Docking-Based 3D-QSAR Studies for 1,3,4-oxadiazol-2-one Derivatives as FAAH Inhibitors

This work aimed to construct 3D-QSAR CoMFA and CoMSIA models for a series of 31 FAAH inhibitors, containing the 1,3,4-oxadiazol-2-one moiety. The obtained models were characterized by good statistical parameters: CoMFA Q2 = 0.61, R2 = 0.98; CoMSIA Q2 = 0.64, R2 = 0.93. The CoMFA model field contributions were 54.1% and 45.9% for steric and electrostatic fields, respectively. In the CoMSIA model, electrostatic, steric, hydrogen bond donor, and hydrogen acceptor properties were equal to 34.6%, 23.9%, 23.4%, and 18.0%, respectively. These models were validated by applying the leave-one-out technique, the seven-element test set (CoMFA r2test-set = 0.91; CoMSIA r2test-set = 0.91), a progressive scrambling test, and external validation criteria developed by Golbraikh and Tropsha (CoMFA r20 = 0.98, k = 0.95; CoMSIA r20 = 0.98, k = 0.89). As the statistical significance of the obtained model was confirmed, the results of the CoMFA and CoMSIA field calculation were mapped onto the enzyme binding site. It gave us the opportunity to discuss the structure–activity relationship based on the ligand–enzyme interactions. In particular, examination of the electrostatic properties of the established CoMFA model revealed fields that correspond to the regions where electropositive substituents are not desired, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one moiety. This highlights the importance of heterocycle, a highly electronegative moiety in this area of each ligand. Examination of hydrogen bond donor and acceptor properties contour maps revealed several spots where the implementation of another hydrogen-bond-donating moiety will positively impact molecules’ binding affinity, e.g., in the neighborhood of the 1,3,4-oxadiazol-2-one ring. On the other hand, there is a large isopleth that refers to the favorable H-bond properties close to the terminal phenoxy group of a ligand, which means that, generally speaking, H-bond acceptors are desired in this area

Développement de nouvelles approches protéo-chimiométriques appliquées à l'étude des interactions et de la sélectivité des inhibiteurs de kinases

The human kinome contains 518 proteins. They share a common mechanism of protein phosphorylation known to play an important role in cellular signaling pathways. Impaired kinase function is recognized to be involved in severe diseases like cancer. Due to high structural similarity between protein kinases, development of potent and selective kinase inhibitors is a challenging task. The selectivity of kinase inhibitors may lead to side effects potentially harmful. In this thesis, we first developed new selectivity metrics to determine inhibitor selectivity directly from biological inhibition data. Compared to existing metrics, the new selectivity scores can be applied on diverse inhibition data types. Second, we developed a proteometric approach in order to understand why some protein kinases are never inhibited by Type II inhibitors. The statistical model built for this purpose allowed us to identify several discriminant residues of which few of them correspond to experimentally described residues of interest. Third, using a new 3D protein kinase descriptor, we developed and validated novel proteo-chemometrics approaches to study and discover new kinase inhibitors.Le kinome humain comprend 518 protéines. Elles participent au processus de phosphorylation des protéines qui joue un rôle important dans les voies de signalisation cellulaire. Leur dérégulation est connue comme étant une cause de nombreuses maladies graves telle que les cancers. Du fait de leur grande similarité structurale des protéines kinases, il est difficile de développer des inhibiteurs qui soient à la fois efficaces et sélectifs. L’absence de sélectivité conduit le plus souvent à des effets secondaires particulièrement néfastes pour l’organisme. Au cours de cette thèse, nous avons d’abord développé de nouvelles métriques dont le but est de déterminer la sélectivité d’inhibiteurs à partir de données d’inhibition. Elles présentent l’avantage, comparées à d’autres métriques, d’être applicables sur n’importe quel type de données. Dans un deuxième temps, nous avons développé une approche protéométrique dans le but de comprendre pourquoi certaines protéines kinases ne sont jamais inhibées par des inhibiteurs de Type II. Le modèle statistique mis en place nous a permis d’identifier plusieurs résidus discriminants dont certains déjà décrits expérimentalement dans la littérature. Dans un troisième temps, nous avons développé un nouveau descripteur 3D de protéines kinases avec lequel nous avons mis en place et validé des modèles protéo-chimiométriques visant à étudier et découvrir de nouveaux inhibiteurs

Studying the Conformational Changes of Protein Kinases Using In Silico Structural Approaches

Protein kinases were brought to the scientific community's attention with the remarkable approval of imatinib more than 20 years ago. Since then, enormous efforts have been made to identify, characterise, and investigate the dynamic and function of this broad protein family. Nowadays, protein kinases are associated with numerous human diseases, including the origin of cancer and beyond.Extensive research of protein kinases led not only to the steady development and approval of new inhibitors and therapeutics but also to the awareness that there are still plenty of questions to be answered about these dynamic proteins. Hence, the In Silico methods come in hand to study their highly dynamic nature. Offering a wide range of novel computational techniques, they provide a possibility to lift the veil of secrecy and glance into the minor details of kinase behaviour. The presentation will cover the introduction to the main research focus – protein kinases, and the central methodology – computer-aided drug design. Firstly, the classification, function, drug discovery trends and a detailed review of the kinase domain’s structural features will be presented. Next, the In Silico drug discovery methods, encompassing the sample workflows and analyses with the main emphasis on the theory and application of molecular dynamics will be discussed. The application of the computational chemistry approaches in kinase drug research will be illustrated in two scientific publications. The first is dedicated to the application of long-scale molecular dynamics in understanding the impact of phosphorylation and mutation on the autoinhibition and dimerization of mitogen- activated protein kinase kinase 4 (MKK4). The second publication focuses on the investigation of the statistical trends and patterns related to protein kinase regulatory hydrophobic spine (R-spine), emphasizing the alfa- helical hydrophobic spine residue three (RS3). The aforementioned publications demonstrate the successful application of In Silico approaches, particularly molecular dynamics in the field of drug discovery, and furthermore offer a framework modification for the investigation of geometrical motions within the protein structures during molecular dynamics simulation

Small Molecule Investigation of KCNQ Potassium Channels: A Dissertation

Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels