Identification of 4-Amino-Thieno[2,3- <i>d</i> ]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis

Abstract

Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by <i>Mycobacterium tuberculosis</i> Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of <i>M. tuberculosis</i> resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-<i>d</i>]pyrimidines, that potently inhibit the growth of <i>M. tuberculosis</i> To elucidate the mechanism by which these compounds inhibit <i>M. tuberculosis</i>, we selected for mutants resistant to a representative 4-amino-thieno[2,3-<i>d</i>]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene <i>qcrB</i>, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome <i>bc</i>₁ oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-<i>d</i>]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-<i>d</i>]pyrimidines to <i>M. tuberculosis</i> cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the <i>M. tuberculosis</i> ETC. Furthermore, 4-amino-thieno[2,3-<i>d</i>]pyrimidines had enhanced activity against a mutant of <i>M. tuberculosis</i> deficient in cytochrome <i>bd</i> oxidase, which is a hallmark of cytochrome <i>bc</i>₁ inhibitors. Therefore, 4-amino-thieno[2,3-<i>d</i>]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome <i>bc</i>₁ complex.<b>IMPORTANCE</b> The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in <i>Mycobacterium tuberculosis</i>, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome <i>bc</i>₁ has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-<i>d</i>]pyrimidines, that potently inhibit <i>M. tuberculosis</i> growth <i>in vitro</i> These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-<i>d</i>]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.