Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb’s cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

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The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins

Archives of Microbiology ◽

10.1007/s00203-015-1179-z ◽

2016 ◽

Vol 198 (2) ◽

pp. 193-197 ◽

Cited By ~ 2

Author(s):

Yue-Yue Wang ◽

Hong-Dou Luo ◽

Xiao-Sheng Zhang ◽

Tao Lin ◽

Hui Jiang ◽

...

Keyword(s):

Coenzyme A ◽

Phosphopantetheinyl Transferase ◽

Carrier Proteins ◽

Acyl Carrier Proteins ◽

Substrate Promiscuity

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Conformational flexibility of coenzyme A and its impact on the post‐translational modification of acyl carrier proteins by 4′‐phosphopantetheinyl transferases

FEBS Journal ◽

10.1111/febs.15273 ◽

2020 ◽

Vol 287 (21) ◽

pp. 4729-4746

Author(s):

Minh Chau Nguyen ◽

Olivier Saurel ◽

Coralie Carivenc ◽

Sabine Gavalda ◽

Stéphane Saitta ◽

...

Keyword(s):

Coenzyme A ◽

Conformational Flexibility ◽

Carrier Proteins ◽

Post Translational Modification ◽

Acyl Carrier Proteins ◽

Phosphopantetheinyl Transferases

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Streptomyces coelicolor phosphopantetheinyl transferase: a promiscuous activator of polyketide and fatty acid synthase acyl carrier proteins

Journal of the Chemical Society Perkin Transactions 1 ◽

10.1039/b204633b ◽

2002 ◽

pp. 1644-1649 ◽

Cited By ~ 25

Author(s):

Russell J. Cox ◽

John Crosby ◽

Oliver Daltrop ◽

Frank Glod ◽

Malgorzata E. Jarzabek ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Streptomyces Coelicolor ◽

Phosphopantetheinyl Transferase ◽

Carrier Proteins ◽

Acyl Carrier Proteins

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Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

Pharmaceutics ◽

10.3390/pharmaceutics13071066 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1066

Author(s):

Alistair S. Brown ◽

Jeremy G. Owen ◽

James Jung ◽

Edward N. Baker ◽

David F. Ackerley

Keyword(s):

Mycobacterium Tuberculosis ◽

High Throughput ◽

Coenzyme A ◽

Critical Role ◽

Binding Pocket ◽

Blue Pigment ◽

Phosphopantetheinyl Transferase ◽

Primary And Secondary Metabolism ◽

Drug Leads

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

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Inhibition of Indigoidine Synthesis as a High-throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

10.20944/preprints202106.0116.v1 ◽

2021 ◽

Author(s):

Alistair S Brown ◽

Jeremy G Owen ◽

James Jung ◽

Edward N Baker ◽

David F Ackerley

Keyword(s):

Mycobacterium Tuberculosis ◽

High Throughput ◽

Coenzyme A ◽

Critical Role ◽

Binding Pocket ◽

Blue Pigment ◽

Phosphopantetheinyl Transferase ◽

Primary And Secondary Metabolism ◽

Peptide Synthetase

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colorimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection, and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Structure, function and dynamics in acyl carrier proteins

PLoS ONE ◽

10.1371/journal.pone.0219435 ◽

2019 ◽

Vol 14 (7) ◽

pp. e0219435 ◽

Cited By ~ 3

Author(s):

Rohit Farmer ◽

Christopher Morton Thomas ◽

Peter James Winn

Keyword(s):

Structure Function ◽

Carrier Proteins ◽

Acyl Carrier Proteins

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Computational exploration and anti-mycobacterial activity of potential inhibitors of Mycobacterium tuberculosis acetyl coenzyme A carboxylase as anti-tubercular agents

SAR and QSAR in Environmental Research ◽

10.1080/1062936x.2021.1882563 ◽

2021 ◽

Vol 32 (3) ◽

pp. 191-205

Author(s):

J. Kuldeep ◽

S.K. Sharma ◽

B.N. Singh ◽

M.I. Siddiqi

Keyword(s):

Mycobacterium Tuberculosis ◽

Coenzyme A ◽

Acetyl Coenzyme A ◽

Acetyl Coenzyme ◽

Computational Exploration ◽

Potential Inhibitors ◽

Acetyl Coenzyme A Carboxylase

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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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