ABSTRACT
Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by in vitro testing of the best virtual hits in recombinant human Caspase-6 activity assays identified novel allosteric Caspase-6 inhibitors with IC50 and Ki values ranging from ~2 to 13 µM. This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.
Subject(s)
Caspase 6/chemistry , Caspase 6/metabolism , Caspase Inhibitors/pharmacology , Mutation, Missense , Small Molecule Libraries/pharmacology , Allosteric Regulation/drug effects , Amino Acid Substitution , Caspase 6/genetics , Caspase Inhibitors/chemistry , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Humans , Models, Molecular , Polymorphism, Single Nucleotide , Protein Binding , Protein Conformation , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator that promotes the transcription of cytoprotective genes in response to oxidative/electrophilic stress. Various Michael-type compounds were designed and synthesized, and their potency to activate the Keap1/Nrf2/ARE pathway was evaluated. Compounds bearing two Michael-type acceptors proved to be the most active. Tether length and rigidity between the acceptors was crucial. This study will help to understand how this feature disrupts the interaction between Keap1 and Nrf2.
