Mycobacterium tuberculosis cytochrome P450 enzymes: a cohort of novel TB drug targets

2012 ◽  
Vol 40 (3) ◽  
pp. 573-579 ◽  
Author(s):  
Sean A. Hudson ◽  
Kirsty J. McLean ◽  
Andrew W. Munro ◽  
Chris Abell
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Drug Resistant ◽  
Cytochrome P450 Enzymes ◽  
Front Line ◽  
Exciting Potential ◽  
Tuberculosis Disease ◽  
Fragment Based Drug Discovery

TB (tuberculosis) disease remains responsible for the death of over 1.5 million people each year. The alarming emergence of drug-resistant TB has sparked a critical need for new front-line TB drugs with a novel mode of action. In the present paper, we review recent genomic and biochemical evidence implicating Mycobacterium tuberculosis CYP (cytochrome P450) enzymes as exciting potential targets for new classes of anti-tuberculars. We also discuss HTS (high-throughput screening) and fragment-based drug-discovery campaigns that are being used to probe their potential druggability.

scholarly journals Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis

2019 ◽  
Vol 24 (7) ◽  
pp. 745-754
Author(s):  
Sandra Ortega Ugalde ◽  
Dongping Ma ◽  
James J. Cali ◽  
Jan N. M. Commandeur
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
High Signal ◽  
Cytochrome P450 Enzymes ◽  
High Enzyme ◽  
Endogenous Substrates ◽  
Selection Of

Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z’-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.

scholarly journals Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

2019 ◽  
Vol 103 (9) ◽  
pp. 3597-3614 ◽  
Author(s):  
Sandra Ortega Ugalde ◽  
Maikel Boot ◽  
Jan N. M. Commandeur ◽  
Paul Jennings ◽  
Wilbert Bitter ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Cytochrome P450 Enzymes ◽  
Redox Partners

Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis

2003 ◽  
Vol 31 (3) ◽  
pp. 625-630 ◽  
Author(s):  
A.W. Munro ◽  
K.J. McLean ◽  
K.R. Marshall ◽  
A.J. Warman ◽  
G. Lewis ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Cytochromes P450 ◽  
Tight Binding ◽  
Binding Constants ◽  
Multidrug Resistant ◽  
Cytochrome P450 Enzymes ◽  
Global Threat ◽  
Novel Drug

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

scholarly journals Label-Free Comparative Proteomics of Differentially Expressed Mycobacterium tuberculosis Protein in Rifampicin-Related Drug-Resistant Strains

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 607
Author(s):  
Nadeem Ullah ◽  
Ling Hao ◽  
Jo-Lewis Banga Ndzouboukou ◽  
Shiyun Chen ◽  
Yaqi Wu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Control Strategies ◽  
Multidrug Resistant ◽  
Differentially Expressed ◽  
Effective Control ◽  
Drug Resistant ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Candidate Target

Rifampicin (RIF) is one of the most important first-line anti-tuberculosis (TB) drugs, and more than 90% of RIF-resistant (RR) Mycobacterium tuberculosis clinical isolates belong to multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. In order to identify specific candidate target proteins as diagnostic markers or drug targets, differential protein expression between drug-sensitive (DS) and drug-resistant (DR) strains remains to be investigated. In the present study, a label-free, quantitative proteomics technique was performed to compare the proteome of DS, RR, MDR, and XDR clinical strains. We found iniC, Rv2141c, folB, and Rv2561 were up-regulated in both RR and MDR strains, while fadE9, espB, espL, esxK, and Rv3175 were down-regulated in the three DR strains when compared to the DS strain. In addition, lprF, mce2R, mce2B, and Rv2627c were specifically expressed in the three DR strains, and 41 proteins were not detected in the DS strain. Functional category showed that these differentially expressed proteins were mainly involved in the cell wall and cell processes. When compared to the RR strain, Rv2272, smtB, lpqB, icd1, and folK were up-regulated, while esxK, PPE19, Rv1534, rpmI, ureA, tpx, mpt64, frr, Rv3678c, esxB, esxA, and espL were down-regulated in both MDR and XDR strains. Additionally, nrp, PPE3, mntH, Rv1188, Rv1473, nadB, PPE36, and sseA were specifically expressed in both MDR and XDR strains, whereas 292 proteins were not identified when compared to the RR strain. When compared between MDR and XDR strains, 52 proteins were up-regulated, while 45 proteins were down-regulated in the XDR strain. 316 proteins were especially expressed in the XDR strain, while 92 proteins were especially detected in the MDR strain. Protein interaction networks further revealed the mechanism of their involvement in virulence and drug resistance. Therefore, these differentially expressed proteins are of great significance for exploring effective control strategies of DR-TB.

scholarly journals Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes

2005 ◽  
Vol 10 (1) ◽  
pp. 56-66 ◽  
Author(s):  
Olga V. Trubetskoy ◽  
Jasmin R. Gibson ◽  
Bryan D. Marks
Keyword(s):  
Cytochrome P450 ◽  
High Throughput Screening ◽  
Cytochrome P450 Enzymes ◽  
Fluorogenic Substrates ◽  
Detailed Characterization ◽  
Human Cytochrome ◽  
Screening Assays ◽  
Well Assay

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid® fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid® fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC50 values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z′ factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format. ( Journal of Biomolecular Screening 2005:56-66)

scholarly journals The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

2014 ◽  
Vol 59 (2) ◽  
pp. 880-889 ◽  
Author(s):  
Wei Gao ◽  
Jin-Yong Kim ◽  
Jeffrey R. Anderson ◽  
Tatos Akopian ◽  
Seungpyo Hong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput Screening ◽  
Cyclic Peptide ◽  
Genome Mining ◽  
Subcutaneous Administration ◽  
Multiple Drug ◽  
Drug Resistant ◽  
Content Type

ABSTRACTDrug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition ofMycobacterium tuberculosisviability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity againstM. tuberculosisin vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains ofM. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition ofM. tuberculosisgrowth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistantM. tuberculosisstrains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target againstM. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

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