Caspase-1 is an inflammatory cysteine protease that is essential for the proteolytic activation of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), and therefore represents an important target aimed at preventing pathological inflammation and neuroinflammation. Covalent inhibitors can, in comparison with non-covalent inhibitors, achieve prolonged enzyme inhibition when equipped with an appropriately selected electrophilic warhead. Their selectivity and efficacy depend on the proper positioning of the ligand within the active site.
In this master’s thesis, we designed and synthesized a series of inhibitors bearing carbamoyl fluoride warheads, systematically varying the size and nature of substituents, and investigating their impact on the inhibition of caspase-1. Reaction products were isolated using normal-phase and reversed-phase chromatography, and their identity was confirmed by mass spectrometry and nuclear magnetic resonance. For nine final compounds, carbamoyl fluorides 13–18, 42, 47 and 50, an in vitro biochemical assay using human, recombinant caspase-1 was used to evaluate how structural modifications affected the inhibitory activity. Results were expressed as residual caspase-1 activity (RA) at compound concentrations of 100 µM and 10 µM. To gain deeper insight into binding and interactions between ligand and caspase-1, molecular docking was also performed. Biochemical evaluation showed that among the tested compounds, indoline derivative 42 was the most potent inhibitor of caspase-1 (RA = 10.5% at 100 µM), followed by indoline 50 (RA = 33.9% at 100 µM). The pyrrolidine and piperidine analogues 13–18 were practically inactive, suggesting that compared to indoline derivatives, the absence of phenyl moiety limits the effective positioning of the warhead toward the catalytic cysteine Cys285. Docking of selected compounds 42, 47 and 50 showed deviations from the expected binding poses and the positioning of functional groups within the S1 and S4 binding pockets were observed. Molecular docking therefore did not fully elucidate the relationship between the structural features and the inhibitory activity of the compounds. In subsequent studies, it would be appropriate to perform molecular dynamics simulations, which could provide insight into the dynamics of the binding interactions. These findings support the initial premise that, in addition to the choice of electrophilic warhead, appropriate noncovalent anchoring and complementarity of the remainder of the molecular scaffold are critical determinants of covalent inhibitor performance.
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