As the world population ages, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease impose a growing burden on healthcare systems. The pathology of disease onset and progression are complex and not yet fully elucidated, with mounting evidence pointing to a central role of neuroinflammation. Microglia and astrocytes initiate inflammatory responses through multiple signaling pathways, among which multiprotein complexes, inflammasomes, play a significant role. The NLRP3 inflammasome is the most extensively studied, and includes the inflammatory protease caspase-1, which cleaves the pro-forms of inflammatory cytokines interleukin-1β and interleukin-18 into their biologically active forms, and triggers a form of programmed cell death known as pyroptosis. Design of caspase-1 inhibitors has often relied on structural mimicry of the IL-1β substrate, with the most clinically advanced being the peptidomimetic belnacasan. In mouse models of Alzheimer’s disease in vivo, belnacasan improved episodic and spatial memory and attenuated neuronal degeneration.
In the thesis, we synthesized analogs of the active belnacasan metabolite, aldehyde VRT-043198, incorporating various bioisosteres of aspartate on the (S)-prolinamide scaffold. The synthesis used protected amino acid derivatives to which we attached modified belnacasan core structures designed to target the S1 pocket of caspase-1. Our aim was to evaluate the contribution of belnacasan's non-covalent interactions to its inhibitory activity. During synthesis and isolation, significant racemization compromised compound purity, and the final products were often obtained as mixtures of stereoisomers. Using in silico modeling of the compounds in complex with caspase-1, we demonstrated that alterations in absolute configuration can substantially affect binding conformation and predicted binding affinity. Finally, we assessed the inhibitory activity using an in vitro biochemical assay with the fluorogenic substrate Ac-YVAD-AMC. After a 30-minute incubation of recombinant human caspase-1 with each compound, residual activity was measured. The most potent inhibitor was compound 11 (residual activity 9.9% at 100 μM), which differs from VRT-043198 only by the absence of the aldehyde warhead. Five additional derivatives also inhibited caspase-1; however, due to insufficient compound purity, these results are unreliable.
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