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. 2014 Jul 25;289(30):21142-52.
doi: 10.1074/jbc.M114.572636.

Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis

Aeshna Nigam, Khaled H Almabruk, Anjali Saxena, Jongtae Yang, Udita Mukherjee, Hardeep Kaur, Puneet Kohli, Rashmi Kumari, Priya Singh, Lev N Zakharov, Yogendra Singh, Taifo Mahmud, Rup Lal

Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis

Aeshna Nigam et al. J Biol Chem. .

Abstract

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

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Figures

FIGURE 1.
FIGURE 1.
Chemical structures of rifamycin B, its semisynthetic derivatives, and rapamycin.
FIGURE 2.
FIGURE 2.
Genetic organization of polyketide synthase gene cluster of rifamycin B biosynthesis. The chain is assembled from a starter unit (AHBA), two acetate, and eight propionate extender units. rifF, amide synthase catalyzes the cyclization of the polyketide backbone. Post-PKS modification and cyclization of polyketide backbone eventually leads to the formation of rifamycin B.
FIGURE 3.
FIGURE 3.
Construction of AT6 mutant of A. mediterranei S699. A, partial polyketide assembly lines in rifamycin and rapamycin biosynthesis. AT6 domain of rifamycin PKS adds propionate, whereas AT2 domain of rapamycin PKS adds acetate to the growing polyketide chains. Proansamycin X is a precursor of rifamycin B. B, schematic overview of homologous recombination events postulated to occur in A. mediterranei S699 to give an AT6 mutant.
FIGURE 4.
FIGURE 4.
Mass spectrometry and NMR spectroscopy analyses of rifamycin B and 24-desmethylrifamycin B. A, (−)-ESI-MS analysis of ethyl acetate extract of the AT6 mutant strain of A. mediterranei S699 that shows prominent peaks at m/z 740 (desmethylrifamycin B) and 682 (desmethylrifamycin SV). B, (−)-ESI-MS analysis of ethyl acetate extract of the wild-type strain that shows prominent peaks at m/z 754 (rifamycin B) and 696 (rifamycin SV). C, partial 1H NMR spectrum of rifamycin B. D, partial 1H NMR spectrum of 24-demethylrifamycin B. Red stars show the four pendant methyl group in rifamycin B, and blue stars show three pendant methyl groups in 24-desmethylrifamycin B.
FIGURE 5.
FIGURE 5.
X-ray crystal structure of 24-desmethylrifamycin S dimer in complex with Ca2+. Broad red arrows indicate the missing methyl groups.
FIGURE 6.
FIGURE 6.
Synthetic scheme for 24-desmethylrifampicin.
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References

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