural flexibility of capsid proteins allows the binding of small molecules to Current Opinion in Virology 2014, 5:63–71
64 Virus structure and function
different binding sites and substantial differences in biological phenotype can be observed even for compounds that bind to the same binding site on the protein.
HIV capsid protein is structurally flexible The HIV capsid (CA) protein is structurally flexible and creates multiple different protein–protein interaction surfaces with itself, other viral and host proteins at different times during the viral life cycle [12,13]. CA is synthesized as part of the Gag polyprotein and consists of two independently folded domains, the N-terminal domain (CA-NTD) and the C-terminal domain (CACTD). CA is the main driver of Gag oligomerization in the formation of the immature capsid, and CA forms the mature, cone-shaped capsid after it is released from Gag by proteolytic processing in the virion [12,13,14]. Drug discovery for this target is now greatly facilitated by structural information that has been generated using native HIV cores and a variety of different protein constructs and methods, including cryo-EM, NMR and crystallography [15,16,17–21]. The structural information and molecular capsid models suggest very different protein–protein interactions of CA in the immature spherical capsid, as compared to the mature cone-shaped capsid [12,13]. To ability of CA to undergo such dramatically different protein interactions forming two different types of hexameric building blocks is facilitated by overall weak interactions between CA dimer (Kd 10–20 mM) and CA hexamers, and by the modulation of CA interactions through other Gag protein domains, especially SP1, which change after cleavage by the HIV protease. Mutational analyses are consistent with the structural models. Importantly, most single point mutations result in replication deficiency, consistent with the critical role that most amino acids play to maintain the ability of the CA protein to adapt a number of different conformations and protein–protein interactions throughout the viral life cycle [22,23,24,25].
HIV replication inhibitors targeting capsid
hydrophobic pocket at the junction of 5 a-helices at the base of the CA-NTD domain [26,27] (Table 1, Figure 1, binding site highlighted in purple). This discovery provided the first indication that small molecule inhibition of HIV capsid function was possible. It encouraged further drug discovery efforts, even though the two pioneer compounds did not bind to the site that had been targeted by the in silico docking method, and despite the fact that one was too toxic to use in antiviral assays, while the other was so weakly binding to CA-NTD, that density for the compound was not visible in the crystals. From a number of efforts in different groups, the most potent compounds to date that bind to the CAP-1 binding site came from research performed at Boehringer Ingelheim. Using a fluorescence based assembly assay with CA-NC fusion protein, a number of different series of HIV replication inhibitors were identified. Two series, named the BD (benzodiazepine) and the BM (benzimidazole) series, were further investigated and delivered compounds with mean antiviral potencies of 70 and 62 nM, respectively [28,29,30] (Table 1). The binding of compounds from these series to the CAP-1 binding site on the HIV CANTD was determined by NMR and crystallography, and binding affinity determined by NMR and ITC. The binding of compound BD3 is shown in Figure 1 (purple color) and indicates a different binding mode as compared to the CAP-1 compound (compare top left zoom view, CAP-1, to the top right, BD3). Compounds from the BD series inhibited virion release from HIV producing cells, consistent with the inhibition of immature capsid assembly. In contrast, the BM series allowed virus budding, but prevented capsid maturation. Interestingly, the biological phenotype of the two series was strikingly different, despite of the fact that both were binding to the same binding site [30]. Virus passaging resulted in the selection of different resistance mutations for both series. The selected mutations were consistent with important interactions of the compounds in the binding site. Most mutations significantly reduced HIV replication capacity [30].
CAP-1, BD, BM
A number of independent small molecule screens have been performed, most of them looking for compounds that could interfere with capsid assembly in vitro. The key compounds identified in this way are summarized in Table 1, and many of them have also been included in another recent review on this topic [24]. Figure 1 shows three major and well separated binding sites of small molecule inhibitors targeting the HIV CA-NTD. The first small molecule binding site on HIV CA was identified from a NMR screen. The screen identified two compounds that bound to CA-NTD and could inhibit salt induced CA aggregation in vitro. One compound was toxic, while the other compound, named CAP-1, inhibited HIV replication in cell culture at a concentration of 100 mM. 1H–15N HSQC NMR and crystallography data were consistent with binding of CAP-1 to an induced Current Opinion in Virology 2014, 5:63–71
PF-74
