Alzheimer’s disease represents one of the greatest challenges in modern medicine, with neuroinflammation playing a key role in its onset and progression. Caspase-1 is an enzyme that activates the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18), and has emerged as a promising therapeutic target, as its inhibition may reduce neuroinflammation and thereby slow the disease progression. In this master’s thesis, we focused on the development of novel covalent caspase-1 inhibitors based on the core structure of belnacasan (VX-765). Studies in mice have shown that belnacasan exhibits neuroprotective effects and improves episodic and spatial memory.
Various bioisosteric replacements of the aspartate carboxylate were introduced onto belnacasan core. Additionally, a carbamoyl fluoride warhead was incorporated into the secondary amine. Using established synthetic procedures, we synthesized and isolated 12 compounds, confirming their identity and purity through appropriate analytical methods. The biological activity of the compounds was evaluated in vitro using recombinant human caspase-1. Following a 30-minute incubation with the inhibitors, enzymatic activity was measured using the fluorogenic substrate Ac-YVAD-AMC and expressed as a percentage of enzyme’s residual activity. The evaluation revealed that the synthesized compounds generally did not inhibit caspase-1, with the most potent inhibitor being a mixture of carbamoyl fluorides 43 and 44, which exhibited 62.1% residual activity at a concentration of 100 μM. Molecular docking indicated that some carboxylate mimetics could successfully occupy the S1 pocket of the enzyme, however, this did not translate into effective inhibition of caspase-1 enzymatic activity.
Our research has demonstrated that the successful development of covalent inhibitors requires a precise balance between structural complementarity, warhead electrophilicity, and the preservation of key interaction points. In the future, it would be reasonable to retain the (S)-prolinamide scaffold and focus on further optimization of bioisosteric approaches that promote both strong binding and improved selectivity over other caspases. Although our compounds did not inhibit caspase-1, the results of this work represent an important step toward the development of effective inhibitors of this pro-inflammatory caspase, which is highly relevant to neurodegenerative diseases.
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