Lenacapavir

HIV-1 co-opts various host factors during its replicative cycle, including during host cell entry, nuclear integration, replication, and virion assembly. Following the initial fusion with the host cell membrane, the viral capsid is released into the host cell cytoplasm. The capsid comprises approximately 250 hexamers and exactly 12 pentamers, each composed of monomeric capsid proteins (CA). Each CA monomer has an N-terminal and C-terminal domain (NTD/CTD) and offers an interaction surface for host cell machinery. Several important protein-protein interaction interfaces occur between CA monomers in the assembled multimers; the binding constants of these proteins are substantially lower for assembled multimers than individual capsid monomers.

To facilitate HIV-1 genomic integration, the capsid must cross the nuclear envelope, for which it utilizes the nuclear pore complex (NPC). Two host proteins shown to be essential for capsid nuclear entry that directly bind to the capsid are cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and nucleoporin 153 (Nup153, an NPC protein present on the nucleoplasmic face of the complex). Both proteins bind the same phenylalanine-glycine binding pocket between the NTD and CTD of neighbouring CA monomers in multimeric CA assemblies.

Lenacapavir contains a difluorobenzyl ring that occupies the same binding pocket as CPSF6/Nup153, overlapping with the benzyl group of F321 in CPSF6 and F1417 in Nup153 in the overlayed structures. Crystal structures of lenacapavir bound to CA hexamers reveal that six lenacapavir molecules bind to each hexamer, establishing extensive hydrophobic interactions, two cation-π interactions, and seven hydrogen bonds, contacting ~2,000 Å2 of buried protein surface area. Strong binding of lenacapavir, therefore competitively interrupts capsid interactions with CPSF6 and Nup153. In vitro HIV-1 replication inhibition experiments in a variety of cell lines show EC50 values of ~12-314 pM, with greater efficacy against early steps over later steps. At very low concentrations (0.5 nM), lenacapavir inhibits viral nuclear entry, while at higher concentrations (5-50 nM), it additionally inhibits viral DNA synthesis and reverse transcription. As CPSF6 and Nup153 are essential for nuclear entry, it is likely that lenacapavir binding inhibits these interactions and blocks capsid nuclear entry.

Lenacapavir may have additional effects beyond blocking interactions with host cell factors. Lenacapavir increases the rate and extent of CA assembly, dramatically extends the lifetime of assembled CA structures, even at high salt concentrations, and alters assembled capsid morphology. The stabilizing concentration is ~1:1, closely mimicking the observed binding stoichiometry to isolated CA hexamers. Further analysis suggests that lenacapavir binding alters intra- and inter-hexamer interactions, altering the structure and stability of the resulting assemblies.

Serial passage of HIV-1 in increasing concentrations of lenacapavir resulted in the appearance of major resistance mutations Q67H and N74D, which remain sensitive to other antiretroviral drugs. Extended passage resulted in the additional mutations L56I, M66I, K70N, N74S, and T107N. All identified resistance mutations map to the lenacapavir binding site, and all but the Q67H variant show reduced replication capacity in vitro. Additional studies have shown no lenacapavir resistance in variants associated with resistance to other antiretrovirals or naturally occurring polymorphisms, suggesting a very low potential for cross-resistance in combination therapy.