Clinical targeting of HIV capsid protein with a long-acting small molecule

Abstract

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis^1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance⁶. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections^1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence¹⁰. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log₁₀-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

Figure 1:. GS-6207 is a potent CA-targeting inhibitor of HIV replication.

a, GS-6207. b, Light scattering (absorbance at 350 nm) responses showing the rate and extent of in vitro CA (20 μM) assembly in 2M NaCl in the presence and absence of GS-6207. Data are representative of four independent experiments (n = 2 biological replicates each). c, Representative thin-section electron micrograph images of HIV-1 produced in the presence of 0.2% DMSO, GS-6207 or the HIV-1 protease inhibitor atazanavir (ATV). Scale bars, 50 nm. d, Quantitation for c. Data are mean ± s.d. from representative images of HIV-1 produced in one of two independent experiments (DMSO, n = 737 virions; GS-6207, n = 591; ATV, n = 618). All P values, here and throughout, by unpaired two-tailed Student’s t-test with Welch’s correction. ****P < 0.0001. e, Inhibition of HIV-1_IIIb in MT-4 cells. Data are mean ± s.d. from four biological replicates in each of 8 to 115 independent experiments: GS-6207 (n = 8), rilpivirine (RPV, n = 113), efavirenz (EFV, n = 113), dolutegravir (DTG, n = 115), bictegravir (BIC, n = 20), ATV (n = 113), darunavir (DRV, n = 60), and tenofovir alafenamide (TAF, n = 15). ****P < 1 x 10⁻¹⁵. f, Inhibition of HIV-2 and HIV-1 Group M (subtypes A-G, CRFs), N and O clinical isolates. Data represent individual isolates (n = 3 biological replicates each). CRFs, circulating recombinant forms. g, X-ray crystal structure of GS-6207-CA hexamer complex. Top and side views of CA hexamer (individual CA monomers colored cyan and gray). The GS-6207 binding site, located between the NTD of one CA monomer and the CTD of an adjacent monomer, is boxed. h, Space-filling view of GS-6207 in its binding site (X-ray structure). i, Hydrogen bonds (dashed black lines, n = 7) and cation-π interactions (dashed yellow lines, n = 2) are shown between GS-6207 and CA residues.

Figure 2:. GS-6207 inhibits multiple capsid-dependent HIV-1 replication steps.

a, Antiviral activity of GS-6207 throughout a full replication cycle or when selectively present during target cell infection (early events) or virus production (late events). Data are mean ± s.d. from four independent experiments (n = 3 biological replicates each). b, Time-of-addition study indicating when GS-6207 inhibits HIV-1 replication relative to rilpivirine (RT inhibitor) and bictegravir (IN inhibitor). Data are means from one of two representative independent experiments (n = 8 biological replicates per group in each experiment). c, Effect of GS-6207 (1.25 nM) on the intracellular abundance of various HIV-1 DNA forms. Data are mean ± s.d. from one of two representative independent experiments (n = 3 biological replicates each). d, Overlay of GS-6207 with CA-binding peptides from nuclear import factors CPSF6 and Nup153 (ref. 25) in their shared binding pocket. e, Effect of GS-6207 on HEK293T producer cell viability, HIV-1 particle production and infectivity. Data are means from one of three representative independent experiments (n = 3 biological replicates each). M66I, GS-6207 resistance-associated CA binding site variant. f, Effect of GS-6207 on intracellular CA levels. Data are mean ± s.d. from three independent experiments (n = 3 biological replicates each). For gel source data, see Supplementary Figure 1.

Figure 3:. Resistance to GS-6207 maps to CA.

a, Emergent CA substitutions denoted during resistance selection in HIV-1_HXB2D-infected MT-2 cells by GS-6207 dose-escalation. Data are representative of one of two biological replicates from a single selection experiment. b, Fold resistance of GS-6207-selected viral isolate passage 3 to 10 (P3 – P10) to GS-6207 and control antiretrovirals. Data are mean ± s.d. from three independent experiments (n = 3 biological replicates each). Red dotted line defines the cut-off for drug resistance. WT, wild-type HIV-1; EFV, efavirenz; DTG, dolutegravir; ATV, atazanavir. c, Frequency of GS-6207-selected CA variants observed at fixed GS-6207 concentrations in PBMCs infected with clinical HIV-1 isolates.

Figure 4:. Clinical testing of GS-6207 in humans.

a, Mean plasma concentration-time profile of GS-6207 after a single subcutaneous administration of 30-450 mg of drug to uninfected participants (n = 8 within each dosing arm). b, Mean plasma concentration-time profile of GS-6207 after a single subcutaneous administration of 20-450 mg to HIV-infected participants (n = 6 within each dosing arm). Red dotted line defines paEC₉₅ for GS-6207. c, Mean log₁₀ change in plasma HIV-1 RNA after a single subcutaneous administration of 20-450 mg GS-6207 to eight participants with untreated HIV-1 infection randomized to drug (n = 6) or placebo (n = 2) within each dosing arm.