A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells

Abstract

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection

“…In contrast, Wang et al discovered that HIV PR is sensed by CARD8 using NNRTIs to enforce HIV PR cytosolic activity as a means to clear latently HIV-infected cells 21 . We speculate that therapeutic strategies that leverage HIV PR -dependent CARD8 inflammasome activation may be bolstered by adjuvants that induce TLR signaling 21,45,46 . Our finding that HIV-1 infection is sufficient to induce inflammasome activation, along with the presence of CARD8 in relevant T cell populations 16,33 , also suggests that CARD8 may contribute to HIV pathogenesis.…”

Potent targeted activator of cell kill molecules eliminate cells expressing HIV-1

“…For infection, PBMCs were centrifuged at 200 g for 5 min, and the supernatant was removed by aspiration. PBMCs were resuspended at about 10 × 10 6 cells/ml in complete medium with interleukin-2 (IL-2) (10 U/ml; Roche) and VSV-G pseudotyped HIV-1 virus stock (VSV-G/pNLG1-P2A-∆Env) ( 36 ). The pNLG1-P2A-ΔEnv plasmid was derived from the NL4.3 strain sequence ( 37 ), with a GFP-encoding sequence and a picornaviral 2A–like sequence (P2A) inserted 5′ of the negative regulatory factor (Nef)–encoding sequence.…”

Optimized M24B Aminopeptidase Inhibitors for CARD8 Inflammasome Activation

“…Selective CARD8 inflammasome activators are not only useful research tools to study CARD8 biology but also have potential therapeutic values. For example, such agents can in theory kill CARD8-expressing cancer cells without triggering the highly inflammatory NLRP1 response in normal tissues. , In addition, these compounds could potentially enhance the elimination of HIV-1 reservoirs by stimulating pyroptosis in HIV-1 infected cells . In our original report describing the discovery of CQ31, we did not attempt to improve the activity of this lead molecule.…”