Computational investigation of small-molecule human tissue transglutaminase inhibitors

Abstract

Human tissue transglutaminase (TG2) catalyses transamidation and deamidation reactions through a nucleophilic cysteine residue (CYS277). TG2 activity was found to increase in celiac disease, cystic fibrosis, neurodegenerative disorders and cancer. For this, TG2 has received much focus as a target for drug discovery and many inhibitors have been designed and tested. The most important of these have an electrophilic warhead that reacts covalently with CYS277 resulting in an irreversible inhibition of TG2. The work presented in this thesis aimed at the development of computational methods that could aid in the design and testing of potential TG2 inhibitors. 3-D models of TG2 active site were developed starting from published X-ray crystal structures by means of docking experiments with known irreversible inhibitors followed by molecular dynamics (MD) simulations. The models were validated by additional docking runs and MD simulations involving a larger set of compounds with a range of activities against TG2. The models performed reasonably well in the validation process and were, therefore, chosen as active site models of TG2. No straightforward correlation could be found to rank the compounds based on their activities. This was the rationale for the next stage of the work, where the mechanism of inhibition of TG2 by two classes of inhibitors was studied. The covalent-bond-forming events for the inhibitors bearing acrylamide warheads were followed by applying quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations to the reaction. The produced activation energies correlated well with the biological activities for the inhibitors and a mechanism with an oxyanion intermediate was proposed. The mechanism of inhibition by compounds having sulfonium ion warheads was investigated using reaction path experiments, where a transition state was first identified and verified and was used as a starting point for the reaction path. The activation energies again produced a reasonable correlation with biological activity and an SN2 mechanism was suggested for this inhibition.On a different level, two allosteric inhibitors proposed in the literature were docked into an allosteric site in TG2 predicted by a collaborator from the University of Strathclyde, and docking complexes were subjected to accelerated MD (aMD) to inspect whether the binding would induce significant conformational changes in TG2. The binding of one inhibitor in the predicted site caused bending in TG2 structure that could be a starting event for complete TG2 inactivation. The other inhibitor seemed to produce a similar effect when bound to the original GDP binding site. An even more profound conformational change was reported due to the binding of GDP in its original binding site. aMD, for the simulation times used (400-1000 nanoseconds), was able to represent some large conformational changes in TG2 brought about by the binding of allosteric inhibitors. To sum up, the work presented in this thesis was successful in applying various computational approaches to the analysis of inhibition of TG2 with irreversible and allosteric inhibitors.