Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

Long-acting antiretroviral therapy would substantially improve the care of people living with HIV and would mitigate a number of challenges including the necessity of daily administration of current HIV medications, suboptimal treatment adherence, and emergence of drug resistance. GS-6207 (Lenacapavir, Gilead Sciences) is the first-in-class long-acting ultrapotent HIV capsid (CA) inhibitor. Recently completed phase 1 clinical trials (NCT03739866) have suggested that a 6-month dosing interval may be possible.

On the basis of these results, GS-6207 has advanced into phase 2/3 clinical trials (NCT04143594/NCT04150068). Initial mechanistic studies with GS-CA1, an archetypal predecessor of GS-6207, revealed its multistage mechanism of antiviral action (1). GS-CA1 potently [half-maximal effective concentration (EC50) = 87 pM] inhibited early steps of HIV-1 replication and also exhibited a second, less potent (EC50 = 240 pM) antiviral activity during virus egress. Molecular modeling studies predicted that both GS-CA1 and GS-6207 bind to the hydrophobic pocket formed by two adjoining CA subunits within the hexamer (2). HIV-1 genotyping, after selection in cell culture in the presence of the inhibitor, identified a number of CA mutations positioned near the potential inhibitor binding site that conferred substantial resistance to GS-CA1 (1). However, the structural and mechanistic bases for how this class of compounds binds and alters the biological functions of HIV-1 CA remain unclear.

We synthesized GS-6207 (Fig. 1A) and examined its antiviral activities. GS-6207 inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and various cell lines, with EC50 values in the range of ~12 to 314 pM (Fig. 1B and table S1). PBMCs and MT4 T cells were fully viable in the presence of 50 mM GS-6207 (the highest concentration tested), indicating a selectivity index of >106 (Fig. 1B). GS-6207 exhibited higher potency during early (EC50 ≈ 55 pM) versus late (EC50 ≈ 314 pM) steps of HIV-1 replication (Fig. 1B and table S1). Our subsequent efforts focused on understanding the structural and mechanistic bases for inhibition of incoming HIV-1 by GS-6207.

To dissect HIV-1 post-entry infection steps targeted by GS-6207, we monitored viral DNA intermediates, including total reverse transcripts, two–long terminal repeat (2-LTR) circles (a surrogate for nuclear import), and integrated proviruses (the viral copy DNA incorporated into the host cell DNA) (Fig. 1C). In parallel, we examined the effects of GS-6207 on viral DNA levels in the cytoplasm and nuclei of infected cells (fig. S1). In control experiments, the reverse transcriptase inhibitor azidothymidine (AZT) impaired viral DNA synthesis, whereas the integrase (IN) inhibitor dolute-gravir (DTG) specifically blocked integration, as evidenced by marked reduction of proviral DNA and increased levels of 2-LTR circles. In contrast, GS-6207 affected multiple sequential steps of virus ingress in a dose-dependent manner. At a comparatively high concentration (50 nM), GS-6207 effectively inhibited reverse transcription. At the pharmacologically relevant concentration of 5 nM (3), the inhibitor partly impaired viral DNA synthesis and effectively blocked formation of 2-LTR circles and integrated HIV-1 DNA. In line with these results (Fig. 1C), 5 nM GS-6207 markedly reduced viral DNA levels in both the cytoplasm and nucleus (fig. S1). At 0.5 nM, GS-6207 inhibited integration without detectably affecting reverse transcription. Although 0.5 nM GS-6207 and 1 mM DTG similarly inhibited integration, the former failed to increase 2-LTR circle formation, likely as a result of concomitant inhibition of nuclear import (Fig. 1C). Results of cellular fractionation indeed support this interpretation of the population-specific polymerase chain reaction assays (fig. S1). Relative to the dimethyl sulfoxide (DMSO) control, 0.5 nM GS-6207 increased and decreased viral DNA levels in the cytoplasm and nucleus, respectively (fig. S1). The multistep inhibition, which depends on the concentration of GS-6207, is likely due to the inhibitor affecting the multifaceted roles of CA during virus ingress (4).

We considered the following two scenarios to account for the observed inhibitions of viral DNA replication intermediates: (i) GS-6207 could adversely affect functional disassembly of the CA shell through stabilizing or destabilizing its architecture, which in turn would adversely affect reverse transcription, nuclear import, and integration; (ii) GS-6207 could interfere with CA interactions with cognate cellular cofactors needed for nuclear import, and/or could interfere with trafficking of pre-integration complexes inside the nucleus to preferred sites of integration.