A different binding site was identified by researchers at Pfizer based on a hit from a phenotypic cell based antiviral assay screen. In this case, the antiviral target was first identified by virus passaging and resistance selection in cell culture, which resulted in the selection of a resistant virus variant with a T107N mutation in the CA-NTD coding sequence. The prototype compound from this series is PF-74 (PF-3450074) with a mean antiviral EC50 of 570 nM [31] (Table 1). Crystallography confirmed the binding of PF-74 to a new, pre-existing site on the CA-NTD domain. This binding site is shown in Figure 1 with red highlight. Binding of PF-74 to this site did not induce any apparent conformational change on the NTD domain. Interestingly, PF-74 accelerated CA assembly in vitro, in contrast to the compounds that bind www.sciencedirect.com
Capsid proteins as antiviral drug targets Klumpp and Cre´pin 65
Table 1 Structures and biological activities of selected antiviral compounds that interfere with viral capsid assembly Compound CAP-1
BD 1
Structure
Activity
Reference
CA-NTD Kd 800 mM EC95 100 mM CC50 > 100 mM
[26]
EC50 = 70 30 nM (n = 21) CC50 > 28 mM
[30]
EC50 = 480 nM
[30]
EC50 = 62 23 nM (n = 53) CC50 = 20 mM
[30]
EC50 = 570 260 nM CC50 = 69 15 mM
[31]
Kd = 500 nM (ITC) IC50 = 350 nM EC50 = 950 nM CC50 = 57 mM
[41]
BD 3
BM 1
PF-74
#6
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66 Virus structure and function
Table 1 (Continued ) Compound
Structure
Activity
Reference
#4 [41] #1 [42]
IC50 = 1200 nM
[41] [42]
BI-1 pyrrolo-pyrazolone
Kd = 20 mM (ITC, NMR) EC50 = 7.5 2.1 mM CC50 > 91 mM
[43]
BI-2
Kd = 3 mM (ITC, NMR) EC50 = 1.4 0.66 mM CC50 > 76 mM
[43]
ST-148
EC50 (DENV-1) = 2.8 1.1 mM EC50 (DENV-2) = 0.016 0.01 mM EC50 (DENV-3) = 0.51 0.42 mM EC50 (DENV-4) = 1.2 0.14 mM CC50 > 50 mM
[44]
EC50 = 0.05 mM CC50 = 7 mM
[50]
EC50 = 0.13 mM CC50 > 61 mM
[51]
EC50 = 0.36 0.05 mM
[52]
Bay 41-4109
AT-130
HAP-1
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Capsid proteins as antiviral drug targets Klumpp and Cre´pin 67
Figure 1
70º
90º
90º
Nt
Ct
Current Opinion in Virology
Three structurally defined inhibitor binding sites on the N-terminal domain of the HIV CA protein. The center panel shows two protomers of CA, one in cartoon and one in surface representation, with the N-terminus (Nt) and C-terminus (Ct) indicated. Three small molecule binding sites are indicated by purple (2JPR, 4E91), red (2XDE) and yellow (4E91) color highlights. The zoom-in views show the binding of representative compounds CAP-1 (purple, top left [27]), BD3 (purple, top middle [30]), PF-74 (red, bottom left [31]) and #4 (yellow, bottom right [41]).
to the CAP-1 binding site, which inhibited CA assembly in vitro. Another difference was the fact that PF-74 inhibited both early and late events in the HIV life cycle with similar potency, while the inhibition of late events (virion release or maturation) was driving the antiviral potency of the compounds that bound to the CAP-1 binding site. Early inhibition of HIV replication by PF74 occurred before reverse transcription [31]. Further studies showed that PF-74 could bind to and destabilize mature capsids, while capsids with the PF-74 resistance mutation were not affected by incubation with the compound [32]. CA mutations which stabilized the capsid could confer resistance to PF-74, while capsids with destabilizing mutations were more sensitive to PF-74 [32]. The binding site of PF-74 was separate from that of CAP-1 and also different from the site where the host factor cyclophilin A binds to CA. Interestingly, cyclosporine A, which inhibits cyclophilin A binding to CA, was strongly antagonistic with PF-74 in the antiviral assay. In addition, CA mutations that prevent cyclophilin A binding were also resistant to PF-74. Depletion of cyclophilin A by siRNA treatment reduced HIV sensitivity to PF-74 [32]. The simultaneous presence of cyclophilin A and PFwww.sciencedirect.com
74 therefore increased the antiviral activity of PF-74, consistent with the notion that cyclophilin A and PF74 can bind to CA simultaneously. Depletion of another host factor, transportin SR2/ TNPO3, also decreased the sensitivity of HIV to inhibition by PF-74. In this case, the cooperative effect was likely due to the fact that both TNPO3 and PF-74 are capsid destabilizing factors and the simultaneous presence of TNPO3 and PF-74 can accelerate premature capsid disassembly [33]. The concept that premature capsid disassembly is deleterious for HIV is also consistent with recent data that suggest that the HIV capsid remains intact in the cytoplasm and that uncoating may happen at the nuclear pore [34,35]. Intact capsid has been observed at the nuclear pore, capsid interaction with the nuclear pore protein NUP358 is required for the perinuclear localization of capsid, and NUP358 has been identified as a required co-factor for HIV replication in siRNA screens [34,35,36,37]. These results suggest that HIV, like a number of other viruses, avoids exposure of the viral RNA to RNA sensing antiviral host factors in the cytoplasm of infected cells, or that maintenance of the Current Opinion in Virology 2014, 5:63–71
68 Virus structure and function
pre-integration complex in the context of the capsid may increase reverse transcription efficiency.
mediated by the compound, facilitating nucleation of crystallization.
CPSF6-358, a truncated cytosolic form of the protein CPSF6 (cleavage and polyadenylation specific factor 6) was also recently found to bind to the CA-NTD domain and restrict HIV replication. Interestingly, the binding site of CPSF6-358 on CA overlapped with that of PF-74, and both molecules showed competitive binding to CA [38]. Interestingly, different forms of cytosolic CPSF6 variants could either stabilize or destabilize capsid, in both cases leading to replication inhibition [39,40].
