Mechanism of L,D-transpeptidase inhibition by β-lactams and diazabicyclooctanes Zainab Edoo To cite this version: Zainab Edoo. Mechanism of L,D-transpeptidase inhibition by β-lactams and diazabicyclooctanes. Microbiology and Parasitology. Sorbonne Université, 2019. English. NNT : 2019SORUS565. tel- 03173551 HAL Id: tel-03173551 https://tel.archives-ouvertes.fr/tel-03173551 Submitted on 18 Mar 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Sorbonne Université Ecole doctorale 515 « Complexité du Vivant » Laboratoire de Structures Bactériennes Impliquées dans la Modulation de la Résistance aux Antibiotiques Centre de Recherche des Cordeliers, UMRS 1138, Equipe 12 Mechanism of L,D-transpeptidase inhibition by β-lactams and diazabicyclooctanes. Mécanisme d’inhibition des L,D-transpeptidases par les β-lactamines et les diazabicyclooctanes. Par Zainab Edoo Thèse de doctorat de Biochimie Dirigée par Jean-Emmanuel Hugonnet Présentée et soutenue publiquement le 22 novembre 2019 Devant un jury composé de : Pr. Sandrine Betuing, Sorbonne Université Présidente Dr. Alain Baulard, Institut Pasteur de Lille Rapporteur Dr. Luiz Pedro Carvalho, Francis Crick Institute Rapporteur Dr. Célia Caillet-Saguy, Institut Pasteur Examinatrice Pr. Jean-Marie Frère, Université de Liège Examinateur Dr. Jean-Emmanuel Hugonnet, Sorbonne Université Directeur de thèse Acknowledgement I would like to start by thanking the members of my thesis jury: the rapporteurs, Alain Baulard and Luiz Pedro Carvalho, and the examinateurs, Sandrine Betuing, Célia Caillet-Saguy, and Jean-Marie Frère, for accepting to evaluate my work, and Jean-Emmanuel Hugonnet for supervising my PhD. I am grateful to the Fondation pour la Recherche Médicale for financial support and to the doctoral school for their assistance. Doing a PhD was harder than I thought but more rewarding than I could have imagined. This achievement would not have been possible without the support and guidance of many individuals. Thank you Jean-Emmanuel (Manu) for your unfailing encouragement during my thesis. You have been of great help and advice throughout the years. Thank you for believing in me! I am deeply grateful to the head of the lab, Michel Arthur. Thank you for your invaluable guidance and for being so generous with your time. I have learned so much from you. I would like to thank the members of my thesis committee, Houssain Benabdelhak, Thierry Touzé, Guennadi Sezonov, and Wladimir Sougakoff, for their feedback and warm encouragement. I had the great opportunity to collaborate with these wonderful people during my thesis: Mélanie Etheve-Quelquejeu, Laura Iannazzo, and Flavie Bouchet (Université de Paris); Ines Gallay and Herman van Tilbeurgh (Université Paris-Saclay); Catherine Bougault and Jean-Pierre Simorre (Institut de Biologie Structurale); Lionel Dubost (Muséum National d’Histoire Naturelle); Waldemar Vollmer (Newcastle University); Nadine Bongaerts, Edwin Wintermute, and Ariel Lindner (Centre de Recherches Interdisciplinaires); Landys Lopez Quezada and Carl Nathan (Weill Cornell Medicine). My sincere appreciation goes to the past and current members of Equipe 12. It has been a pleasure to work with all of you. Special thanks to Grazyna for keeping us in line and treating us with cakes, Sébastien for patiently walking me through enzyme kinetics during my Master’s internship, Matthieu for answering my chemistry-related questions, Delphine for her technical help, Antoine for the cheerfulness he brought to our office, Jean-Luc for his excellence, and David for being the best labmate. Cheers to my fellow PhD students with whom I shared the ups and downs of the last three years, and the interns, the youngest being 5-year-old Margot, who joined the lab for brief but fun periods. My heartfelt gratitude goes to the teachers who have inspired and encouraged me over the years. I am forever indebted to my family and friends, near and far. I have been truly blessed! You all hold a very special place in my heart. Henri, thank you for always managing to make me laugh! To my family – Thank you for your unwavering support and love. I owe it all to you. ZAK forever ♥ Foreword Antibacterial chemotherapy is one of the most essential contributions of medicine to human health in the last century. Ever since the discovery of the first antibiotic, penicillin, peptidoglycan biosynthesis has been one of the preferred targets for the discovery of new antibiotics. Peptidoglycan is an essential component of bacterial cells. It is composed of glycan chains, which