Development of novel small-molecule inhibitors of human tissue transglutaminase

Abstract

Tissue transglutaminase (TG2) is the most ubiquitous enzyme among Ca2+-dependent crosslinking-protein enzyme family that catalyses the acyl-transfer reaction between the ɣ-carboxamide group of a peptide-bound glutamine and the Ɛ-amino group of a peptide-bound lysine or a primary amine. The adventitious cross-linking related to unregulated TG2 activity has been implicated in various pathologies. This research focused on the development of both allosteric and irreversible TG2 inhibitors.

LDN27219, a published lead TG2 allosteric inhibitor, whose binding site was predicted by the previous computational modelling, was modified to develop potential allosteric inhibitors. Molecular dynamics (MD) simulations were applied for the complexes of the parent compound and newly designed compounds docked into the predicted allosteric site. Attempts were made to prepare the designed compounds from ethyl 4-oxo-5-phenyl-3H-thieno[2,3-d]pyrimidine-2-carboxylate derivatives. However, the key literature method failed to provide the desired esters. Many attempts were made and the optimised reaction conditions provided the pure desired products in 83% yield, followed by the discovery of novel methods to synthesise 5-phenyl-3H-thieno[2,3-d]pyrimidine-4-ones in up to 84% yields. Consequently, a set of potential allosteric inhibitors was prepared and screened in vitro against human TG2. The highest potency was seen in acylhydrazide derivatives, two of which are nearly two times more potent than the parent compound. In the MD simulations, the binding of the potent inhibitors were observed to stabilise the open conformation of TG2 but also enlarge the catalytic site’s entrance and cause the moving of TRP241, which stabilises the enzyme-thiol intermediates, from the catalytic binding site.

Potential irreversible TG2 inhibitors were designed by modifications of the lead inhibitor developed by the Griffin group (Aston University) including attachment of a carboxyl group on the acrylamide warhead, shortening the aliphatic side chain length, inserting a phenyl ring between the piperazine ring and the acrylamide warhead and changing the whole scaffold structure. The parent and the designed compounds docked into the TG2 catalytic binding site were subjected to MD simulations. The affinity and potency of the ligands were predicted based on stability of the ligand into the catalytic site and the distance between the warhead electrophilic carbon and the catalytic cystein sulphur. A set of acrylamides with short side chains and (2Z)-4-amino-4-oxo-2-butenoic acid derivatives were prepared and tested against human TG2. All the newly synthesised acrylamides are significantly less potent than the parent compound and the potency was halted by the attachment of the carboxyl on cis position of the acrylamide warhead.

Date of Award	Mar 2020
Original language	English
Awarding Institution	Aston University
Supervisor	Dan Rathbone (Supervisor) & Martin Griffin (Supervisor)