To examine these possibilities, we imaged the effects of GS-6207 on incoming HIV-1 by using single-particle detection of virus cores colabeled with CypA-DsRed (a marker for CA) and INmNG (IN fused to NeonGreen protein) (fig. S2) (5). GS-6207 substantially increased levels of virus cores in the cytoplasm, which suggested a stabilizing effect of the inhibitor (Fig. 1D). Conversely, GS-6207 inhibited the formation of IN puncta in the nucleus with concomitant inhibition of HIV-1 infection (Fig. 1, E and F, and fig. S3). These findings indicate that GS-6207 stabilizes virus cores, leading to their accumulation in the cytoplasm and preventing nuclear import.

To explore the stabilizing role of the inhibitor on the CA shell, we conducted in vitro assays with isolated HIV-1 particles (6). In the absence of inhibitor, virus cores fully dissociated within 30 min, whereas picomolar concentrations of GS-6207 markedly enhanced the stability of native cores (Fig. 1G and fig. S4). Next, we tested the effects of GS-6207 on tubular assemblies made in the presence of 2 M NaCl (7) (Fig. 1H). The preassembled tubes dissociated immediately upon exposure to a buffer containing 150 mM NaCl (<1 min; Fig. 1H, lane 2). In sharp contrast, addition of GS-6207 to preassembled CA tubes rendered these tubular assemblies highly resistant to low ionic strength (150 mM NaCl) conditions. Strikingly, in the presence of GS-6207, tubular CA assemblies remained stable even after 96 hours of incubation under physiologically relevant conditions (Fig. 1H). The stabilizing effects correlated with a GS-6207:CA ratio of ~1:1 (fig. S5).

We tested the effects of the cellular CA binding partner CypA on GS-6207’s activities. As expected (8, 9), the addition of increasing concentrations of CypA resulted in effective disassembly of the preformed CA tubes in the absence of the inhibitor (fig. S6). In sharp contrast, GS-6207–stabilized CA tubes remained intact in the presence of CypA (fig. S6). Furthermore, GS-6207’santiviralactivitiesremained unaffected by depletion or overexpression of CypA in Jurkat and MT4 T cells (fig. S7).

Next, we examined whether GS-6207 affects CA interactions with the known cellular cofactors Nup153 and CPSF6 needed for nuclear import (10, 11). GS-6207 substantially reduced binding of cellular Nup153 and CPSF6 to pre-assembled CA tubes (fig. S8). Because CPSF6 is also known to regulate integration site selectivity (12, 13), we tested whether GS-6207 influences sites of HIV-1 integration. The inhibitor substantially reduced integration in gene-dense regions and, conversely, enhanced integration in lamina-associated domains (fig. S9). These GS-6207–mediated effects on integration targeting mimicked the CPSF6 depletion phenotype. However, the extent of inhibitor-induced changes was less than those seen with CPSF6 knockout, which suggests that GS-6207 may not fully displace the cellular cofactor. Taken together, our mechanistic studies reveal stabilizing effects of GS-6207 on viral cores, coupled with the ability of the inhibitor to interfere with CA binding to the cognate cellular cofactors CPSF6 and Nup153.

To understand the structural basis for GS-6207 interaction with CA, we solved a cocrystal structure of the inhibitor bound to a prestabi-lized CAA14C/E45C/W184A/M185A hexamer (14) (Fig. 2 and table S2). The high-resolution structure (2.22 Å) revealed that GS-6207 binds in the hydrophobic pocket formed by two adjacent CA subunits (Fig. 2A and fig. S10) with a stoichiometry of six GS-6207 compounds bound per each CAA14C/E45C/W184A/M185A hexamer. GS-6207 makes extensive van der Waals and hydrogen-bonding interactions with CA1-NTD (the N-terminal domain of CA subunit 1), CA2-CTD (the C-terminal domain of CA subunit 2), and CA2-NTD. Two ring systems, R3 and R4, primarily drive the van der Waals interactions with CA1-NTD and CA2-CTD (fig. S10). R1 and R2 also provide additional interactions with CA1-NTD and CA2-NTD. GS-6207 establishes a hydrogen-bonding network with the side chains of Asn57, Lys70, and Asn74 of CA1-NTD, Ser41 of CA2-NTD, and Gln179 and Asn183 of CA2-CTD (Fig. 2B, fig. S10, and table S3).

