Could Mycobacterial MelF Protein (Rv1936) be used as a Potential Drug Target?

Abstract

Macrophages produce antimicrobial reactive oxygen species (ROS) and reactive nitrogen species (RNS) through NADPH oxidase (NOX2/gp91 phox ) and inducible nitric oxide synthase in response to mycobacterial infections [1]. In general, mycobacteria are resistant to ROS, but RNS inhibit growth and even kill mycobacteria within the activated macrophages. Mycobacterium tuberculosis is also continually exposed to endogenous ROS including the production of superoxide radicals as part of normal aerobic respiration [2]. In fact, M. tuberculosis has developed several defense mechanisms to counteract ROS stress, for example, lipid enriched cell wall, maintenance of cytosolic redox homeostasis, genes involved in repair and protection of DNA/protein as well as several ROS scavenger genes, including katG (Rv1908c), SodA (Rv3846), sodC (Rv0432), ahpC (Rv2428), ahpE (Rv2238c), melF (Rv1936), etc. [3,4,5]. Notably, global regulator oxyR is inactive in M. tuberculosis, but is active in other pathogenic mycobacterial species including M. marinum. Pathways involved in mycobacterial resistance to RNS include the generation of RNS scavenger genes such as noxR1 (Rv2997), noxR3 (Rv1500), dlaT (Rv2215), msrA (Rv0137c) and cysH (Rv2392), DNA repair as well as protein degradation in the proteasome [3]. Moreover, peroxynitrite is produced by SOD in the presence of H 2 O 2 and nitric oxide, connecting the two important mechanisms of oxidative and nitrosative stress-mediated bacterial cell death. M. tuberculosis possesses inherent resistance to peroxynitrite as compared with less pathogenic mycobacteria [3]. Hence, targeting anti-ROS/RNS machinery of mycobacterial cells would facilitate efficient clearance of bacteria from the invading tissue.The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941, size 7.9 kb), originally identified by El-Etr et al. [6], is M. marinum and M. tuberculosis (∼98% sequence homology with M. marinum) specific and is absent in other pathogenic and nonpathogenic mycobacterial species, including M. bovis, M. avium, M. leprae and M. smegmatis, which can tolerate ROS and RNS stress responses. In silico analysis of six genes of mel2 locus, in other words melF, melG, melH, etc. revealed them to be close homologs of lux genes present in Vibrio harveyi [3]. Moreover, the melF within this locus shows high similarity to luxA, a monooxygenase gene involved in resistance to ROS in bioluminescent bacteria. The mel2 locus also confers resistance to ROS/RNS stress in vitro and reveals a similar function in activated murine macrophages [3,4]. Moreover, the M. marinum mel2 mutants do not display growth defect in macrophages in presence of ROS scavengers or nitric oxide synthase inhibitors, thus demonstrating that the growth defect is dependent on the production of both ROS and RNS [4]. Interestingly, the mutation in melF of mel2 locus displays a polar effect on the downstream genes of mel2 locus as the enhanced susceptibility of melF mutant to ROS/RNS could only be partially recovered by melF alone and comp...

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages

“…ROS and RNS are the most effective antimycobacterial molecules generated by the host macrophages during infection. Infection of Phox−/− and NOS−/− mice as well as bone marrow-derived macrophages infected with M. tuberculosis clearly suggest that the mel2 locus imparts pathogenesis through its ability to resist host ROS stress 8,13 . M. tuberculosis is also constantly exposed to endogenous ROS including the production of superoxide radicals during normal aerobic respiration 20 .…”

“…# 6513745 (by inhibiting MelF possessing anti-ROS/RNS machinery), thus leading to high susceptibility of mycobacteria within RAW264.7 macrophages. Moreover, compounds such as clofazimine and plumbagin capable of generating ROS through production of superoxide radicals, were previously shown to reduce the MIC of INH for M. tuberculosis 8,29 . In a similar manner, inhibitor IMBI-3 targeting ICL exhibited a synergistic effect in combination with INH against persistent M. marinum within J774A.1 macrophages 9 .…”

“…Though WCS has been more successful in giving possible hits as TBS does not take into consideration the poor penetration and efflux problems, it lacks precise target knowledge 4 . Virtual ligand screening (VLS) is also considered as an effective tool for in silico screening of large compound libraries and small molecule chemical libraries for obtaining the potential leads against biological targets 7,8 .…”

“…marinum infection model has also been employed for evaluating ATDs in vivo as a HTS system 10 . Interestingly, mycobacterial mel2 locus (Rv1936-1941) is M. marinum and M. tuberculosis specific and absent in other pathogenic and nonpathogenic mycobacterial species, which can tolerate reactive oxygen species (ROS) and reactive nitrogen species (RNS) stress response 8,11,12 . Notably, M. marinum melF of mel2 locus displayed a high similarity to luxA of Vibrio harveyi , which has a significant role in resistance to ROS in bioluminescent bacteria 11 .…”

“…tuberculosis in an in vitro system 7 , however, the effect of these inhibitors against mycobacterium-infected macrophages was not examined. Indeed, macrophages are the key effectors cells to control mycobacterial infections and that also have a niche for their replication 14,15 , which produce antibacterial ROS and RNS molecules through NADPH oxidase and inducible nitric oxide synthase 8,16 . Mostly, mycobacteria are resistant to ROS, but RNS display bacteriostatic/bactericidal effect within activated macrophages.…”