Multiple Approaches to Novel Rifamycin Analogs to Combat MDR-TB Public

http://ir.library.oregonstate.edu/concern/honors_college_theses/n296x1194

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  • According to the Center for Disease Control and Prevention (CDC), in 2015 10.4 million people worldwide became infected with tuberculosis (TB) with 1.8 million TB related deaths. Tuberculosis is a bacterial infectious disease caused by Mycobacterium tuberculosis, a slow growing yet highly infectious bacterium. Among the first line of treatments for TB is the antibiotic rifampin, a synthetic derivative of the natural product rifamycin B. Rifampin selectively inhibits bacterial RNA polymerase by binding to its β-subunit, effectively inhibiting protein synthesis leading to cell death. However, recently the development of multidrug resistant (MDR) and extensively drug resistant (XDR) TB has reduced the effectiveness of commercially available treatments, including rifampin. Bacterial resistance to rifampin is mainly due to mutations in the target protein RNA polymerase. Through a separate bio-computational modeling study, it was hypothesized that making a slight change to the rifamycin backbone may lead to a novel rifamycin analog that more effectively binds and inhibits mutated RNA polymerase. The suggested change, based on the bio-computational modeling study, is the removal of the methyl on C-7, lessening molecular steric hindrance. Methods focused initially on the genetic re-engineering of the bacterial producer of rifamycin, Amycolatopsis mediterranei S699, through the modification of the biosynthetic machinery. This can be achieved by replacing the acyltransferase (AT) domain of module 1 of the rif-PKS (which recognizes methylmalonyl-CoA as the substrate) with the AT domain of module 2 of the rapamycin PKS (which recognizes malonyl-CoA) using a double crossover homologous recombination. However, there were some issues in the transformation of the final construct into the competent A. mediterranei cells leading to the exploration of another approach. By observation, it was recognized that old plates of Amycolatopsis mediterranei S699 began to show evidence of different metabolites, one of which resembled, in mass spectrometry measurements, that of a desmethylrifamycin SV. In order to test this, cells were starved on different media and then tested at various points in time for metabolite production. After analysis, the compound of interest was identified to be 12-desmethylrifamycin SV. This compound did show significant activity against M. tuberculosis, but not against drug resistant forms. The selective removal of the methyl group on C-12 is most likely due to enzyme catalysis. There are two genes, which encode cytochrome P450 monooxygenases (rif-orf13 and rif-orf16), within the rifamycin biosynthetic gene cluster that may be responsible for the oxidation and removal of the methyl group. Therefore, both of them were cloned and heterologously expressed in Escherichia coli. However, the proteins were found to be insoluble. Therefore, detailed investigations of these proteins cannot be done at this point in time, and will have to wait until soluble proteins are available. Future work would include refinement of the heterologous expression methodology and full characterization of Orf13 and Orf16 to determine their involvement in the formation of 12-desmethylrifamycin SV. Keywords: genetics, drug development, tuberculosis, multi-drug resistance
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