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, 6 (12), e1001220

HIV Capsid Is a Tractable Target for Small Molecule Therapeutic Intervention


HIV Capsid Is a Tractable Target for Small Molecule Therapeutic Intervention

Wade S Blair et al. PLoS Pathog.


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.

Conflict of interest statement

CP, SLI, DGB, MA, RB, JC, GC, JI, RH, KW, HW, and SLB are employed by Pfizer, which carries out research and development of antiretrovirals for HIV.


Figure 1
Figure 1. Structures and antiviral activities of inhibitors that target HIV-1 capsid.
Antiviral activity was determined in CPE assays after infection of MT-2 cells with HIV-1 NL4-3. Results represent the mean ± standard deviations from 4–6 experiments, or the mean with individual values from 2 experiments. TI, therapeutic index is the ratio of mean CC50: mean EC50.
Figure 2
Figure 2. Activity of inhibitors against diverse strains of HIV-1.
The spectrum of antiviral potencies was determined for two capsid inhibitors, efavirenz, and zidovudine against a panel of HIV isolates including clinical isolates and laboratory adapted strains. Each dot in the figure represents the geometric mean EC50 value for a single HIV isolate. The variability of the antiviral spectrum is further illustrated for each compound with a superimposed “box whisker” plot. The box represents the inter-quartile range and the whiskers highlight the 95% confidence interval. The vertical line in the box represents the geometric mean of the EC50 values.
Figure 3
Figure 3. Identification of mechanism of action.
a) HIV DNA quantification. A Taqman quantitative PCR assay was performed to determine the effect of test compounds on the levels of total HIV DNA, 2-LTR circles and integrated provirus generated during infection. The data is presented as a percentage of amounts observed in an untreated control tested in parallel. b) Time-of-addition experiment. HeLa CD4 LTR/beta-Gal reporter cells were infected with HIV-1 (NL43) and test compounds (at 2×EC 90) were added at different times after infection. Reporter signal was monitored at 72 h post-infection as a measure of replication. c) Effect of PF-3450074 in a virus production assay. 293T cells transfected with pNL43 were incubated in varying concentrations of PF-3450074. After 48 hours supernatants were transferred to HeLa CD4 LTR/beta-Gal reporter cells. The line graph shows the percentage of β galactosidase production compared with a mock treated control. Solid bars indicate the concentration of HIV capsid protein (p24) secreted by the 293T cells at each concentration tested. d) Kinetics of CA multimerization. HIV CA spontaneously multimerizes in the presence of high salt, causing a change in optical density. Test compounds were incubated with CA protein and multimerization was initiated with the addition of concentrated salt solution.
Figure 4
Figure 4. EM analysis of the effects of PF-3450074 on nascent viral particles.
PBMCs were infected with HIV NL4-3 in medium containing either no compound or 7µM PF-3450074. The cells and resulting viral products were processed and observed by transition electron microscopy. Close in view of an untreated native-like particle (a) reveals the central conical capsid structure, while a wider view (b) shows the high level of uniformity in morphology and size among untreated particles. Close in view of PF-3450074-treated particles (c) shows consistent loss of the central capsid density, and a wider view (d) illustrates a much wider range in particle morphology and size.
Figure 5
Figure 5. Structure of the novel inhibitor binding site and context in the NTD.
a) Overlays of capsid structures with PF-3450074 in blue and CAP-1 in pink bound to capsid N-terminal domain; b) Close up view of PF-3450074 site (binding site residues labelled in black, R1-3 sub-pockets labelled in purple). In the R1 sub-pocket PF-3450074 makes hydrophobic interactions with Ile-73, Ala-105, Thr-107, Tyr-130 and a stacking interaction with the side chain of Asn-53. The benzyl group R2 makes a number of hydrophobic interactions in a sub-pocket formed by the side chains of Met66, Leu69, Val59, Ile-73 and Leu-56. The R3 sub-pocket is only partially occupied by the indole group, forming interactions with the side chains of Met-66, Gln-67, Lys-70 and Gln-63 amide. The indole NH forms a hydrogen bond interaction with the side chain amide of Gln-67 via a water molecule, while an Asn-57 forms a key hydrogen bond with the cis amide bond of PF-3450074. c) Location of resistant mutations (purple) in relation to PF-3450074 capsid binding site. (PDB ID code 2XDE).
Figure 6
Figure 6. Model of inhibitor effects at the NTD-CTD interface.
a) Model of HIV-1 capsid in hexameric complex with PF-3450074 bound in each of the binding pockets (each of the six full-length CA monomers are coloured differently); b) Close up view of model of interface between adjacent N-terminal subunits (green and blue) and the C-terminal domain of an adjacent monomer (pink) in assembled capsid. PF-3450074 highlighted in purple. Residues highlighted Pro38 (adjacent N-terminal domain green), Thr-107, Thr-58, Asn-57, Asn-53, Lys-70 (N-terminal domain blue), Tyr-169, Leu-172, Arg-173, Gln-179, Lys-182 (C-terminal domain pink).

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