An integrative study of the mycobacterial Fatty Acid Synthase type II, a strategic target for novel TB drugs. – FASMY

Tuberculosis (TB) remains today one of the leading causes of death in the world from an infectious agent. Due to misapplication of TB treatment, multidrug-resistant bacilli that fail to respond to conventional treatment have emerged. Thus, according to the WHO, the development of a new generation of drugs effective against Mycobacterium tuberculosis resistant strains is urgently needed. Our project corresponds to basic research which stands upstream of the TB drug development pipeline and aims at providing the TB drug discovery programs with novel relevant drug targets.
Mycolic acids are essential lipids of M. tuberculosis envelope. Many new molecules recently discovered through phenotypic screenings and showing great promise as selective tuberculosis therapeutics inhibit their biosynthesis. The Fatty Acid Synthase type II (FAS-II) involved in this pathway constitutes a highly relevant and validated pharmaceutical target because it has unique features, plays key roles in the virulence and persistence of the tubercle bacillus and is the target of TB drugs.
The objective of the FASMY project is to characterize the mycobacterial FAS-II multienzyme system with a completely new line of attack, at the scale of the entire complex, using advanced technologies. Such a strategy is much more relevant than the classical approach based on isolated target-enzymes since it takes into account the spatial organization of the complex, interactions between partner enzymes and the accessibility of the target-enzyme, which greatly influence the action of drugs. A multidisciplinary and integrative approach will be used to decipher in depth the protein composition, the 3D structure and the enzymatic activity of the whole purified FAS-II complex as well as the mode of action of known TB drugs. The pioneer lab in the field of mycolic acid biosynthesis (Partner 1) will purify the FAS-II complex using two complementary strategies, the Single Step Affinity Purification method lately adapted to mycobacterial complexes and validated, and the immunoprecipitation technique. The FAS-II proteins will be identified by proteomics by an internationally recognized Proteomics laboratory (national IBiSA labelled facility, French Proteomics Infrastructure ProFI, Partner 2). Then, advanced lipidomics technology recently optimized by Partner 1 will be used to accurately determine FAS-II function by analyzing the fine structure of its products. The function of newly identified enzymes of the system will also be characterized by both in vitro activity assays and phenotypic analysis of M. tuberculosis mutants. A group expert in multi-scale structural biology (Partner 3) will use electron microscopy and image processing to study FAS-II 3D structure at intermediate resolution coupled with immunolabeling cartography to localize individual proteins. A European leader group in Structural biophysics (national IBiSA labelled platform, Partner 4) will solve the high-resolution structures of the newly identified enzymes, which will be combined to the microscopy data to gain an insight into the spatial organization of the complex. The acquired knowledge on FAS-II system will then constitute unprecedented tools to decipher the mode of action of known TB drugs targeting this complex, thiacetazone and isoxyl, at the molecular level.
The novelty of the approach adopted here is to consider the whole M. tuberculosis FAS-II complex as a target. The development of an activity assay with the purified system and the thorough knowledge of its composition and 3D structure will mark a major evolution for the high throughput screening of chemical libraries and rational design both in terms of physiological relevance and of time- and cost-effectiveness, three key issues for pharmaceutical companies. The determination of the modes of action of clinically-used drugs will represent a valuable tool for the design of inhibitors of FAS-II, which may lead to the development of novel TB antibiotics.