Pyrrolo-pyrazolones BI-1/BI-2
Apex binding benzimidazoles
The Boehringer Ingelheim group also identified another series of benzimidazoles from their in vitro capsid assembly screen that, according to 1H–15N NMR, affected different sets of residues as compared to the BM series described above. In agreement with the NMR data, a cocrystal structure showed binding of a representative compound from this series to a pocket at the base of the cyclophilin A binding loop on HIV CA. This binding site is located at the apex of the CA-NTD helical bundle and is well separated from the other two binding sites described above [41,42] (Table 1, #1, #4, #6). The apex binding site with compound #4 [41] is highlighted in Figure 1 with yellow color. The compounds from this original hit series showed preliminary SAR with Kd values determined with CA-NTD by ITC between 0.5 and 43 mM, which correlated well with Kd values determined from chemical shift changes by NMR. The compounds inhibited capsid assembly in the oligonucleotide activated CA-NC assembly assay with IC50 values between 0.35 and 6.1 mM and the best compound had an antiviral EC50 value of 0.95 mM. Although the binding site of this series was close to the cyclophilin A binding site, both could bind simultaneously to CA-NTD as determined by NMR. In addition, compounds from the previously described BD series could bind to the CAP-1 binding site in the presence of compounds from this series, consistent with the large separation of the two binding sites (purple and yellow in Figure 1) [41]. There was no significant conformational change induced by binding according to crystallography. Assembly inhibition could result from an interference of CA-NTD-mediated interhexamer contacts that are required in the formation of the mature capsid [16,41]. Interestingly, the presence of a compound from this series was found to improve crystallization of CA-NTD and enabled the generation of ternary co-crystal structures with other compounds binding to the CAP-1 binding site that had failed to generate cocrystal structures with CA-NTD on their own [42]. These results provided further proof that the two compound series could bind simultaneously to CA-NTD. The improved crystallization performance was hypothesized to be related to the facilitation of protein dimerization Current Opinion in Virology 2014, 5:63–71
Another interesting series of compounds targeting HIVCA were identified from a cell based, single cycle HIV infection assay. The representative pyrrolo-pyrazolones BI-1 and BI-2 inhibited HIV replication in single cycle (EC50 = 8.2 (BI-1), 1.8 (BI-2) mM) and multi-cycle (EC50 = 7.5 (BI-1), 1.4 (BI-2) mM) assays [43] (Table 1). But these compounds did not inhibit infectious virus formation in HIV producer cells (EC50 > 43 mM). The target and binding site was determined by resistance selection, ITC, NMR and crystallography as overlapping with the PF-74 binding site on the CA-NTD domain (PF74 binding site highlighted in red in Figure 1). Similar to PF-74, there was no apparent conformational change associated with compound binding observed in crystallography and the compounds were accelerators of CA-NC assembly in vitro. There were, however, major phenotypic differences between this series and PF-74. (a) PF74 inhibited reverse transcription and had a de-stabilizing effect on capsid, while this series did not inhibit reverse transcription and had a stabilizing effect on capsid, while the concentration of 2-LTR circles was reduced, consistent with an inhibition of nuclear import; (b) PF-74 inhibited early and late phases of HIV replication, while this series only inhibited the early phase. HIV capsid inhibitor summary
The characterization of antiviral compounds that interfere with HIV capsid function indicates a striking multitude of binding sites and phenotypic profiles. Three clearly defined small molecule binding sites have been identified on the N-terminal domain of CA-NTD alone. In addition, it has been interesting to learn that the binding of compounds to the same binding site on CANTD could have very different biological consequences in the HIV life cycle. These results are all consistent with the multifunctional role of the capsid protein that involves a number of different protein conformations and interactions with different protein surfaces and host factors. It will be highly interesting to learn more about the potential to increase the antiviral potency by lead optimization and by the combination of compounds binding to different binding sites on CA. In addition, the barrier to resistance remains to be better understood, which may also be affected by combination of compounds binding to different binding sites. Finally, the principles observed with HIV capsid targeting are likely to translate into opportunities for targeting capsid proteins of other enveloped viruses.
Capsid inhibitors demonstrate antiviral activity in vivo There is already ample evidence to suggest the translatability of capsid inhibition principles learned in the HIV www.sciencedirect.com
Capsid proteins as antiviral drug targets Klumpp and Cre´pin 69
field to other viral disease indications, and antiviral activity has been demonstrated in animal models in a number of cases. For example, a group at SIGA Technologies recently published the discovery of a small molecule, ST-148, which targets Dengue virus capsid protein, showed activity across all four Dengue serotypes and reduced viremia in a non-lethal AG129 mouse model [44]. BAY-41-4109, a compound that interferes with HBV capsid assembly has also shown evidence of in vivo antiviral activity in the HBV transgenic mouse model and in Alb-uPA/SCID mice with humanized chimeric liver [45,46]. Unfortunately, treatment in the humanized mouse model was only for 5 days and started already 10 days after infection. These conditions did not provide information on possible antiviral activity of the capsid assembly inhibitor in a chronic HBV infection model. Similar to the HIV case, compounds with potent antiviral activity have been identified that accelerate rather than inhibit HBV capsid assembly in vitro. A recently published crystal structure of the HBV capsid obtained in the presence of the HBV capsid assembly effector AT-130 from the phenylpropenamide class indicates that AT-130 binds to an overlapping binding site with HAP-1, a compound from a different structural class of heteroaryldihydropyrimidines [47]. HAP compounds were shown to stabilize capsid protein dimer interactions and to induce misassembly by changing the geometry of these dimer interactions. In contrast, the primary phenotype of phenylpropenamides is the block of viral RNA packaging and the formation of empty capsids without affecting capsid stability or geometry [48,49]. Similar to the HIV case, therefore, compound binding to an overlapping binding site on the viral capsid protein can result in substantially different biological effects.
