Identification of an antiretroviral small molecule that appears to be a host-targeting inhibitor of HIV-1 assembly

There is an urgent need for development of antiretrovirals that act by novel mechanisms allowing treatment of multidrug resistant HIV-1. Host-targeting drugs are of special interest because they can offer a high barrier to resistance. Here we report identification of potent antiretroviral small molecules that inhibit viral late events, a stage of the HIV-1 life cycle for which potent and specific inhibitors are lacking. These drugs were discovered using cell-free protein synthesis and assembly systems that recapitulate intracellular host-catalyzed viral capsid assembly pathways. The drugs inhibit HIV-1 replication in human T cell lines and PBMCs with nanomolar EC50 values, and are effective against an HIV-1 primary isolate. Drug treatment dramatically reduces virus production, and appears to inhibit a post-translation step HIV-1 Gag assembly. Notably, the drugs colocalize with HIV-1 Gag in situ. However, unexpectedly, drug selection failed to identify drug-specific resistance mutations in Gag or any other viral gene product, despite development of resistance mutations in a parallel Nelfinavir selection control. Thus, we hypothesized that instead of binding to Gag directly, this drug might localize to assembly intermediates, the intracellular multiprotein complexes containing Gag and host factors that are formed during immature HIV-1 capsid assembly. Indeed, imaging of infected cells showed colocalization of the drug with ABCE1 and DDX6, two host enzymes found in assembly intermediates. While the exact target and mechanism of action of the drugs remain to be determined, these findings suggest that they may represent first-in-class, host-targeting inhibitors of intracellular events in HIV-1 assembly.