Identification of Allosteric Inhibitors against Active Caspase-6

Agne Tubeleviciute-Aydin,Ludovic J Deny,Anne Marinier,Andrea C Leblanc,Jeffrey Lynham,Alexandre Beautrait,Naoto Soya,Gyanesh Sharma,Gergely L Lukacs,Bhushan Nagar,Alexei Gorelik

doi:10.1038/s41598-019-41930-7

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.

Highlights

Caspases belong to a highly conserved family of cysteine proteases that have essential roles in programmed cell death, inflammation, differentiation, neuronal development and axon pruning[1]
Like Casp6-WT, Casp[6] variants, underwent complete self-processing at TETD23, and TEVD193 sites, and slight self-cleavage at the DVVD179 site, generating mainly large subunit with a linker (LS-L), small subunit (SS), and a minor amount of large subunit (LS) (Fig. 1c,d)
Casp[6] variants were catalytically active against Ac-VEIDAFC but Casp6-A34E, Casp6-E35K, and Casp6-T182S, displayed lower activity than Casp6-WT in enzyme concentration-dependent manner (Fig. 1e) whereas the activity of Casp6-A109T was comparable to Casp6-WT

Summary

Introduction

Caspases belong to a highly conserved family of cysteine proteases that have essential roles in programmed cell death, inflammation, differentiation, neuronal development and axon pruning[1]. Knock-in mice that express a self-activated form of human Casp[6] in the CA1 region of the hippocampus display age-dependent spatial and episodic memory impairment, in the absence of NFTs and amyloid plaques[23]. Substrate mimetic peptide inhibitors were derived from the Casp[6] cleavage site in transcription factor activator protein 2 isoform alpha[32], lamin A33, Huntingtin (Htt)[34], and synthetic caspase peptide substrates[35]. Most of these inhibitors contain an electrophilic warhead, aldehyde, fluoromethylketone or methyl vinyl sulfone[36], which reacts with a catalytic cysteine. The catalytic site is highly conserved among the caspases, which challenges the development of highly selective Casp[6] active site inhibitors

Methods

Results

Conclusion