Conclusion Viral capsid proteins of enveloped viruses have emerged as promising targets for the design of a new generation of antiviral agents. It has become clear that the processes of viral genome encapsidation and release that are dependent on controlled capsid assembly and disassembly are highly sensitive to even subtle molecular disturbances that increase or decrease capsid stability, increase or decrease the rate of capsid assembly, change the geometry or conformation of capsid building blocks or interfere with host factor engagement. A number of different hit series of small molecule inhibitors of viral replication targeting these processes have already been identified from biochemical and cell based assay screens. Although most of these compounds are early stage tool compounds or hits, they are important as they have demonstrated the potential for multiple compound binding sites on capsid proteins and the potential for multiple mechanistic phenotypes of inhibition. The biological requirement for viral capsid proteins to be structurally flexible for the www.sciencedirect.com
engagement into very different molecular interactions at different phases of the virus life cycle represents a viral weakness and offers clear opportunities for pharmacologic interference.
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26. Tang C, Loeliger E, Kinde I, Kyere S, Mayo K, Barklis E, Sun Y, Huang M, Summers MF: Antiviral inhibition of the HIV-1 capsid protein. J Mol Biol 2003, 327:1013-1020. 27. Kelly BN, Kyere S, Kinde I, Tang C, Howard BR, Robinson H, Sundquist WI, Summers MF, Hill CP: Structure of the antiviral assembly inhibitor CAP-1 bound to the HIV-1 capsid protein. J Mol Biol 2007, 373:355-366. 28. Fader LD, Bethell R, Bonneau P, Bos M, Bousquet Y, Cordingley MG, Coulombe R, DeRoy P, Faucher AM, Gagnon A, Goudreau N et al.: Discovery of a 1,5dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly. Bioorg Med Chem Lett 2011, 21:398404. 29. Tremblay M, Bonneau P, Bousquet Y, DeRoy P, Duan J, Duplessis M, Gagnon A, Garneau M, Goudreau N, Guse I, Hucke O et al.: Inhibition of HIV-1 capsid assembly: optimization of the antiviral potency by site-selective modifications at N1, C2 and C16 of a 5-(5-furan-2-yl-pyrazol-1-yl)-1H-benzimidazole scaffold. Bioorg Med Chem Lett 2012, 22:7512-7517. Current Opinion in Virology 2014, 5:63–71
41. Goudreau N, Lemke CT, Faucher AM, Grand-Maitre C, Goulet S, Lacoste JE, Rancourt J, Malenfant E, Mercier JF, Titolo S, Mason SW: Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography. ACS Chem Biol 2013, 8:1074-1082. 42. Lemke CT, Titolo S, Goudreau N, Faucher AM, Mason SW, Bonneau P: A novel inhibitor-binding site on the HIV-1 capsid N-terminal domain leads to improved crystallization via compound-mediated dimerization. Acta Crystallogr 2013, D69:1115-1123. 43. Lamorte L, Titolo S, Lemke CT, Goudreau N, Mercier JF, Wardrop E, Shah VB, von Schwedler UK, Langelier C, Banik SSR, Aiken C, Sundquist WI, Mason SW: Discovery of novel smallmolecule HIV-1 replication inhibitors that stabilize capsid complexes. Antimicrob Agents Chemother 2013, 57:4622-4631. Demonstrate that binding of compounds to the same binding site on capsid protein can have very different biological phenotypes. 44. Byrd CM, Dai D, Grosenbach DW, Berhanu A, Jones KF, Cardwell KB, Schneider C, Wineinger KA, Page JM, Harver C, Stavale E et al.: A novel inhibitor of dengue virus replication that targets the capsid protein. Antimicrob Agents Chemother 2013, 57:15-25. www.sciencedirect.com
Capsid proteins as antiviral drug targets Klumpp and Cre´pin 71
Demonstration of in vivo antiviral activity of a compound targeting the capsid protein of an enveloped virus. 45. Weber O, Schlemmer KH, Hartmann E, Hagelschuer I, Paessens A, Graef E, Deres K, Goldmann S, Niewohner U, Stoltefuss J, Haebich D: Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antivir Res 2002, 54:69-78. 46. Brezillon N, Brunelle M, Massinet H, Giang E, Lamant C, Da Silva L, Berissi S, Belghiti J, Hannoun L, Puerstinger G, Wimmer E et al.: Antiviral activity of Bay 41-4109 on hepatitis B virus in human Alb-uPA/SCID mice. PLoS ONE 2011, 6:e25096. 47. Katen SP, Tan Z, Chirapu SR, Finn MG, Zlotnick A: Assembly directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure. Structure 2013, 21:1406-1416. First indication that the binding sites for 2 important prototype series of HBV capsid targeting compounds are overlapping. Demonstrate that binding to an overlapping binding site can be associated with different biological phenotypes.
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48. Bourne CR, Finn MG, Zlotnick A: Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1. J Virol 2006, 80:11055-11061. 49. Katen SP, Chirapu SR, Finn MG, Zlotnick A: Trapping of hepatitis B virus capsid intermediates by phenylpropenamide assembly accelerators. ACS Chem Biol 2010, 5:1125-1136. 50. Deres K, Schroder CH, Paessens A, Goldmann S, Hacker HJ, Weber O, Kramer T, Niewohner U, Pleiss U, Stoltefuss J, Graef E et al.: Inhibition of hepatitis B virus replication by drug-induced depletion of nucleocapsids. Science 2002, 299:893-896. 51. Perni RB, Conway SC, Ladner SK, Zaifert K, Otto MJ, King RW: Phenylpropenamide derivatives as inhibitor of hepatitis B virus replication. Bioorg Med Chem Lett 2000, 10:2687-2690. 52. Bourne C, Lee S, Venkataiah B, Lee A, Korba B, Finn MG, Zlotnick A: Small-molecule effectors of hepatitis B virus capsid assembly give insight into the virus life cycle. J Virol 2008, 82:10262-10270.
