Mechanistic basis for a novel dual-function Gag-Pol dimerizer potentiating CARD8 inflammasome activation and clearance of HIV-infected cells

A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside Reverse Transcriptase inhibitors (NNRTIs) that promote Pyroptosis of HIV-1 infected cells. These NNRTIs stimulate dimerization of the Gag-Pol Polyprotein, resulting in premature HIV-1 protease (PR) dimerization and cleavage of intracellular CARD8. A unique cell-based high-throughput screen was developed to identify potent compounds activating the CARD8 inflammasome through Gag-Pol dimerization. Our in-house library of NNRTIs was evaluated, including a series of catechol diethers, which are potent, nontoxic antivirals. JLJ648 was identified as a promising dual-function Antiviral and Gag-Pol dimerizer. Cryo-EM studies of HIV Reverse Transcriptase p66 bound to JLJ648 revealed populations of homodimers and, surprisingly, a homotetramer. This novel homotetramer structure resembling an 'infinity knot' revealed two JLJ648-bound homodimers forming an extensive interface and nucleated around a dimer of JLJ648 molecules. Structure-guided mutagenesis studies indicate that Gag-Pol homotetramerization may play a critical role in facilitating PR self-cleavage and triggering Pyroptosis.

Drug discovery and development; High-throughput screening; Structure-based drug design; Target validation.