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

Abstract

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 Mycobacterium tuberculosis , 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 bc 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- d ]pyrimidines, that potently inhibit M. tuberculosis growth in vitro . These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3- d ]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target. Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis . 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 M. tuberculosis 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- d ]pyrimidines, that potently inhibit the growth of M. tuberculosis . To elucidate the mechanism by which these compounds inhibit M. tuberculosis , we selected for mutants resistant to a representative 4-amino-thieno[2,3- d ]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 qcrB , which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc 1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3- d ]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3- d ]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3- d ]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc 1 inhibitors. Therefore, 4-amino-thieno[2,3- d ]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 bc 1 complex. IMPORTANCE 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 Mycobacterium tuberculosis , 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 bc 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- d ]pyrimidines, that potently inhibit M. tuberculosis growth in vitro . These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3- d ]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.