The interacting helices that predominantly form the GS-6207 binding pocket include aH3 and aH4 from CA1-NTD, aH8 and aH9 from CA2-CTD, and aH2* from CA2-NTD (Fig. 2C). Particularly noteworthy is that GS-6207 strongly influences the conformation and relative positioning of aH9 of CA2-CTD with respect to aH4 of CA1-NTD. For comparison, aH9 is seen to exhibit substantial conformational variation in the absence or presence of different cellular protein partners bound to CAA14C/E45C/W184A/M185A or native CA hexamers (figs. S11 to S20).

Previously reported resistant mutations to predecessor compound GS-CA1 are within close proximity of the GS-6207 binding site (Fig. 2D). Met66 is a key constituent of the hydrophobic pocket and forms strong van der Waals interactions with rings R3 and R4. The M66I substitution had the most profound effects on loss of GS-6207 potency, reducing activity by more than four orders of magnitude (table S4). N57S, Q67H, K70A, and N74D substitutions, which are expected to adversely affect direct interactions of CA with GS-6207, reduced potency by factors of ~60, ~10, ~45, and 14, respectively (table S4). Consistent with a previous report (1), infectivity of the M66I mutant virus, which conferred the greatest extent of GS-6207 resistance, was markedly compromised (fig. S21). The infectivity of N57S and K70A mutant viruses, which exhibited substantial resistance to the inhibitor, was severely and considerably reduced, respectively. Q67H and N74D, which exhibited lower levels of resistance, displayed wild-type HIV-1 infectivity (fig. S21).

Structural comparison of GS-6207 with the substantially less potent HIV-1 CA inhibitor PF74 (15–17) revealed both similarities and marked differences (fig. S22 and table S3). The resemblance between the two compounds is seen with respect to their interactions with CA1-NTD. The phenyl R1 and R2 rings and indole R3 ring of PF74 superimpose onto the indazole (R2), difluorobenzyl (R3), and cyclo-pentapyrazole (R4) rings of GS-6207, respectively. However, unlike PF74, which only makes limited hydrophobic contacts with CA2-CTD, GS-6207 establishes extensive hydrogen-bonding and hydrophobic interactions with adjoining CA2-NTD and CA2-CTD (Fig. 2 and figs. S10 and S22).

We also compared GS-6207 binding to known interactions of CPSF6 and Nup153 with CA hexamers (15, 18). The backbone of Nup153 aligns along R1 and R3 of GS-6207, with Phe1417 of Nup153 closely superimposing on the di-fluorobenzyl moiety (R3) of GS-6207 (fig. S23). Similarly, there is substantial overlap between GS-6207 and the main chain of CPSF6, with Phe321 of CPSF6 superimposing on R3 extremely well (fig. S24). Interestingly, the binding pockets for Nup153 and CPSF6 are more open, with CTD aH9 being positioned farther away from NTD aH4 than in the presence of GS-6207. In turn, the closer aH4-aH9 conformation imposed by GS-6207 creates steric clashes with Nup153 and CPSF6 (figs. S23 and S24). Collectively, these findings provide structural explanations for the displacement of Nup153 and CPSF6 by GS-6207 (fig. S8).

To understand the structural basis for GS-6207’s interactions with curved CA assemblies, we used cryo-EM. GS-6207, but not a DMSO control, stabilized preformed tubes and resulted in well-defined tubular CA assemblies at physiological salt concentration (Fig. 3A and figs. S25 and S26). Imaging these structures allowed us to obtain a 6.3 Å map for GS-6207 bound to A92E CA tubes (Fig. 3B, figs. S27 and S28, and table S5); GS-6207 interacted similarly with wild-type and A92E CA tubes (fig. S25), and the latter protein was successfully used for prior cryo-EM studies (8, 19). A hexamer with pseudo–two-fold symmetry characteristic of CA tubes was readily identified (Fig. 3, C and D) and was further refined by analyzing helical tube patches with RASTR, a single-particle approach independent of helical parameter determination (fig. S29) (20). The mutually independent helical and RASTR approaches produced equivalent maps of a tube hexamer (fig. S30), further validating the map’s accuracy (figs. S31 to S33). Rigid-body docking of individual crystallographic CA monomers in the presence of GS-6207 could account for all features in the cryo-EM hexamer, including the positions of well-defined a helices in the CTD (Fig. 3D and figs. S30 and S34). Density corresponding to bound GS-6207 could be identified by segmentation of the helical or RASTR cryo-EM maps (fig. S35). Thus, we were able to obtain a model of the GS-6207–bound tube hexamers under physiologically relevant conditions.

