Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease represent an increasing healthcare and societal challenge in ageing populations. The pathology underlying their onset and progression is complex and remains incompletely understood, with growing evidence highlighting the important role of neuroinflammation. Key mediators of this process include microglia, astrocytes, and multiprotein complexes known as inflammasomes. Among these, the NLRP3 inflammasome is the most extensively studied and, upon activation, promotes the formation of active caspase-1. Caspase-1 converts the pro-inflammatory cytokines pro-interleukin-1β and pro-interleukin-18 into their active forms and induces pyroptosis through the cleavage of gasdermin D. Owing to its central role in numerous disorders, caspase-1 represents a promising therapeutic target for the development of novel drugs.
Based on the structure of the caspase-1 inhibitor CZL80, we designed and synthesized a series of novel 3-benzamidobenzoic acid derivatives bearing different electrophilic warheads and bioisosteric replacements of the carboxylate group. The carboxylate of CZL80 is positioned within the S1 pocket of the caspase-1 active site, and the compound itself does not form a covalent bond with the catalytic residue Cys285, therefore we investigated how modifications of the carboxylate structural element affect inhibitory activity and explored its potential conversion into covalent inhibitors. Compounds containing carbamoyl fluoride, vinyl sulfonamide, acrylamide, and nitrile warheads were synthesized, together with several analogues designed to examine the effect of substitution pattern on inhibitory activity. Using in silico modelling of the derivatives in complex with caspase-1, we investigated their predicted binding modes within the enzyme active site and evaluated the impact of structural modifications on binding poses and protein-ligand interactions. The inhibitory activity of the synthesized compounds was subsequently assessed in an in vitro biochemical assay using recombinant human caspase-1 and the fluorogenic substrate Ac-YVAD-AMC. The results demonstrated that inhibitory activity strongly depended on both the nature and position of the electrophilic warhead. Vinyl sulfonamides 22 and 29, and carbamoyl fluoride 28 were the most potent inhibitors of caspase-1. These findings indicate that 3-benzamidobenzoic acid derivatives represent a promising starting point for the further development of novel covalent caspase-1 inhibitors.
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