New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis

Abstract

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02-0.09 μg/mL) and low toxicity (TD 50 � 5μg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc 1 by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc 1 complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc 1 complex.

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

“…Even if an initial infection could be first achieved by regulating the expression of one of the terminal oxidases, the absence of both terminal oxidases led to severe persistence defects, with a decrease of five orders of magnitude in bacterial load in 35 days [35]. These findings are consistent with previous reports, whereby the joint inactivation, either pharmacologically or genetically, of both cytochrome bcc and bd oxidases resulted in bactericidal effects on M. tb in vitro and in vivo [65,76,77,160]. Exploiting the synthetic lethal interaction between these two terminal oxidases is an extremely attractive approach to eradicate M. tb.…”

“…These characteristics would indicate a role of this terminal oxidase in bacterial survival in environments of low oxygen tension and protection against oxidative stress, even though it appears to function efficiently under normoxic conditions as well [65]. The upregulation of the cydAB operon has been reported for M. tb under hypoxia and in the presence of NO, as well as during the chronic phase of infection in mice [72][73][74], and when the function of the Cyt-bcc-aa3 is compromised [32,[59][60][61][75][76][77].…”

“…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.…”

“…The series was extensively profiled and optimised, with the lead of this series having an MIC 90 of 0.24 µM against M. tb, a high-selective index and improved microsomal stability [134]. In an acute model of TB infection, the lead compound The most recent class of Cyt-bcc-aa3 inhibitors identified are the quinazoline derivatives, 2-ethylthio-4-methylaminoquinazolines [77]. The most potent derivatives have activity against in vitro and intracellular M. tb in the micromolar range, and have low cytotoxicity in human hepatocytes.…”

“…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. However, with the deletion of Cyt-bd, mycobacterial respiration is effectively blocked, and cytochrome bcc inhibitors become bactericidal [65,76,77]. Taken altogether, these concerted efforts have generated a potential pool of backup cytochrome bcc inhibitors as Q203 continues in clinical trials.…”