STRUCTURE BASED IDENTIFICATION OF INHIBITORS OF FASR, A KEY TRANSCRIPTIONAL REGULATOR OF CELL WALL SYNTHESIS IN Mycobacterium tuberculosis.

COLACCINI F ; QUIROGA R; VILLARREAL MA; GRAMAJO H; GAGO G

Congreso; SAIB 2021; 2021

STRUCTURE BASED IDENTIFICATION OF INHIBITORS OF FASR,A KEY TRANSCRIPTIONAL REGULATOR OF CELL WALL SYNTHESISIN Mycobacterium tuberculosisColaccini F 1, Quiroga R2, Villarreal MA 2, Gramajo H1, Gago G11 Instituto de Biología Molecular y Celular de Rosario, Argentina.2 Instituto de Investigaciones en Fisicoquímica de Córdoba (INFIQC-CONICET), Departamento de Química Teórica y Computacional, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. E-mail: colaccini@ibr-conicet.gov.arMycobacterium tuberculosis (Mtb) has a very complex lifestyle. The flexibility in its metabolism allows it to adapt and survive in the infected host. During this process, one of the most affected pathways is lipid metabolism, both in the host and in the pathogen. Despite there is a lot of information about the biosynthesis, structure and biological function of the main lipids present in Mtb envelope, little is known about the mechanisms that allow the bacteria to modulate and adapt the biosynthesis of the components of the cell wall in response to changes in environment. Thus, the study of the processes involved in the regulation of the biosynthesis of lipids in Mtb represents a crucial step in the comprehension of the physiology of this pathogen, as well as to find potential drug targets and contribute to combat tuberculosis. The biosynthesis of fatty acids in Mtb involves two different systems of fatty acid synthases (FAS I and FAS II). Both synthases are involved in the biosynthesis of membrane fatty acids and several lipid components of the cell wall, like mycolic acids (essential for viability and pathogenesis). The multi domain single protein FAS I catalyses de novo biosynthesis of acyl-CoAs in a bimodal fashion rendering long-chain acyl-CoAs that are used as primers by the FAS II multiprotein system for the synthesis of mycolic acids. The transcription factor that we study, FasR, plays a key role in this process by positively regulating the expression of fas and acpS genes. These genes, coding for FAS I and AcpS (essential to produce functional ACP), form a single operon in Mtb. FasR:DNA binding is regulated by long-chain acyl-CoAs (products of FAS I) which disrupt the interaction of FasR with its cognate DNA. Although FasR is not essential for in vitro growth, regulation of lipid biosynthesis mediated by FasR is critical for macrophage infection and essential for virulence in vivo using a mouse model of infection that is why it could be an interesting drug target. In this work, an in silico screening of a library of thousands of compounds was carried out, from which we obtained a set of 25 candidates to promote the uncoupling of the FasR:DNA binding. This set of candidate compounds was tested through electrophoretic mobility shift assays (EMSA), analyzing the ability of FasR to bind to the DNA probe. We defined as best hits those compounds which, at concentrations of the order of 20 μM, were able to inhibit the binding. The data obtained by docking the selected hits in the crystallographic structure of FasR, helped us to further refine the search and identification of new compounds within the primary library. Overall, the results of these studies have provided strong bases toward understanding which are the most relevant features of the more active compounds and some of their key interactions with FasR, that in the future should allow the identification of more potent compounds in this or other chemical libraries.