Emergence of resistance to multiple antibiotics requires the indentification and chartacterization of new targets for drug developement. The aim of our project is to characterize a promising target for the tretament of multidrug resistant tuberculosis
Tuberculosis is a re-emerging infectious disease caused by the bacterium Mycobacterium tuberculosis. Usually treated by the association of rifampin, isoniazide, pirazinamide, and ethambutol for six months, problems resulting from poor adherence to therapy by the patients in developing countries have resulted in the emergence of multidrug resistant (MDR) extensively drug resistant (XDR) strains. Since the patients infected by the latter strains are in a therapeutic deadlock, there is an urgent need to develop new active molecules. <br />We have recently shown that the cell wall of M. tuberculosis is polymerized by a novel enzyme family, the L,D-transpeptidases, and that this enzyme family is inactivated by carbapenems, a class of antibiotics belonging to the beta-lactam family (which also includes penicillin). Since these unexpected observations open up new prospects for the treatment of XDR-TB, our project aims at characterizing the molecular mechanism of L,D-transpeptidase inactivation by beta-lactams. The challenge here is to understand the molecular bases of target inactivation to generate the knowledge base required for the development of new drugs. <br />
Our consortium is composed of four teams specialized in the study of the bacterial cell wall and in the enzymes involved in its synthesis (biochemistry), in the synthesis of bioactive molecules (organic chemistry), in the determination of protein structure and dynamics (by NMR), and in silico modeling of chemical and enzymatic reactions (biophysics).
In the first six months of the contract, we have developed the methods required to achieve the aims of the project, including the methods required for purification of large amounts of L,D-transpeptidases and the development of experimental and computational tools for kinetic analyses of target inactivation and demonstration of its irreversible nature. We have also developed the synthesis routes for obtaining molecules that will be used to investigate the mechanism of inactivation of L,D-transpeptidases by beta-lactams.
Among infectious diseases, tuberculosis (TB) remains the second infectious disease leading to mortality, after AIDS, despite the World Health Organization (WHO) programs. It is estimated that one third of the world’s population is infected with M. tuberculosis. According to the WHO report 2011, there are 8.8 million new cases and 1.45 million deaths in 2010. Inappropriate use of essential antituberculosis agents leads to emergence of bacilli resistant to one or more of these drugs and widespread dissemination of these bacilli represents an obstacle to the control of tuberculosis. In 2010, the WHO estimated that there were 650,000 new cases of infections due to multidrug resistant M. tuberculosis (MDR-TB). Unfortunately, the extensive use of second-line drugs has led to the emergence of extensively drug resistant M. tuberculosis (XDR-TB) that show a very poor prognosis with mortality rates as high as 65 to 100%, due to the lack of an efficient therapy. Except for fluoroquinolones, all drugs used to treat tuberculosis have been approved more than 45 years ago, illustrating the complexity and of TB drug development. The aim of our project is to generate the knowledge base required for the development of new drugs belonging to the beta-lactam family.
Publication of the first results of consortium is anticipated for the end of the second year of the contract.
Beta-lactams are the most widely used drugs for treatment of bacterial infections. The family comprises the penicillins, cephalosporins, and carbapenems, which have been developed to combat resistance. Until recently, all beta-lactams were thought to exclusively act on the active-site serine D,D-transpeptidases (PBPs) that catalyze the final cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Recently, the team of the coordinator has shown that these D,D-transpeptidases can be by-passed by a novel class of enzymes, the L,D-transpeptidases (Ldt), in beta-lactam-resistant enterococci and in Mycobacterium tuberculosis. These enzymes are promising targets for development of anti-tuberculous drugs belonging to the carbapenem class since the peptidoglycan of M. tuberculosis is predominantly cross-linked by L,D-transpeptidases, which are inactivated by these beta-lactams. In addition, carbapenems are bactericidal against both replicative and ‘dormant’ forms of the bacilli in association with clavulanic acid, which inactivates the beta-lactamase. There is an urgent medical need to discover efficient therapies to eradicate ‘dormant’ bacilli, which are only eliminated by prolonged (=6-months) polychemotherapy involving toxic drugs, and to combat the emergence of extensively-resistant strains, which are increasingly responsible for therapeutic failure in developed countries with mortality rates as high as 50%. The main objective of the proposal is to understand the molecular mechanism of inactivation of L,D-transpeptidase LdtMT1 from M. tuberculosis by beta-lactams of the carbapenem class in a multidisciplinary consortium with expertise in biochemistry, organic synthetic chemistry, structural biology, and theoretical chemistry. Preliminary results obtained by members of the consortium have shown that L,D-transpeptidases are inactivated by formation of an acylenzyme in a two-step reaction involving formation of a non-covalent complex prior to acylation of the active site cysteine residue of the enzyme. In addition, 3D NMR structures have revealed that acylation is associated with substantial conformational alteration and increased flexibility of the enzyme. Our first specific objective will be to synthesize various carbapenems in order to decipher the role the side chains of the drugs have for binding and proper orientation of the beta-lactam ring within the active-site. A second aspect concerns the reactivity of the bi-cyclic carbapenem ring. Theoretical chemistry will be used to model the path of the reaction and propose hypotheses for the catalytic mechanism that will be tested by modification of active site residues of the enzyme and the use of beta-lactams with different bi-cyclic rings. In the third objective, we will explore by NMR the structure and dynamics of the enzyme to assess the role played by conformational flexibility in the efficiency of the acylation reaction. The last part of the project will concern comparison of the inhibitor and the physiological substrates to determine how carbapenems mimic the peptidoglycan precursors and to understand the basis for the stability of the drug-LdtMt1 adduct, which is essential for antibacterial activity. Together, these analyses will provide insight into the mechanism of action of beta-lactams on a novel type of targets that have not been previously investigated at the molecular level. Although drug development is not an objective of the current proposal, the knowledge base built on LdtMt1 will clearly be an asset for rational design of L,D-transpeptidase inhibitors. In a broader perspective, structural modification of naturally occurring beta-lactams and beta-lactones is thought to be a highly effective strategy for generating drugs for treating cancer, obesity, and hyperlipidemia. Our project will contribute to further the understanding of the features required to combine high affinity, chemoselective reactivity, and high stability of the acylated target protein.
Monsieur MICHEL ARTHUR (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION REGIONALE PARIS VI) – email@example.com
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UNIVERSITE DE PARIS DESCARTES UNIVERSITE PARIS V
CEA, CNRS, Univ. Joseph Fournier CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
UNIVERSITE PARIS VI [PIERRE ET MARIE CURIE]
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION REGIONALE PARIS VI
Help of the ANR 569,998 euros
Beginning and duration of the scientific project: June 2011 - 48 Months