Study On The Target Of New Anti - Tuberculosis Compound

Tuberculosis is a contagious disease caused by Mycobacterium tuberculosis, the recent co-infection of tuberculosis (TB) and human immunodeficiency virus (HIV) makes the prevention and control of TB become more and more difficult, thus to find new anti-TB drug targets and develop new anti-TB drugs become more and more urgent.3277-3, a potent anti-TB candidate with a new structure, which is screened by our lab, possesses good inhibitory activity against both the sensitive and the drug-resistant Mycobacterium tuberculosis strains. To find and confirm the target of3277-3, we created a random gene highly expressing library of Mycobacterium marinum, and sequenced two mutants’genome of3277-3-resistant strains. This work comprises two parts:In the first part, we select the Mycobacterium marinum which shares high homology with TB and with good safety as a model bacterium, to create a random high gene expression library for the target screening of3277-3. In the study, we constructed a double cos-sites cosmid with single cos-site cosmid pJRD215and pJDC16; By utilizing double cos-sites and phage assembling, the random gene highly expressing library of Mycobacterium marinum is obtained, which contains about3000strains. To further validate the feasibility of using the library to find drug targets,5anti-TB drugs with accurate mechanisms of action were exploited. The targets ddlA and gyrA of D-cycloserine and ciprofloxacin, respectively, have been screened in this way, which were consistent with their proved targets. These preliminary evidences indicate that the library can be used in looking for the targets of some anti-TB compounds.In the second part, we screened the mutants of Mycobacterium marinum which were resistant to3277-3. By sequencing the genome of the drug-resistant mutants, we intended to find and validate the target of3277-3. In the study, we used UV cross linker to mutagenize the Mycobacterium marinum, and two mutant strains Mm3277-36and Mm3277-48which were resistant to3277-3were obtained. By sequencing the mutants’ genome and blasting the mutants’genome with that of the standard Mycobacterium marinum, we found16mutant genes which maight encode the potential drug targets. The results from bioinformatics suggest that4of the16genes involved are members of PE/PPE family,2of which are PKSs.3are NRPSs,3are hypothetical protein,1is kinase,2are membrane proteins. Most of the mutant genes are relevant with the biosynthesis of cell wall. These results further substantiate the target validation of3277-3.