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Structure guided generation of thieno[3,2-d]pyrimidin-4-amine Mycobacterium tuberculosis bd oxidase inhibitors.

ABSTRACT: Cytochrome bd oxidase (Cyt-bd) is an attractive drug target in Mycobacterium tuberculosis, especially in the context of developing a drug combination targeting energy metabolism. However, currently few synthetically assessable scaffolds target Cyt-bd. Herein, we report that thieno[3,2-d]pyrimidin-4-amines inhibit Cyt-bd, and report an initial structure-activity-relationship (SAR) of 13 compounds in three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis clinical isolate N0145 in an established ATP depletion assay with or without the cytochrome bcc : aa ₃ (QcrB) inhibitor Q203. All compounds displayed activity against M. bovis BCG and the M. tuberculosis clinical isolate strain N0145 with ATP IC₅₀ values from 6 to 54 μM in the presence of Q203 only, as expected from a Cyt-bd inhibitor. All derivatives were much less potent against M. tuberculosis H37Rv compared to N0145 (IC₅₀'s from 24 to >100 μM and 9-52 μM, respectively), an observation that may be attributed to the higher expression of the Cyt-bd-encoding genes in the laboratory-adapted M. tuberculosis H37Rv strain. N-(4-(tert-butyl)phenethyl)thieno[3,2-d]pyrimidin-4-amine (19) was the most active compound with ATP IC₅₀ values from 6 to 18 μM against all strains in the presence of Q203, making it a good chemical probe for interrogation the function of the mycobacterial Cyt-bd under various physiological conditions.

SUBMITTER: Hopfner SM

PROVIDER: S-EPMC8130631 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

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Structure guided generation of thieno[3,2-d]pyrimidin-4-amine Mycobacterium tuberculosis bd oxidase inhibitors.

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