Abstract 1126 Generative Artificial Intelligence in Molecular Design and Virtual Screening of Novel Caspase-1 Inhibitors

Recently, caspase-1 has emerged as an anti-inflammatory target for therapeutic development due to its functions associated with releasing pro-inflammatory cytokines and initiating pyroptosis. Caspase-1 is activated by inflammasomes that detect cytosolic contamination or perturbation of viral or bacterial infections. It subsequently activates pro-inflammatory cytokines, such as Il-1β and Il-18, and cleaves gasdermin D, which induces the inflammatory form of lytic programmed cell death, or pyroptosis. In this study, we explore the hypothesis that caspase-1 inhibition can prevent inflammatory damage in the blood-brain barrier (BBB). Using the real-time RT-qPCR (reverse transcription-quantitative polymerase chain reaction) technique, we studied the gene expression of the death-inducing signaling complex (DISC) in the apoptotic pathway and inflammasome proteins and cytokines in brain microvascular endothelial cells (BMVECs) treated with substances of abuse and HIV-1 Tat protein. Our initial results suggested that the illicit drugs and HIV-1 Tat protein activate inflammasome and promote the expression of pro-inflammatory cytokines while suppressing both apoptotic and pyroptotic forms of cell death. In particular, caspase-1, which is activated by inflammasome and the initiator of pyroptosis and pro-inflammatory cytokines, is significantly up-regulated. Utilizing a generative graph-based machine learning (ML) algorithm, 3D Scaffold, developed by our collaborators at the Pacific Northwest National Laboratory (PPNL), thousands of novel candidates that are potential inhibitors of caspase-1 were generated. A virtual screening process narrowed down to a dozen molecules that possess favorable drug properties, such as low toxicity, bioavailability, and feasibility for synthesis, for validation. We also expressed and purified the human caspase-1 catalytic domain (p20/p10) from E. coli bacterial cells. In the enzyme activity assay, the purified enzyme demonstrated hydrolysis activity. The AI-generated caspase-1 inhibitors will be further validated experimentally, including enzymatic inhibition assay (IC50) and cell-based assay, including gene expression, cell viability, and BBB permeability. This research is partially supported by NIH Grant # R21DA046223 and the Department of Education Grant On PACE-3500381.