Caspase-6 is a cysteine protease from the family of effector caspases that is involved in programmed cell death – apoptosis, pathological axonal degeneration, and the development of neurodegenerative diseases. Increased caspase-6 activity in the brain, resulting from enzyme self-activation, is associated with early pathological changes in Alzheimer’s and Huntington’s diseases, in which the enzyme cleaves neuronal proteins. Inhibition of caspase-6 therefore represents a promising therapeutic strategy for slowing or preventing the progression of neurodegenerative diseases. Caspase-6 can be inhibited by various types of inhibitors that target eaither allosteric binding sites or catalytic amino acid residue (Cys163). The most recent approach targeting caspase-6 is based on covalent vinylsulfonamide inhibitors, which form a covalent thioether bond with the non-catalyic cysteine residue Cys264 in the active site of the enzyme.
As a part of the master’s thesis, various literature-known electrophilic warheads were introduced onto the piperidine nitrogen of (R)-piperidine-3-carboxamide. A total of 21 compounds were successfully synthesized, and their identities were confirmed by spectroscopic methods (nuclear magnetic resonance – NMR and high-resolution mass spectrometry – HRMS), while their purity was assessed by ultra-high-performance liquid chromatography. The compounds were evaluated by in vitro biochemical assay on recombinant human caspase-6 to determine residual activity, and for the most potent inhibitors, half-maximal inhibitory concentrations (IC50 values) were also established. The in vitro results showed that most of the synthesised analogues were inactive. The most potent inhibitors were the control inhibitors, vinyl sulfonamide 19 and its 5-phenyl analogue 25, carbamoyl fluoride 20, and chloroacetamide 36. Weaker inhibition of caspase-6 was observed for but-3-ynamide 33, propiolamide 32, and N-allylpiperidine 30. The results indicate that replacement of the vinyl sulfonamide warhead of (R)-piperidine-3-carboxamide significantly reduces inhibition. Further work should focus on determining whether the active derivatives inhibit caspase-6 via covalent or non-covalent mechanisms, followed by optimisation of caspase-6 inhibition, introduction of additional covalent warheads, and evaluation of selectivity versus structurally related caspases.
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