Comparisons of our cryo-EM structure with published cryo-EM– and cryo–electron tomography–derived structures of CA hex-amers from tubes and native HIV-1 particles (19, 21) reveal the principal differences in formation of curved hexameric lattices in the absence and presence of GS-6207 (figs. S36 to S41). Normally, CA CTDs move away from the adjacent NTDs to accommodate inter-hexamer contacts in the context of a curved topology (19, 21). In sharp contrast, GS-6207 strongly restricts changes in the CTD position with respect to the adjoining NTD, and requirements for establishing inter-hexamer interactions on a curved surface are satisfied by repositioning of the comparatively rigid GS-6207–bound CA monomers in each hexamer (see movies S1 to S4; also compare movie S5 with movies S7 and S9, and movie S6 with movies S8 and S10). Accordingly, NTD aH4 and CTD aH9 from adjacent subunits are farther apart and closer together in the absence and presence of the inhibitor, respectively (figs. S37 and S39).

To further understand how GS-6207 affects tubular CA assemblies, we used hydrogen-deuterium exchange (HDX) (figs. S42 to S46). HDX experiments revealed strong protection in CA segments that directly interact with the inhibitor (figs. S47 and S48). Unexpectedly, we observed strong protection beyond the direct inhibitor binding sites. The NTDs that form the inner hexamer core and provide the binding site for IP6 [a natural cellular cofactor of CA that also stabilizes virus cores (22)] showed strong protection (figs. S47 and S49) despite a lack of direct contacts with GS-6207. These findings suggest that GS-6207 stabilizes individual CA hexamers. This notion is further supported by thermal shift assays, which show that GS-6207 substantially increases the melting temperature of isolated CA hexamers (fig. S50). Collectively these biochemical findings are consistent with our cocrystal structure (Fig. 2), which shows that each GS-6207 connects two adjoining monomers in a hexamer, with the binding of six inhibitors resulting in a more stable hexamer.

Strikingly, the strongest GS-6207–induced protections were seen in aH9 (figs. S47 and S51), which suggests that the inhibitor stabilizes inter-hexamer aH9-aH9 contacts essential for curved lattice formation (Fig. 3, E and F) (19, 21). The E45A and E180A CA substitutions, which influence intra- and inter-hexamer interfaces, respectively (23–25), but do not directly interact with GS-6207 (fig. S47), conferred partial resistance to the inhibitor (fig. S52).

Pliability of intra- and inter-hexamer interactions is essential for both proper assembly of the CA shell during virion maturation and its subsequent disassembly during virus ingress (8, 23). GS-6207 disrupts this delicately balanced interplay by rigidifying the CTD conformation and stabilizing both intra-hexamer and aH9-aH9 inter-hexamer interactions (Figs. 2 and 3 and fig. S47). These findings provide structural clues as to how GS-6207 inhibits functional disassembly of virus cores and blocks incoming HIV-1 in infected cells (Fig. 1). Taken together, our results elucidate the structural and mechanistic bases for the multimodal, potent antiviral activity of GS-6207 and provide a platform for rationally developing improved long-acting therapies.

We note that during the revision of the present manuscript, an article describing clinical targeting of HIV CA by GS-6207, which also includes synthesis of the inhibitor and a crystal structure of GS-6207 bound to CA hexamer (fig. S53), was published (26).

ACKNOWLEDGMENTS

We thank S. Rebensburg, P. Koneru, and other members of the participating laboratories for their help with data analysis and valuable suggestions. All cryo-EM data were collected at the Anschutz Medical Campus Cryo-EM Core Facility. Funding: Supported by NIH grants R01 AI062520 and R01 AI143649 (M.K.), R01 AI157802 (M.K., F.J.A., and J.R.F.), U54 AI150472 (M.K., P.R.G., A.C.F., G.B.M., and A.N.E.), R01 AI129862 (G.B.M.), R01 AI052014 (A.N.E.), and R01 AI77344 and DP1 DA043915 (E.M.P.). Author contributions: The following authors conducted experiments and interpreted the results: S.M.B. (x-ray crystallography); G.W., A.S.A., and J.M. (virology); N.S. (biochemistry); H.Z., N.I., P.V.B., and F.J.A (cryo-EM); D.A.-A. and J.R.F. (medicinal chemistry); A.C.F. and G.B.M. (cell imaging); V.V.C. and P.R.G. (HDX); P.K.S. and A.N.E. (integration site sequencing). Separate aspects of the study were designed and supervised by E.M.P., A.N.E., A.C.F., G.B.M., P.R.G., J.R.F., F.J.A., and M.K. The entire project was conceived by M.K. with all authors providing intellectual input and contributing to preparation of the manuscript. Competing interests: A.N.E. declares fees from ViiV Healthcare Co. for work unrelated to this project. No other authors declare competing interests. Data and materials availability: The cocrystal structure and cryo-EM–derived atomic model are deposited in the Protein Data Bank under accession numbers 6VKV and 6VWS, respectively. The EM maps are deposited in the Electron Microscopy Data Bank under accession codes EMD-21423 and EMD-21424. DNA sequences for integration site mapping are deposited in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) under accession code PRJNA608802. All other data are available in the manuscript or the supplementary materials. Materials are available from M.K.

