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New Conformations of Acylation Adducts of Inhibitors of ?-Lactamase from Mycobacterium tuberculosis.

ABSTRACT: Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ?-lactam antibiotics due to the production of the extended spectrum ?-lactamase BlaC. ?-Lactam/?-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the ?-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 S? atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other ?-lactamases are discussed.

SUBMITTER: Tassoni R

PROVIDER: S-EPMC6383187 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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New Conformations of Acylation Adducts of Inhibitors of β-Lactamase from Mycobacterium tuberculosis.

Tassoni Raffaella R Blok Anneloes A Pannu Navraj S NS Ubbink Marcellus M

Biochemistry 20190130 7

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid ...[more]

PMID: 30632739

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Project description:New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosis in vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc 1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc 1 -aa 3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc 1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc 1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

Dataset Information

New Conformations of Acylation Adducts of Inhibitors of ?-Lactamase from Mycobacterium tuberculosis.

Publications

New Conformations of Acylation Adducts of Inhibitors of β-Lactamase from Mycobacterium tuberculosis.

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