Pyroptosis is a type of programmed cell death that occurs in macrophages and neutrophils during caspase-1 driven inflammatory responses. During this process, caspase-1 is activated via a large protein scaffold known as an inflammasome. Disruption of this pathway can lead to chronic inflammation, and so inhibitors of this pathway are of clinical interest. A specific inhibitor of the NLRP3 inflammasome, MCC950, has shown promise in animal models for, among others, atherosclerosis, Alzheimer’s disease and heart disease, however until recently the target protein of this drug was unknown.
This thesis reports the use of a powerful chemical proteomic technique for MCC950 target and off-target protein identification, and discusses the design, synthesis and validation of two photo-crosslinkable probes. These probes utilize diazirine photo-reactivity and alkyne-azide ‘click’ reactivity, enabling labelled proteins to be distinguished through mass spectrometry and proteomic analysis. Affinity-based protein profiling (AfBPP) is
used here in intact cells to confirm NLRP3 as the target protein of MCC950, and to identify novel off-target proteins for the molecule. In parallel, this thesis reports the development of a proteomics-led cellular thermal
shift assay (CETSA) technique which identifies proteins stabilised upon small molecule binding. This method is used in intact cells with MCC950 to validate novel off-target proteins discovered through affinity-based protein profiling.