Development Of New Chemotherapeutics For Tuberculosis

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Currently this project focusses on five key areas: (1) the chemical synthesis of derivatives of a natural product called thiolactomycin that targets an enzyme involved in the biosynthesis of the mycobacterial cell wall, (2) the chemical synthesis of analogs of nitroimidazoles such as PA-824, (3) the synthesis and evaluation of inhibitors of synthesis of the mycobacterial siderophore, Mycobactin, (4) the synthesis and evaluation of substrates for, and inhibitors of, transpeptidases that are responsible for remodeling the TB cell envelope, and (5) the synthesis of inhibitors of NAD synthetase. Project (1) targets mycolic acids which are complex alpha-branched, beta-hydroxy fatty acids that are unique to mycobacteria which are heavily modified by a variety of functional groups. Mycolic acids are biosynthetically produced through an extension of normal fatty acid metabolism. In mycobacteria this is initiated by a "eukaryotic"-like Type I fatty acid synthase, a large multifunctional enzyme that produces primarily short-chain (16-24 carbons) fatty acids that are then substrates for a second fatty acid synthase system that is more typically associated with bacteria. This Type II system appears to be the molecular target for isoniazid as well as other inhibitors such as triclosan. Thiolactomycin is a low molecular weight natural product isolated from a soil Nocardia species that specifically inhibits one component of the bacterial Type II fatty acid synthase system. Although it is a modest inhibitor against most bacteria it has shown in vivo activity in various experimental infections of animals. Studies this year, however, have uncovered some serious metabolic stability issues with the core thiolactone ring that have resulted in termination of this series of molecules. In Project (2) we are synthesizing analogs of nitroimiadazooxazines and nitroimidazooxazoles related to PA-824. PA-824 is currently in Phase II studies in humans for the treatment of tuberculosis. Working with scientists at the Novartis Institute for Tropical Diseases, TBRS scientists have synthesized and characterized a large collection of analogs of these compounds and are advancing these through preclinical studies. Two candidate molecules were identified this year using computational methods (QSAR modeling) and were synthesized and found to be extremely potent (fourty fold better than PA-824) with improvements noted in solubility and toxicological profiling. These are currently in advanced preclinical testing and may advance to IND-enabling preclinical studies within the first quarter of 2009. In addition the enzyme responsible for activating these drugs has been conclusively identified and the mechanism of the drug class has been established. In this work we established the mechanism of action of PA-824 by synthesis of analogs and metabolites and in the process discovered an entire new family of enzymes and an entirely novel and unexpected chemical mechanism of drug action. These agents function as "Trojan horses" and intracellularly release Nitric oxide (NO) after activation by a bacterial enzyme. NO is a molecule intimately involved in natural human defense against tuberculosis and many other pathogens and this project suggests a new general paradigm for antibacterials development, as a result this work was just accepted for publication in Science. There are also immediate practical applications, in collaboration with scientists at the Genomics Institute of the Novartis Foundation (GNF) who have reengineered this protein into a soluble form we hope to have preliminary crystals that diffract x-rays for structural work. We have synthesized the deazaflavin cofactor of this enzyme in mg quantities (a 14 step synthesis) for cocrystalization studies and hope to initiate structure-guided synthesis of analogs this year. In Project (3) we are evaluating approaches to the inhibition of the biosynthesis of the iron-acquiring siderophore of Mycobacterium tuberculosis, Mycobactin. In collaboration with scientists at the Unvieristy of Minnesota's Center for Drug Design we are testing inhibitors of one of the earliest biosynthetic steps in this biosynthetic pathway. Targettinng iron acquisition builds upon a strong historical interest in TBRS in understanding the biosynthesis of this molecule which is required for bacterial growth during infection. we have designed and synthesized many analogs and several are being tested in animal models presently. In project (4) we are exploring the biological role of a unique family of L,D-transpeptidases that are thought to contribute to the inherent beta-lactam resistance of TB. By a combination of biochemical and chemical studies we have established the function of two of the five members of this family and we have synthesized a panel of inhibitors and substrates in the hopes of both elucidating the underlying biology/enzymology as well as developing proof of concept molecules for a TB-specific family of penicillin-like compounds. Working with scientists at the Albert Einstein College of medicine who have solved the x-ray Crystal structure of the TB Beta lactamase in complex with clavulanic acid we have now also initiated a structure-guided program for designing better inhibitors. In project (5) we are working with the Gerratana laboratory who have solved the x-ray crystal structure of NadE which performs the last step of Nicotinamide biosynthesis to design and synthesize inhibitors of this enzyme. Both computation and structure-based approaches are being applies to elaborate fragments bound at the enzyme active site. In addition a high-throughput screening assay for this enzyme has been designed and implemented together with the NIH Chemical Genomics Center and will be run this year.