Identification of 4-Amino-Thieno[2,3- d ]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 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 bc1 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 bc1 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 bc1 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 bc1 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.

SAR Analysis of Small Molecules Interfering with Energy-Metabolism in Mycobacterium tuberculosis

“…Another screening of 78 small-molecule nucleotide mimetics [ 75 ] identified 4-amino-thieno[2,3- d ]-pyrimidines which inhibit the growth of M. smegmatis. The improved analogue CWHM-728 ( Figure 12 ) inhibits Mtb growth with an IC 50 of 2.7 ± 0.84 μM [ 75 ], and it was then selected as the lead compound for initial SAR studies focusing on the side chain. The introduction of a 3-phenylpropyl side chain led to an increase of 38-fold in potency versus Mtb (IC 50 = 83 ± 5.4 nM).…”

Terminal Respiratory Oxidases: A Targetables Vulnerability of Mycobacterial Bioenergetics?

“…In combination with Q203, CWHM-1023 efficiently decreased the ATP levels in M. smegmatis and Mtb. Also, upon the deletion of cydA, bacteria become more sensitive towards this compound, proving its efficacy as an anti-TB drug molecule (Harrison et al, 2019).…”

Oxidative Phosphorylation—an Update on a New, Essential Target Space for Drug Discovery in Mycobacterium tuberculosis

“…The Qp site of the Cyt-bcc-aa3 complex is particularly susceptible to chemical inhibition, as evident from the multitude of structurally diverse and distinct compounds detailed above. It has been consistently observed across several of the studies that characteristic consequences of cytochrome bcc inhibition include a general depletion of intracellular bacterial ATP levels, an upregulation of Cyt-bd oxidase, and an increase in OCR and bacterial respiration due to Cyt-bd [12,76,77,[128][129][130]134,135,155,158]. This compensation by the alternate terminal oxidase leads to an incomplete respiratory shutdown in M. tb, resulting in the bacteriostatic nature of cytochrome bcc inhibitors alone.…”