SUPPLEMENTARY MATERIALS science.sciencemag.org/content/370/6514/360/suppl/DC1 Materials and Methods Figs. S1 to S53 Tables S1 to S5 1H, 13C, and HSQC NMR Spectra of GS-6207 and Synthetic Intermediates References (27–58)

Movies S1 to S10 MDAR Reproducibility Checklist

26 February 2020; accepted 25 August 2020 10.1126/science.abb4808

DIAGRAM: Fig. 3. Cryo-EM structure of GS-6207–stabilized CA tubes. (A) Cryo-EM image of A92E CA tubes stabilized by GS-6207 in 150 mM NaCl. Inset shows a subset of the averages obtained by 2D clustering of tube segments. (B) Cryo-EM map at 6.3 Å resolution from helical processing of GS-6207– stabilized A92E CA tubes. (C) Diagram showing the pseudo–two-fold symmetric arrangement of monomers in a tube hexamer. (D) Atomic model of a hexamer in the GS-6207–stabilized CA tube generated by rigid-body fitting of six copies of the x-ray structure of a GS-6207– bound CA monomer into the RASTR map. (E) A portion of a tube showing interactions between seven hexamers. Coloring corresponds to HDX protection levels. The cyan, green, and orange lines indicate three helical directions. (F) Close up of aH9-aH9 interactions involving a central hexamer. All six H9 helices in the central hexamer (dark blue) were superimposed. aH9 helices in neighboring hexamers are shown in cyan, green, and orange, matching the coloring of helical directions in (E). The absence of true two-fold symmetry results in slight differences in the positioning of the two helices along a specific helical direction. The visible side chain is Glu180.

DIAGRAM: Fig. 2. Structural basis for GS-6207 interaction with CA hexamer. (A) X-ray crystal structure of GS-6207 (orange) bound to the prestabilized CAA14C/E45C/W184A/M185A hexamer (PDB ID 6VKV). GS-6207 binds at the pocket formed by two adjoining CA subunits CA1 (light gray) and CA2 (pale yellow). Relative positions of CA1-NTD, CA2-NTD, and CA2-CTD are indicated. (B) Cartoon representation of the structure indicating GS-6207’s interactions with the two subunits that form the binding pocket, CA1 and CA2. Hydrogen bonds are denoted by black dashed lines. (C) The main helices (aH2*, aH3, aH4, aH8, and aH9) that interact with GS-6207 are indicated. (D) Reported resistance mutations (green) for GS-CA1 (1) are shown in the context of GS-6207 bound to CA1-NTD.

DIAGRAM: Fig. 1. Multimodal mechanism of action of GS-6207. (A) Chemical structure of GS-6207. (B) Antiviral activities and cytotoxicity of GS-6207 (see also table S1). (C) Effects of GS-6207 on formation of total reverse transcripts, 2-LTR circles, and proviruses. Error bars indicate SD for three independent experiments. (D) Effect of GS-6207 on the number of post-fusion HIV-1 cores in the cytoplasm (see also fig. S2). (E) Inhibition of nuclear import of HIV-1 (see fig. S3A). (F) Effect of GS-6207 on HIV-1 infectivity (see fig. S3B). eGFP, enhanced green fluorescent protein. (G) GS-6207 increases the stability of isolated HIV-1 cores in vitro (see fig. S4). Error bars in (D) to (G) represent SEM from four fields of view for a representative experiment of two independent experiments (*P < 0.05, **P < 0.005 ***P < 0.0001). (H) Effects of GS-6207 on the stability of recombinant CA tubes. Only pelleted fractions of CA from each reaction are shown. CA tubes were assembled in 2 M NaCl in the absence (top) or presence of GS-6207 (bottom) and then either directly pelleted (lane 1) or exposed to low ionic strength (150 mM NaCl) buffer for increasing periods of time (0, 1, 4, 24, 48, and 96 hours; lanes 2 to 7) and then pelleted.