Establishment And Application Of Screening Model Of Mycobacterium Tuberculosis Protein Kinase B Inhibitor From Microorganism

Tuberculosis (TB) is one of the most serious threat to human health caused by Mycobacterium tuberculosis (Mtb). WHO "2013Global TB Report" showed that the number of TB patients in2012had been reached860millionworldwide,1.3million of which have died. Although the number of infections and deaths showed a slow decline, there is still another serious crisis due to drug-resistance on TB. Meanwhile, the slow progress in the development of anti-TB drugs also make it harder to win the battle against TB. Therefore, the development of novel targets for anti-TB drugs is an urgent need.PknB is one of11eukaryote-like serine/threonine protein kinases encoded by Mycobacterium tuberculosis, which is extensively involved in signal transduction pathways required for the growth and reproduction of M. tuberculosis. Since PknB plays an important role for the survival of M. tuberculosis, it is considered as a target for anti-TB drug screening.In this study, we firstly expressed and purified the N-terminal kinase domain of PknB protein, and then we used the purified PknB kinase domain to established and optimized a high throughput screening assay for PknB inhibitors from microbial fermentation products.56positive samples were identified from20,000fermentation sample. Among them,5samples including I10AA-00359, I10AA-00827, I11AA-01925, I11AA-02040, I11AA-02296, were able to inhibit the growth of Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium tuberculosis. Moreover, I10AA-00359, II1AA-01925and II1AA-02296showed a low cytotoxicity.We further investigated the specificity of activity of the five positive samples, using PknH, PknF of Mycobacterium tuberculosis and human serine/threonine protein kinase AKT-1inhibition assay established in this study. The results showed that I10AA-00359, I10AA-00827, II1AA-02040and II1AA-02296showed better selective activity. Finally, each sample subjected to preliminary separation, and the fractions were assessed for their enzyme inhibition, cytotoxicity assays, antibacterial activity and selectivity of activity. The results showed that, fraction H and D of I11AA-02040, fraction D of I10AA-00827exhibit potent inhibitory activity and antimicrobial activity, low cytotoxicity and high specificity.