Current Opinion in Virology 2014, 5:63–71
ScienceDirect Capsid proteins of enveloped viruses as antiviral drug targets Klaus Klumpp1 and Thibaut Cre´pin2 Viral proteins have enabled the design of selective and efficacious treatments for viral diseases. While focus in this area has been on viral enzymes, it appears that multifunctional viral proteins may be even more susceptible to small molecule interference. As exemplified by HIV capsid, small molecule inhibitors can bind to multiple binding sites on the capsid protein and induce or prevent protein interactions and conformational changes. Resistance selection is complicated by the fact that the capsid proteins have to engage in different protein interactions at different times of the life cycle. Viral capsid assembly and disassembly have therefore emerged as highly sensitive processes that could deliver a new generation of antiviral agents across viral diseases. Addresses 1 Novira Therapeutics, Inc., 3805 Old Easton Road, Doylestown, PA 18902, United States 2 University of Grenoble Alpes-EMBL-CNRS, Unit for Virus Host-Cell Interactions, 6 rue Jules Horowitz, 38042, France Corresponding author: Klumpp, Klaus ([email protected], [email protected]) and
Current Opinion in Virology 2014, 5:63–71 This review comes from a themed issue on Virus structure and function Edited by Wah Chiu and Thibaut Crepin
1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2014.02.002
Introduction Viral diseases are the source of significant morbidity and mortality worldwide [1]. More than 20 virus families contain known human pathogens. The development of prophylactic vaccines and efficacious antiviral treatments has been very successful against a few of these pathogens, but large medical needs remain un-addressed and major challenges remain even in those diseases for which treatments are available. For example, the current treatment options for HIV or Hepatitis B infections have had a major impact on survival of chronically infected patients. Optimal treatment can significantly delay the onset of severe disease, but ongoing low level virus replication, safety and tolerability issues and the difficulty to maintain adherent to life-long daily drug administration are main causes of treatment failure. Chronic inflammation, long term drug administration and complexities associated with polypharmacy and drug–drug interactions in many www.sciencedirect.com
patients present additional health issues that need to be addressed ([2–9]; http://aidsinfo.nih.gov/guidelines). It is also of concern that only very few options are available to manage severe acute infections that are caused by a number of different viruses. Treatments are needed that provide very fast onset of antiviral activity and highly efficient shut-down of viral replication in these cases [9– 11]. There is therefore an urgent need for additional classes of antiviral agents to address serious unmet medical need across a range of viral diseases. Viral core or capsid proteins are emerging as interesting targets for the development of new potent antiviral agents. The process of encapsidation of viral nucleic acid to enable the formation of virions and the infection of new cells is complicated by the fact that the capsid forming protein subunits have to interact with each other, and with other viral and host proteins to form the capsid at the right time during the viral life cycle and at the right location in the infected cell. The capsid has to be stable to protect the genome in extracellular environments, but not too stable as to prevent efficient genome release after entry into new host cells. The typical icosahedral or conical capsid structures are formed from hexameric and pentameric building blocks that emerge from slightly different interaction of the same capsid protein subunits. The capsid proteins have therefore evolved to be conformationally flexible to allow different functional interactions with themselves (to form hexamers or pentamers) and with other proteins. Even small interferences with the ability of the capsid forming proteins to undergo required conformational changes or changes in the stability of protein interactions can disrupt critical steps in the process between genome encapsidation and release. In addition, many capsid forming proteins are performing additional regulatory functions in infected cells, such as modulation of host gene expression and interference with immune recognition. Not surprisingly therefore, the sequences of capsid proteins show high sequence conservation levels and many single point mutations are associated with replication deficiency. Recent studies have described the identification of structurally diverse small molecule inhibitors of viral replication that target these sensitive processes of genome encapsidation and release. This short review will focus on examples of inhibitors of HIV replication, which exemplify the principle that targeting capsid forming proteins with small molecules is feasible and can result in different biological phenotypes, depending which step in the encapsidation–release–host factor interaction network is primarily inhibited. The structural flexibility of capsid proteins allows the binding of small molecules to Current Opinion in Virology 2014, 5:63–71
64 Virus structure and function
different binding sites and substantial differences in biological phenotype can be observed even for compounds that bind to the same binding site on the protein.
