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The cytochrome bc1 complex or complex III of the electron transport chain is an integral membrane protein that forms a key component in the bacterial respiratory system. The complex functions as a ubiquinol-cytochrome C reductase, utilizing a catalytic core of three highly conserved components namely the cytochrome c1, cytochrome b and the Rieske iron sulfur protein. A Q-cycle mechanism couples electron transfer to proton translocation, adding to the proton electrochemical gradient that is used to generate adenosine triphosphate. The mechanism of electron transfer within the cytochrome bc1 complex has previously been described. There are a number of well-characterized inhibitors of the bc1 complex. The elucidation of their mechanism and crystallographic data identifying binding sites has lead to the development of compounds that inhibit the function of the cytochrome bc1 complex for therapeutic purposes. These inhibitors tend to target the two catalytic domains utilizing an analogous structure to either quinone or quinol. von Jagow and colleagues first characterized the most widely understood inhibitor of the cytochrome bc1 complex, myxothiazol, an antibiotic from Myxococcus fulvus. Myxothiazol was found to inhibit the oxidant-induced reduction of b cytochromes by competitively displacing quinone from the Rieske iron sulfur protein at the high affinity binding site Qo with a Kd,161029 M. Many other inhibitors have been identified that function by the same mechanism as myxothiazol, such as mucidin and strobilurin A. Another inhibitor, antimycin, has been shown to inhibit the cytochrome bc1 complex at a different location to myxothiazol, as it functions by binding to the Qi site, proximal to the BH heme, inhibiting oxidation of the cytochrome b subunit. Here we have reported QcrB as the target of the IP family of compounds of the cytochrome bc1 complex, which was identified by whole genome sequencing of resistant mutants. Resistant mutants raised against all three compounds IP 1, IP 3 and IP 4 carried an SNP in the qcrB gene where a single base change translated to the substitution of a threonine at position 313 for an alanine in the cytochrome b subunit. In silico mapping of this amino acid substitution utilizing the structure of a myxothiazol-bound cytochrome bc1 complex found the substitution did not fall within the myxothiazol binding site, and therefore resistance is likely conferred by a conformational change as opposed to a mechanistic alteration. SNPs were also identified in other genes. However, due to their inconsistent locations in the genomes of the IP resistant mutants generated and their reported non-essentiality, these SNPs were assumed to be non-consequential and not investigated further. In order to investigate the mechanistic similarities of the three IP compounds in the series, cross-resistance of the genetically dissimilar mutants was established. It was found that all mutants generated were resistant to each of the IP compounds, confirming T313A as the common factor in the resistant phenotype and suggesting all three IP inhibitors function identically. Despite the high potency of the inhibitors, there may be a requirement for future re-engineering and lead optimization now the QcrB target has been identified in this study. Nevertheless, the highly efficient bacterial clearance and novelty of the target as a major component of the electron transport chain shows considerable promise for IP compounds in the treatment of both active and latent phase mycobacterial infection. The latter has been shown to be particularly susceptible to inhibitors of the electron transport chain. Further evidence to support and validate our findings came from the over-expression study of QcrB in M. bovis BCG using the mycobacterial vector pMV261, which approximately exerts a 5 times copy number. Three varying length inserts were selected for this study so as to ensure the synthesis of native QcrB, as depicted in Figure 5. M. bovis BCG containing an empty pMV261 vector exhibited no change in tolerance to IP 3 in comparison to the wild-type strain, with an MIC of 0.5 mM. On the contrary, there was a marked OTX015 increase in MIC to.8 mM for M. bovis BCG transformants containing all three inserts encompassing qcrB. At 166MIC, we found ample growth from all three inserts, including pMV261::qcrB, despite the reported finding that QcrB was only expressed to 10% efficiency if lacking functional QcrC in overexpression vectors. This result clearly confirms QcrB as the target of IP 3 and the other IP compounds: increased expression levels of the IP target enable higher concentrations of IP to enter the cell and bind QcrB without a fatal impact on cell survival.

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