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Capsid: New Class of HIV Drugs
  PLoS Pathogens: HIV Capsid is a Tractable Target for Small ...
(2010) HIV Capsid is a Tractable Target for Small Molecule Therapeutic Intervention. PLoS Pathog 6(12): e1001220. doi:10.1371/journal.ppat.1001220;
"We report a new class of antiretrovirals that targets HIV-1 capsid and demonstrate that it is active at two critical stages in the viral replication cycle........PF-1385801 was identified as a hit in a high throughput screen for inhibitors of HIV replication.....Here we describe a novel series of antiviral compounds that target HIV-1 CA in infected cells and appear to interfere with both the viral uncoating process and the formation of infectious particles......PF-3450074 and PF-3759857 were active against all strains of HIV-1 tested with median EC50 values of 0.207 (range 0.113 to 0.362 ÁM) and 1.17 (range 0.51 to 3.17) ÁM, respectively (Fig. 2 and Tables S1 and S2). In addition, PF-3759857 was active against HIV-2 with an EC50 of 4.7 ÁM. This tight spectrum of activities against HIV-1 compared well with the marketed drugs, AZT, a nucleoside analog, and efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI)......Although additional work is needed to determine the individual contributions of the mutations identified in addition to T107N, it appears that more than one mutation is required to generate high levels of resistance to this inhibitor class.....The capsid protein (CA) of HIV-1 plays critical roles in both late and early stages of the viral replication cycle and is widely viewed as an important unexploited therapeutic target......At the earliest stages of particle assembly, the interactions between CA domains of the Gag polyprotein help drive the formation of immature particles at the membrane of host cells [7]. After the release of immature particles from infected cells, proteolytic processing of the Gag polyprotein is completed, leading to capsid assembly and formation of the mature virus. During assembly, the viral RNA genome is packaged into a capsid particle composed of a lattice of CA protein hexamers that form a distinct fullerene cone shaped particle [8]. After virus fusion with a target cell, the core is released into the cytoplasm and CA is thought to undergo a controlled disassembly reaction in order for reverse transcription of the viral genome to occur properly [9]."

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.
We describe a novel class of inhibitors that target HIV CA by a unique mechanism that interferes with both early and late events in the viral replication cycle. HIV CA plays an essential role in several stages of viral replication and is viewed as an important, yet unexploited target for therapeutic intervention [4], [5], [6]. This new series demonstrates that small molecules targeting HIV CA can have potent broad-spectrum antiviral activity. We demonstrate directly that HIV CA is the antiviral target of these inhibitors in infected cells by showing that mutations in HIV CA confer resistance to several members of the series. EM analysis shows that the series profoundly affects the morphology of nascent HIV particles. We demonstrate that the compounds affect CA protein multimerization in vitro and we have elucidated details of the novel compound binding site on HIV CA by solving a co-crystal structure of a compound from the series bound to the NTD of the protein. Our data strongly suggest that this new series of inhibitors targets HIV CA function during both the virion uncoating and viral core assembly processes.
Previous studies have described two molecules that target HIV-1 CA assembly in vitro, CAP-1 (a small molecule) and CAI (a dodecapeptide) [10], [11], [12]. CAP-1 acts in the late stage of viral replication and does not inhibit HIV-1 infection when added to pre-formed HIV-1 particles. A cell-permeable derivative of CAI, NYAD-1, inhibits formation of both immature and mature HIV-1 virus particles as well as early events in the replication cycle at low micromolar concentrations. The properties of the new class of CA inhibitors described in this study are clearly distinguished from those of other HIV-1 inhibitors, including previously described CA inhibitors. Unlike CAP-1, the small molecules described here inhibit both early and late events in the HIV replication cycle. In addition, PF-3450074 did not inhibit Gag particle production from HIV-1 transfected cells, suggesting that the compound series does not affect immature particle assembly. This is in contrast to the effects on immature particle assembly reported for NYAD-1. A co-crystal structure of a representative compound (PF-3450074) demonstrated a new binding site on HIV-1 CA distinct from those described for CAP-1 or CAI. Furthermore, PF-3450074 increased the rate of HIV-1 CA multimerization in vitro, while CAI and CAP-1 decreased the rate of CA multimerization in the same assay. While this does not necessarily define the action of these compounds on replicating virus, it does suggest a fundamentally different mechanism of inhibition from that of previously described CA inhibitors.
The proposed mechanism of action for both the early and late stage activities of this new class of inhibitors involves a direct effect on higher-order structures of HIV CA, in assembly and uncoating. Although the present data do not indicate whether the compounds enhance or inhibit the uncoating process, either effect is likely to interfere with proper reverse transcription [25]. HIV-1 capsid mutations proximal to the PF-3450074 binding pocket have been described that either destabilize or enhance the stability of viral cores and result in specific postentry defects in virus replication [24]. It is possible that such mutations and the compounds described in this study have analogous effects on inter-subunit capsid interactions. To gain further insights into the mode of action of PF-3450074, we generated a model of an assembled capsid hexamer in complex with PF-3450074 (Fig. 6a) based on superpositioning of published assembled capsid structures [13], [14] with the structure of the PF-3450074/CA complex. In the model, the R3 indole group which protrudes from the NTD in our structure localizes to the interface between capsid monomers in an assembled capsid and sits directly between the NTD of one capsid monomer and the C-terminal domain of another, making contacts to Tyr-169, Leu-172, Arg-173, Gln-179, and Lys-182 (Fig. 6b). This suggests the R3 indole group of PF-3450074 could play a critical role in modulating inter-subunit interactions. Both the CA NTD contact residues described by the co-crystal structure and these putative C-terminal contacts are well conserved across viral strains (Tables S5 and S6). This is consistent with the broad-spectrum antiviral activity observed for this series.
Although the sum of our results suggests a mechanism that affects interactions between capsid monomers, the early stage activity is consistent with other models. Cyclophilin A plays a critical role in the early stages of HIV-1 replication through interactions with the viral capsid [26]. Also, capsid-binding restriction factors such as the tripartite motif containing (TRIM) proteins prevent the infection of many primate cells with HIV or SIVs from other species [5], [9]. Thus, based on our data, we cannot dismiss the possibility that, during early infection, this new series might affect specific capsid-host protein interactions that mediate the viral uncoating process. A detailed study of the early stage activity of PF-3450074 has demonstrated direct destabilization of the HIV-1 capsid and a dependence on cyclophilin A, indicating that the compound induces premature uncoating of the virus, potentially through a mechanism similar to that of TRIM restriction [27].
In this study, we identify a new binding site on HIV-1 CA that can be targeted by small molecule inhibitors resulting in broad-spectrum antiviral activity. In addition, we describe the discovery and characterization of a novel series of compounds that act at this site and inhibit the virus at two points in the replication cycle. This series should serve as a good starting point for the development of a new class of HIV therapeutics through structure-based drug design or other approaches. The broad spectrum activity of this series is particularly exciting and highlights this novel mechanism as a significant therapeutic opportunity.
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