Computational Study on the Reactivity and Inhibition of Arginine Gingipain B, a Potential Target for the Treatment of Alzheimer's Disease

Resumen

Since its identification in 1901 by the German psychiatrist Alois Alzheimer, Alzheimer's disease has emerged as one of the greatest challenges in pharmaceutical research. Being the most common type of dementia, it presents severe effects such as loss of memory, cognitive abilities and physical dexterity, symptoms that occur mainly in the elderly population. Classified as a multifactorial disease, the attempt to develop a treatment for Alzheimer's disease has transited over a large number of molecular targets. In 2019, the cysteine protease RgpB was identified as a possible new pharmacological target for the development of neuroprotective treatments using small drugs-like molecules. In fact, a family of irreversible inhibitors was discovered and patented, and they are nowadays in advanced stages of clinical trials. All of the above highlights the need for a thorough and detailed understanding of possible targets for the design of drugs for Alzheimer's disease treatments. This thesis represents an effort to shed light on atomic-level details of the mechanisms by which the cysteine protease RgpB works and the processes of its inhibition by small-molecules drugs candidates. For this purpose, a comprehensive computational study combining methods based on classical molecular dynamics and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics was carried out. These methods were employed in different methodological schemes to obtain reaction free energies and binding free energies. This, allowing us to elucidate the mechanism by which the proteolysis reaction proceeds on RgpB, to characterize non-covalent interaction profiles between putative inhibitors and RgpB and to unravel the covalent binding mechanisms of such irreversible inhibitors. In more detail, a preliminary step using classical molecular dynamics and principal component analysis (PCA) determined the most likely protonation state of the Cys/His catalytic dyad of RgpB and its influence in promoting the reactive arrangement of catalytic residues and peptide. Next, umbrella sampling QM/MM calculations were employed to evaluate all possible pathways of RgpB-catalyzed proteolysis. The most favorable mechanism was found to proceed through three steps. For acylation, initially, the sulfur atom of the Cys244 residue attacks the carbonyl carbon of the peptide and the proton of the Cys244 residue is transferred to the amino group of the peptide in a concerted manner. Then, the peptide bond is cleaved and a fragment is released. Finally, in the deacylation step a water molecule attacks the carbonyl carbon of the peptide and a proton from the water is transferred to the Cys244 residue. For the mechanism found, the rate-limiting step free energy barrier is in very good agreement with the available experimental evidence. Notably, it should be emphasized that our hypothesized mechanism shows an unusual role of the His211 residue and a crucial role of the peptide in activating catalysis. For the study of the inhibition of RgpB, a set of previously reported irreversible inhibitors was used as a reference. From these, by means of alchemical transformations and Poisson-Boltzmann surface area molecular mechanics (MM/PBSA) calculations supplemented with interaction entropies, the binding energies were calculated. The interactions governing the affinity of the inhibitors to the binding pocket were characterized by means of contact maps and interaction energies decomposed by residue. Notably, residues Ser213, Glu214, Asp158 and Asp281 were found to crucially interact via hydrogen bonds with the inhibitors. Finally, following the same methodology as for the wild-type substrate, the reaction mechanism by which these drug candidates lead to covalently bound complexes inhibiting the RgpB protease was evaluated. This process is performed in a single step, that consists of the activation/deprotonation of Cys244 by the oxygen atom of the carbonyl group concertedly with the attack of the Sγ:Cys244 atom to the carbon atom of the same group. In a final stage of the thesis, and in order to explore methodologies to enrich our findings on the activity and inhibition of RgpB, the use of metadynamics in enzyme reactivity studies was performed. This incursion was carried out on the investigation of the ATPase activity of Prp2 and its RNA regulation. We found that hydrolysis proceeds by a four-step mechanism in which the rate-determining step is the nucleophilic attack on the gamma phosphate. Complementary, MD simulations disclosed the molecular terms of RNA-driven activation of ATP hydrolysis. These results demonstrated the potential and applicability of DFT-based metadynamics in the study of biological systems, and in particular on systems that lower-level Hamiltonians can represent a source of error during the conformational sampling. All in all, the work presented in this thesis contributes to the understanding of the reaction mechanism and inhibition of an enzyme that is a potential target for the treatment of Alzheimer's disease. This understanding will allow to rationalize and optimize future inhibitors in the pharmaceutical race for the development of new potential treatments.