HIV capsid protein is structurally flexible The HIV capsid (CA) protein is structurally flexible and creates multiple different protein–protein interaction surfaces with itself, other viral and host proteins at different times during the viral life cycle [12,13]. CA is synthesized as part of the Gag polyprotein and consists of two independently folded domains, the N-terminal domain (CA-NTD) and the C-terminal domain (CACTD). CA is the main driver of Gag oligomerization in the formation of the immature capsid, and CA forms the mature, cone-shaped capsid after it is released from Gag by proteolytic processing in the virion [12,13,14]. Drug discovery for this target is now greatly facilitated by structural information that has been generated using native HIV cores and a variety of different protein constructs and methods, including cryo-EM, NMR and crystallography [15,16,17–21]. The structural information and molecular capsid models suggest very different protein–protein interactions of CA in the immature spherical capsid, as compared to the mature cone-shaped capsid [12,13]. To ability of CA to undergo such dramatically different protein interactions forming two different types of hexameric building blocks is facilitated by overall weak interactions between CA dimer (Kd 10–20 mM) and CA hexamers, and by the modulation of CA interactions through other Gag protein domains, especially SP1, which change after cleavage by the HIV protease. Mutational analyses are consistent with the structural models. Importantly, most single point mutations result in replication deficiency, consistent with the critical role that most amino acids play to maintain the ability of the CA protein to adapt a number of different conformations and protein–protein interactions throughout the viral life cycle [22,23,24,25].
HIV replication inhibitors targeting capsid
hydrophobic pocket at the junction of 5 a-helices at the base of the CA-NTD domain [26,27] (Table 1, Figure 1, binding site highlighted in purple). This discovery provided the first indication that small molecule inhibition of HIV capsid function was possible. It encouraged further drug discovery efforts, even though the two pioneer compounds did not bind to the site that had been targeted by the in silico docking method, and despite the fact that one was too toxic to use in antiviral assays, while the other was so weakly binding to CA-NTD, that density for the compound was not visible in the crystals. From a number of efforts in different groups, the most potent compounds to date that bind to the CAP-1 binding site came from research performed at Boehringer Ingelheim. Using a fluorescence based assembly assay with CA-NC fusion protein, a number of different series of HIV replication inhibitors were identified. Two series, named the BD (benzodiazepine) and the BM (benzimidazole) series, were further investigated and delivered compounds with mean antiviral potencies of 70 and 62 nM, respectively [28,29,30] (Table 1). The binding of compounds from these series to the CAP-1 binding site on the HIV CANTD was determined by NMR and crystallography, and binding affinity determined by NMR and ITC. The binding of compound BD3 is shown in Figure 1 (purple color) and indicates a different binding mode as compared to the CAP-1 compound (compare top left zoom view, CAP-1, to the top right, BD3). Compounds from the BD series inhibited virion release from HIV producing cells, consistent with the inhibition of immature capsid assembly. In contrast, the BM series allowed virus budding, but prevented capsid maturation. Interestingly, the biological phenotype of the two series was strikingly different, despite of the fact that both were binding to the same binding site [30]. Virus passaging resulted in the selection of different resistance mutations for both series. The selected mutations were consistent with important interactions of the compounds in the binding site. Most mutations significantly reduced HIV replication capacity [30].
CAP-1, BD, BM
A number of independent small molecule screens have been performed, most of them looking for compounds that could interfere with capsid assembly in vitro. The key compounds identified in this way are summarized in Table 1, and many of them have also been included in another recent review on this topic [24]. Figure 1 shows three major and well separated binding sites of small molecule inhibitors targeting the HIV CA-NTD. The first small molecule binding site on HIV CA was identified from a NMR screen. The screen identified two compounds that bound to CA-NTD and could inhibit salt induced CA aggregation in vitro. One compound was toxic, while the other compound, named CAP-1, inhibited HIV replication in cell culture at a concentration of 100 mM. 1H–15N HSQC NMR and crystallography data were consistent with binding of CAP-1 to an induced Current Opinion in Virology 2014, 5:63–71
PF-74
A different binding site was identified by researchers at Pfizer based on a hit from a phenotypic cell based antiviral assay screen. In this case, the antiviral target was first identified by virus passaging and resistance selection in cell culture, which resulted in the selection of a resistant virus variant with a T107N mutation in the CA-NTD coding sequence. The prototype compound from this series is PF-74 (PF-3450074) with a mean antiviral EC50 of 570 nM [31] (Table 1). Crystallography confirmed the binding of PF-74 to a new, pre-existing site on the CA-NTD domain. This binding site is shown in Figure 1 with red highlight. Binding of PF-74 to this site did not induce any apparent conformational change on the NTD domain. Interestingly, PF-74 accelerated CA assembly in vitro, in contrast to the compounds that bind www.sciencedirect.com
Capsid proteins as antiviral drug targets Klumpp and Cre´pin 65
Table 1 Structures and biological activities of selected antiviral compounds that interfere with viral capsid assembly Compound CAP-1
BD 1
Structure
Activity
Reference
CA-NTD Kd 800 mM EC95 100 mM CC50 > 100 mM
[26]
EC50 = 70 30 nM (n = 21) CC50 > 28 mM
[30]
EC50 = 480 nM
[30]
EC50 = 62 23 nM (n = 53) CC50 = 20 mM
[30]
EC50 = 570 260 nM CC50 = 69 15 mM
[31]
Kd = 500 nM (ITC) IC50 = 350 nM EC50 = 950 nM CC50 = 57 mM
[41]
BD 3
BM 1
PF-74
#6
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66 Virus structure and function
Table 1 (Continued ) Compound
Structure
Activity
Reference
#4 [41] #1 [42]
IC50 = 1200 nM
[41] [42]
BI-1 pyrrolo-pyrazolone
Kd = 20 mM (ITC, NMR) EC50 = 7.5 2.1 mM CC50 > 91 mM
[43]
BI-2
Kd = 3 mM (ITC, NMR) EC50 = 1.4 0.66 mM CC50 > 76 mM
[43]
ST-148
EC50 (DENV-1) = 2.8 1.1 mM EC50 (DENV-2) = 0.016 0.01 mM EC50 (DENV-3) = 0.51 0.42 mM EC50 (DENV-4) = 1.2 0.14 mM CC50 > 50 mM
[44]
EC50 = 0.05 mM CC50 = 7 mM
[50]
EC50 = 0.13 mM CC50 > 61 mM
[51]
EC50 = 0.36 0.05 mM
[52]
Bay 41-4109
AT-130
HAP-1
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Capsid proteins as antiviral drug targets Klumpp and Cre´pin 67
Figure 1
70º
90º
90º
Nt
Ct
Current Opinion in Virology
Three structurally defined inhibitor binding sites on the N-terminal domain of the HIV CA protein. The center panel shows two protomers of CA, one in cartoon and one in surface representation, with the N-terminus (Nt) and C-terminus (Ct) indicated. Three small molecule binding sites are indicated by purple (2JPR, 4E91), red (2XDE) and yellow (4E91) color highlights. The zoom-in views show the binding of representative compounds CAP-1 (purple, top left [27]), BD3 (purple, top middle [30]), PF-74 (red, bottom left [31]) and #4 (yellow, bottom right [41]).
