HIGH THROUGHPUT SCREENING AND IDENTIFICATION OF INHIBITORS OF FASR, A KEY TRANSCRIPTIONAL REGULATOR OF CELL WALL SYNTHESIS IN Mycobacterium tuberculosis

COLACCINI, F; QUIROGA, RODRIGO; DESCUTTER, S; VILLARREAL, M; GRAMAJO, H; GAGO, G

Congreso; XVII Congreso Argentino de Microbiología General, Sociedad Argentina de Microbiología General (SAMIGE); 2022

Mycobacterium tuberculosis (Mtb) has a complex lifestyle and its flexible metabolism allows it to adapt and survive in the infected host. During this process, one of the most affected pathways is lipid metabolism and 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 cell wall. 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 involved in the biosynthesis of mycolic acids, essential components for viability and pathogenesis. FAS I catalyses the de novo biosynthesis of long chain acyl-CoAs that are used by the FAS II 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, we obtained a set of 25 candidates through an in silico screening of a library of thousands of compounds that fit into the FasR hydrophobic tunnel. The ability to uncouple FasR:DNA binding of this set of candidate compounds was tested in vitro using electrophoretic mobility shift assays (EMSA). We selected the best hits and the data obtained by docking these compounds in the crystal structure of FasR helped us to further refine the search and identify new compounds within the library. A second set of 25 candidates was generated through this in silico selection and then tested by EMSA. As results of both screenings, 18 active compounds were selected out of 50 candidates. The in vivo validation of these hits was carried out by testing the selected compounds against a M. smegmatis bioreporter strain, which has the pfas sequence (promoter region of the fas-acpS operon) fused with lux genes.