Caspase-6 is an apoptotic caspase involved in the activation of programmed cell death pathways and functions as an important regulator of innate immunity. Its activity is closely associated with pathological changes in neurodegenerative diseases, particularly in Alzheimer’s disease.
Within the master’s thesis, we developed an in vitro biochemical assay on microtiter plates, and identified novel inhibitors of the cysteine protease caspase-6, using a fragment-based drug design approach. In the first step, we determined the optimal conditions for caspase-6 activity, namely the concentration of the fluorogenic substrate based on Michaelis-Menten kinetics, as well as the effect of the solvent – dimethyl sulfoxide – on enzyme activity. As a reference compound, we used peptide inhibitor, N-acetyl-Val-Glu-Ile-Asp-CHO, and confirmed the reliability of the assay. Subsequently, we carried out screening of the fragment library to examine their ability to inhibit the enzymatic activity of caspase-6, which was expressed as residual activity. For compounds with the lowest values of residual activity, we further determined the concentration at which they inhibit half of the enzymatic activity, the IC50 value. In addition, for the most potent inhibitors, we monitored the course of substrate hydrolysis over time and assessed kinetic parameters that comprehensively describe their inhibitory potency. The results showed that fragments with electrophilic warheads, such as γ-butyrolactone, carbamoyl fluoride, aldehyde, and sulfonyl fluoride, strongly inhibit caspase-6. Among them are derivatives with nanomolar IC50 values, which have not yet been described as inhibitors of this enzyme. Based on the literature, caspases can be inhibited in various ways, either through non-covalent or covalent binding to the catalytic site, or via allosteric binding sites. To interpret the exact mechanism or the binding site of covalent fragments, mass spectrometry or X-ray crystallography would have to be performed.
In summary, we developed a robust in vitro assay that enabled the discovery of several covalent fragments with strong inhibition of caspase-6. These compounds represent promising candidates for further optimization and development of caspase-6 inhibitors that could be useful in the therapy of neurodegenerative diseases in which this protease plays a central role.
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