to the CAP-1 binding site, which inhibited CA assembly in vitro. Another difference was the fact that PF-74 inhibited both early and late events in the HIV life cycle with similar potency, while the inhibition of late events (virion release or maturation) was driving the antiviral potency of the compounds that bound to the CAP-1 binding site. Early inhibition of HIV replication by PF74 occurred before reverse transcription [31]. Further studies showed that PF-74 could bind to and destabilize mature capsids, while capsids with the PF-74 resistance mutation were not affected by incubation with the compound [32]. CA mutations which stabilized the capsid could confer resistance to PF-74, while capsids with destabilizing mutations were more sensitive to PF-74 [32]. The binding site of PF-74 was separate from that of CAP-1 and also different from the site where the host factor cyclophilin A binds to CA. Interestingly, cyclosporine A, which inhibits cyclophilin A binding to CA, was strongly antagonistic with PF-74 in the antiviral assay. In addition, CA mutations that prevent cyclophilin A binding were also resistant to PF-74. Depletion of cyclophilin A by siRNA treatment reduced HIV sensitivity to PF-74 [32]. The simultaneous presence of cyclophilin A and PFwww.sciencedirect.com
74 therefore increased the antiviral activity of PF-74, consistent with the notion that cyclophilin A and PF74 can bind to CA simultaneously. Depletion of another host factor, transportin SR2/ TNPO3, also decreased the sensitivity of HIV to inhibition by PF-74. In this case, the cooperative effect was likely due to the fact that both TNPO3 and PF-74 are capsid destabilizing factors and the simultaneous presence of TNPO3 and PF-74 can accelerate premature capsid disassembly [33]. The concept that premature capsid disassembly is deleterious for HIV is also consistent with recent data that suggest that the HIV capsid remains intact in the cytoplasm and that uncoating may happen at the nuclear pore [34,35]. Intact capsid has been observed at the nuclear pore, capsid interaction with the nuclear pore protein NUP358 is required for the perinuclear localization of capsid, and NUP358 has been identified as a required co-factor for HIV replication in siRNA screens [34,35,36,37]. These results suggest that HIV, like a number of other viruses, avoids exposure of the viral RNA to RNA sensing antiviral host factors in the cytoplasm of infected cells, or that maintenance of the Current Opinion in Virology 2014, 5:63–71
68 Virus structure and function
pre-integration complex in the context of the capsid may increase reverse transcription efficiency.
mediated by the compound, facilitating nucleation of crystallization.
CPSF6-358, a truncated cytosolic form of the protein CPSF6 (cleavage and polyadenylation specific factor 6) was also recently found to bind to the CA-NTD domain and restrict HIV replication. Interestingly, the binding site of CPSF6-358 on CA overlapped with that of PF-74, and both molecules showed competitive binding to CA [38]. Interestingly, different forms of cytosolic CPSF6 variants could either stabilize or destabilize capsid, in both cases leading to replication inhibition [39,40].
