GS-6207 (Lenacapavir, Gilead Sciences) is an experimental long-acting and highly potent HIV-1 capsid (CA) inhibitor. Viral breakthrough assays in cell culture identified a number of HIV-1 CA substitutions including M66I, Q67H, N74D and Q67H/N74D that confer substantial resistance to the inhibitor. Furthermore, the Q67H capsid substitution has emerged in HIV-1 infected patients receiving GS-6207.
We have determined high-resolution x-ray crystal structures of cross-linked HIV-1 CA hexamers containing the following drug-resistant substitutions: M66I, Q67H, N74D and Q67H/N74D. In addition, we have determined the structure of CA[sub]Q67H/N74D[/sub] in the complex with GS-6207.
Our x-ray crystal structures have uncovered the following three distinct mechanisms for drug-resistance: i) the structure of the CAM66I hexamer reveals that the isoleucine’s ?-branched side chain extends further from the main chain and creates steric hindrance with respect to GS-6207; ii) the structures of the CAQ67H and CAQ67H/N74D hexamers show markedly different positioning of the histidine side chain compared with the glutamine in wild type CA hexamer + GS-6207. Specifically, the side chain of His67 is positioned into the inhibitor binding pocket and would therefore sterically hinder the binding of GS-6207 unless repositioned. The structure of the CAQ67H/N74D hexamer + GS-6207 reveals that the side chain of His67 is in fact repositioned to a similar conformation as Gln67 in wild type CA hexamer to accommodate GS-6207 within the binding pocket; iii) while the structure of the CAN74D hexamer resembles its wild type counterpart, the Asn to Asp substitution compromises a hydrogen bonding interaction with a nearby oxygen of the sulfonyl group of GS-6207.
Our studies reveal the structural bases for how the HIV-1 CA substitutions confer resistance to the experimental drug GS-6207 and provide the means for rationally developing second-generation inhibitors.