are connected by short peptide stems. In most bacteria, the synthesis of the cross-links between the peptide stems is catalyzed by the Penicillin-Binding Proteins (PBPs). As their name implies, the PBPs are targeted by penicillin, which was the first discovered member of the β-lactam family. β-lactams have since been extensively developed for antibacterial activity against Gram-positive and Gram- negative bacteria. However, their use is threatened by numerous resistance mechanisms, the most important of which is the production of β-lactamases. The development of β-lactams that are not hydrolyzed by β-lactamases and of β-lactamase inhibitors have nonetheless contributed towards defeating emerging resistance. Peptidoglycan cross-links are not only formed by the classical PBPs but also by an unrelated family of enzymes, the L,D-transpeptidases (LDTs). This unusual mode of cross-linking is predominant in a few pathogenic bacteria, including Mycobacterium tuberculosis. PBPs and LDTs are structurally unrelated, function with different catalytic nucleophiles (Ser vs. Cys, respectively), and use different peptidoglycan precursors for the cross-linking reaction. In contrast to PBPs, which are potentially inactivated by all β-lactam classes, LDTs are efficaciously inactivated only by the carbapenem class. The two objectives of the thesis are to investigate the mechanism of inhibition of LDTs by carbapenems and to explore whether the diazabicyclooctane scaffold, recently developed for β-lactamase inhibition, also inactivates LDTs. The manuscript will begin with an introduction on the role and synthesis of peptidoglycan, the mode of action of β-lactams, and the catalytic activities of PBPs and LDTs. The introduction is followed by a description of the specific objectives of the thesis. The result section is based on published papers and is divided into two parts focused on the mechanism of inhibition of LDTs by β-lactams and diazabicyclooctanes. The last section consists of a discussion on the implications of the findings regarding the catalytic mechanism of LDTs for optimization of inhibitors. In parallel to the main objectives of the thesis, I contributed to two studies investigating the LDTs from Clostridium difficile and inhibition of LDTs by copper(II) ions. These two publications appear in an annex at the end of the manuscript. 3 Table of Content ACKNOWLEDGEMENT 2 FOREWORD 3 TABLE OF CONTENT 4 LIST OF FIGURES 6 LIST OF TABLES 7 LIST OF ABBREVIATIONS 8 INTRODUCTION 9 1. PEPTIDOGLYCAN BIOSYNTHESIS 10 1.1 ROLE OF PEPTIDOGLYCAN 10 1.2 PEPTIDOGLYCAN STRUCTURE AND SYNTHESIS 11 1.3 ANTIBIOTICS TARGETING PEPTIDOGLYCAN SYNTHESIS 27 2. THE β-LACTAM ANTIBIOTICS 28 2.1 MODE OF ACTION OF β-LACTAMS 28 2.2 CLASSES OF β-LACTAMS 29 2.3 RESISTANCE TO β-LACTAMS 32 3. β-LACTAMASES 34 3.1 CLASSIFICATION OF β-LACTAMASES 34 3.2 β-LACTAMASE INHIBITORS 37 4. PENICILLIN-BINDING PROTEINS 41 4.1 CATALYTIC MECHANISM 42 4.2 CLASSIFICATION OF PBPS 45 4.3 INHIBITION OF PBPS BY β-LACTAMS 50 5. L,D-TRANSPEPTIDASES 53 5.1 IDENTIFICATION OF LDTS 53 5.2 ROLE OF LDTS IN VARIOUS BACTERIA 56 5.3 STRUCTURE OF LDTS 64 5.4 CATALYTIC MECHANISM OF L,D-TRANSPEPTIDATION 68 5.5 INHIBITION OF LDTS BY β-LACTAMS 70 OBJECTIVES 81 RESULTS 82 1. MECHANISM OF INHIBITION OF L,D-TRANSPEPTIDASES BY β-LACTAMS 83 PUBLICATION 1 85 PUBLICATION 2 85 PUBLICATION 3 85 2. INHIBITION OF L,D-TRANSPEPTIDASES BY A NOVEL DIAZABICYCLOOCTANE SCAFFOLD AND MODE OF ACTION OF THE MOLECULES IN M. TUBERCULOSIS 87 PUBLICATION 4 89 4 DISCUSSION 91 1. MODELS ACCOUNTING FOR ACYLATION OF L,D-TRANSPEPTIDASES BY β-LACTAMS 92 2. BASIS FOR FLUORESCENCE QUENCHING OBSERVED UPON L,D-TRANSPEPTIDASE ACYLATION BY β-LACTAMS 95 3. THE REACTIVITY OF β-LACTAMS HAS A KEY ROLE IN THE EFFICACY OF L,D-TRANSPEPTIDASE ACYLATION 95 4. MULTIPLE POTENTIAL TARGETS OF DBOS IN M. TUBERCULOSIS 96 5. OPTIMIZATION OF INHIBITORS TARGETING L,D-TRANSPEPTIDASES 100 ANNEX 102 ANNEX 1 103 ANNEX 2 103 REFERENCES 104 5 List of Figures Figure 1. Structure of the bacterial cell wall. ........................................................................................ 10 Figure 2. The peptidoglycan subunit and its synthesis. ........................................................................ 12 Figure 3. Common variations found in the peptidoglycan subunit of E. coli, E. faecium, and M. tuberculosis. .......................................................................................................................................... 15 Figure 4. Structure of MurJ and the conformational