Two projects are envisaged, both of which will benefit from a chemical biology approach.
(i) NLRP3/MCC950. The inflammasome is a cytosolic apparatus that mediates sterile inflammation in response to exogenous and endogenous insults that disrupt cellular homeostasis. Inflammasome activation sounds the immunological alarm and initiates an appropriate host response to tissue injury by activating the protease caspase-1. Substrates of caspase-1 include pro-IL-1? and pro-IL-18, and the inhibited form of the pore-forming protein Gsdmd. Cleavage of Gsdmd results in a lytic form of cell death termed pyroptosis that contributes to the release of active IL-1? and IL-18. Proteins that detect specific inflammatory insults nucleate inflammasome complexes. For example, NLRP3 detects insoluble aggregates such as cholesterol crystals, uric acid crystals, and beta-amyloid clumps, but it also responds to prolonged activation of the purinergic P2X7 receptor. The tool compound, MCC950, can inhibit NLRP3-dependent inflammasome activation and has shown remarkable efficacy in a bewildering array of animal models ranging from Alzheimer's to Non-alcoholic Steatohepatitis. Our preliminary studies suggest that MCC950 binds to a discrete domain within NLRP3, but with a very high off rate. The goal will be to synthesize more potent compounds using a combination of chemical synthesis and structure aided design.
(ii) Fragment-based screen for pyroptosis inhibitors. These tool compounds will used to identify and characterize critical components of the pyroptosis cell death pathway. Such an experimental setting is highly effective as the unparalleled power of positive selection is unleashed: one is choosing survivors in the face of an otherwise lethal stimulation. This relatively facile approach should identify functional binders, in our case, components of the pyroptosis pathway. By Q4 of this year, we will have a non-covalent fragment library of ~ 1,000 cysteine-reactive members, each with a terminal alkyne and diazirine photoreactive group. Prior work has established that such probes are cell permeable and hence suitable for cell-based assays. Exposure to UV will covalently link the probe to bound protein(s), which can then be tagged with azide-biotin through the terminal alkyne. Subsequent IP enrichment and mass spectroscopy-based proteomics should lead to identification of the target. It is worth emphasizing that cysteine reactive probes are particularly attractive for the interrogation of the pyroptotic pathway as caspase-1 is a cysteine protease and the terminal effector Gsdmd possesses critical free cysteines. Hence it is conceivable that the project will result in the discovery of inhibitors of these previously designated undruggable targets.
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