Pyrrolo-pyrazolones BI-1/BI-2
Apex binding benzimidazoles
The Boehringer Ingelheim group also identified another series of benzimidazoles from their in vitro capsid assembly screen that, according to 1H–15N NMR, affected different sets of residues as compared to the BM series described above. In agreement with the NMR data, a cocrystal structure showed binding of a representative compound from this series to a pocket at the base of the cyclophilin A binding loop on HIV CA. This binding site is located at the apex of the CA-NTD helical bundle and is well separated from the other two binding sites described above [41,42] (Table 1, #1, #4, #6). The apex binding site with compound #4 [41] is highlighted in Figure 1 with yellow color. The compounds from this original hit series showed preliminary SAR with Kd values determined with CA-NTD by ITC between 0.5 and 43 mM, which correlated well with Kd values determined from chemical shift changes by NMR. The compounds inhibited capsid assembly in the oligonucleotide activated CA-NC assembly assay with IC50 values between 0.35 and 6.1 mM and the best compound had an antiviral EC50 value of 0.95 mM. Although the binding site of this series was close to the cyclophilin A binding site, both could bind simultaneously to CA-NTD as determined by NMR. In addition, compounds from the previously described BD series could bind to the CAP-1 binding site in the presence of compounds from this series, consistent with the large separation of the two binding sites (purple and yellow in Figure 1) [41]. There was no significant conformational change induced by binding according to crystallography. Assembly inhibition could result from an interference of CA-NTD-mediated interhexamer contacts that are required in the formation of the mature capsid [16,41]. Interestingly, the presence of a compound from this series was found to improve crystallization of CA-NTD and enabled the generation of ternary co-crystal structures with other compounds binding to the CAP-1 binding site that had failed to generate cocrystal structures with CA-NTD on their own [42]. These results provided further proof that the two compound series could bind simultaneously to CA-NTD. The improved crystallization performance was hypothesized to be related to the facilitation of protein dimerization Current Opinion in Virology 2014, 5:63–71
Another interesting series of compounds targeting HIVCA were identified from a cell based, single cycle HIV infection assay. The representative pyrrolo-pyrazolones BI-1 and BI-2 inhibited HIV replication in single cycle (EC50 = 8.2 (BI-1), 1.8 (BI-2) mM) and multi-cycle (EC50 = 7.5 (BI-1), 1.4 (BI-2) mM) assays [43] (Table 1). But these compounds did not inhibit infectious virus formation in HIV producer cells (EC50 > 43 mM). The target and binding site was determined by resistance selection, ITC, NMR and crystallography as overlapping with the PF-74 binding site on the CA-NTD domain (PF74 binding site highlighted in red in Figure 1). Similar to PF-74, there was no apparent conformational change associated with compound binding observed in crystallography and the compounds were accelerators of CA-NC assembly in vitro. There were, however, major phenotypic differences between this series and PF-74. (a) PF74 inhibited reverse transcription and had a de-stabilizing effect on capsid, while this series did not inhibit reverse transcription and had a stabilizing effect on capsid, while the concentration of 2-LTR circles was reduced, consistent with an inhibition of nuclear import; (b) PF-74 inhibited early and late phases of HIV replication, while this series only inhibited the early phase. HIV capsid inhibitor summary
The characterization of antiviral compounds that interfere with HIV capsid function indicates a striking multitude of binding sites and phenotypic profiles. Three clearly defined small molecule binding sites have been identified on the N-terminal domain of CA-NTD alone. In addition, it has been interesting to learn that the binding of compounds to the same binding site on CANTD could have very different biological consequences in the HIV life cycle. These results are all consistent with the multifunctional role of the capsid protein that involves a number of different protein conformations and interactions with different protein surfaces and host factors. It will be highly interesting to learn more about the potential to increase the antiviral potency by lead optimization and by the combination of compounds binding to different binding sites on CA. In addition, the barrier to resistance remains to be better understood, which may also be affected by combination of compounds binding to different binding sites. Finally, the principles observed with HIV capsid targeting are likely to translate into opportunities for targeting capsid proteins of other enveloped viruses.
Capsid inhibitors demonstrate antiviral activity in vivo There is already ample evidence to suggest the translatability of capsid inhibition principles learned in the HIV www.sciencedirect.com
Capsid proteins as antiviral drug targets Klumpp and Cre´pin 69
field to other viral disease indications, and antiviral activity has been demonstrated in animal models in a number of cases. For example, a group at SIGA Technologies recently published the discovery of a small molecule, ST-148, which targets Dengue virus capsid protein, showed activity across all four Dengue serotypes and reduced viremia in a non-lethal AG129 mouse model [44]. BAY-41-4109, a compound that interferes with HBV capsid assembly has also shown evidence of in vivo antiviral activity in the HBV transgenic mouse model and in Alb-uPA/SCID mice with humanized chimeric liver [45,46]. Unfortunately, treatment in the humanized mouse model was only for 5 days and started already 10 days after infection. These conditions did not provide information on possible antiviral activity of the capsid assembly inhibitor in a chronic HBV infection model. Similar to the HIV case, compounds with potent antiviral activity have been identified that accelerate rather than inhibit HBV capsid assembly in vitro. A recently published crystal structure of the HBV capsid obtained in the presence of the HBV capsid assembly effector AT-130 from the phenylpropenamide class indicates that AT-130 binds to an overlapping binding site with HAP-1, a compound from a different structural class of heteroaryldihydropyrimidines [47]. HAP compounds were shown to stabilize capsid protein dimer interactions and to induce misassembly by changing the geometry of these dimer interactions. In contrast, the primary phenotype of phenylpropenamides is the block of viral RNA packaging and the formation of empty capsids without affecting capsid stability or geometry [48,49]. Similar to the HIV case, therefore, compound binding to an overlapping binding site on the viral capsid protein can result in substantially different biological effects.
Conclusion Viral capsid proteins of enveloped viruses have emerged as promising targets for the design of a new generation of antiviral agents. It has become clear that the processes of viral genome encapsidation and release that are dependent on controlled capsid assembly and disassembly are highly sensitive to even subtle molecular disturbances that increase or decrease capsid stability, increase or decrease the rate of capsid assembly, change the geometry or conformation of capsid building blocks or interfere with host factor engagement. A number of different hit series of small molecule inhibitors of viral replication targeting these processes have already been identified from biochemical and cell based assay screens. Although most of these compounds are early stage tool compounds or hits, they are important as they have demonstrated the potential for multiple compound binding sites on capsid proteins and the potential for multiple mechanistic phenotypes of inhibition. The biological requirement for viral capsid proteins to be structurally flexible for the www.sciencedirect.com
engagement into very different molecular interactions at different phases of the virus life cycle represents a viral weakness and offers clear opportunities for pharmacologic interference.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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