High throughput screening and identification of inhibitors of FasR, a key transcriptional regulator of cell wall synthesis in Mycobacterium tuberculosis

COLACCINI FACUNDO; VILLARREAL MARCOS ARIEL; QUIROGA RODRIGO; DESCHUTTER MARÍA SOFÍA

Los Cocos, Córdoba

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Sociedad Argentina de Microbiología General (SAMIGE)

Mycobacterium tuberculosis (Mtb) has a complex lifestyle and its flexible metabolism allows it toadapt and survive in the infected host. During this process, one of the most affected pathways is lipidmetabolism and despite there is a lot of information about the biosynthesis, structure and biologicalfunction of the main lipids present in Mtb envelope, little is known about the mechanisms that allowthe bacteria to modulate and adapt the biosynthesis of the cell wall. Thus, the study of the processesinvolved in the regulation of the biosynthesis of lipids in Mtb represents a crucial step in thecomprehension of the physiology of this pathogen, as well as to find potential drug targets andcontribute to combat tuberculosis. The biosynthesis of fatty acids in Mtb involves two differentsystems 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 longchain acyl-CoAs that are used by the FAS II for the synthesis of mycolic acids. The transcription factorthat we study, FasR, plays a key role in this process by positively regulating the expression of fas andacpS genes. These genes, coding for FAS I and AcpS (essential to produce functional ACP), form asingle 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 vitrogrowth, regulation of lipid biosynthesis mediated by FasR is critical for macrophage infection andessential for virulence in vivo using a mouse model of infection; that is why it could be an interestingdrug target. In this work, we obtained a set of 25 candidates through an in silico screening of a libraryof thousands of compounds that fit into the FasR hydrophobic tunnel. The ability to uncoupleFasR:DNA binding of this set of candidate compounds was tested in vitro using electrophoreticmobility shift assays (EMSA). We selected the best hits and the data obtained by docking thesecompounds in the crystal structure of FasR helped us to further refine the search and identify newcompounds within the library. A second set of 25 candidates was generated through this in silicoselection and then tested by EMSA. As results of both screenings, 18 active compounds wereselected out of 50 candidates. The in vivo validation of these hits was carried out by testing theselected compounds against a M. smegmatis bioreporter strain, which has the pfas sequence(promoter region of the fas-acpS operon) fused with lux genes. We identified several compoundsthat showed a large drop in the luminescence signal, indicating the ability to uncouple FasR:DNAbinding in vivo. Overall, these studies have provided relevant information on compound-protein keyinteractions that should allow the identification of more potent compounds.