Novel Small Molecule Inhibitors of MDR <i>Mycobacterium tuberculosis</i> by NMR Fragment Screening of Antigen 85C

Scheich, Christoph; Puetter, Vera; Schade, Markus

doi:10.1021/jm100993z

Cited by 32 publications

(36 citation statements)

References 25 publications

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“…Ag85C also showed chemical shift changes with OSG, again concordant with published results (Fig. 4B) (39). The interaction of Ag85C with OSG and I3-AG85 was also observed in physiological PBS buffer at pH 7.0 with a low DMSO concentration of 0.5% but without the detergent CHAPS (see Fig.…”

Section: I3-ag85 Binds To Ag85c I1-ag85 Binding To Ag85c Has Beensupporting

confidence: 91%

“…Binding of I3-AG85 or OSG to Ag85A, -B, and -C was monitored by two series of 15 N-1 H heteronuclear singlequantum correlation spectroscopy (HSQC) experiments using either (i) PBS buffer with 0.5% DMSO at pH 7.0 or (ii) 5 mM citrate buffer (pH 6.0) containing 1 mM dithiothreitol (DTT), 0.5% DMSO, and 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) to achieve better resolution. With Ag85C, an additional experiment was performed with the latter buffer modified using 5% DMSO to generate results comparable to those obtained by Scheich et al (39). The protein concentration was 30 M. The compounds were added from a stock solution in DMSO to yield a concentration of 600 M and a DMSO concentration of 0.5%.…”

Section: Methodsmentioning

confidence: 97%

“…demonstrated by recent NMR studies (39). Here, the binding of the derivative I3-AG85 to Ag85C was investigated by 15 N-1 H HSQC using three different buffer systems containing different amounts of DMSO or including/excluding CHAPS (see Materials and Methods).…”

Section: I3-ag85 Binds To Ag85c I1-ag85 Binding To Ag85c Has Beenmentioning

confidence: 99%

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Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

Warrier

Tropis

Werngren

et al. 2012

Antimicrob Agents Chemother

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ABSTRACTThe antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital forMycobacterium tuberculosisdue to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential forM. tuberculosisvirulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition ofM. tuberculosisdivision in broth culture. I3-AG85 also inhibitedM. tuberculosissurvival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistantM. tuberculosisstrains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of theM. tuberculosisenvelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of theM. tuberculosisenvelope. I3-AG85 inhibited the division of and reduced TDM synthesis in anM. tuberculosisstrain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.

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Section: I3-ag85 Binds To Ag85c I1-ag85 Binding To Ag85c Has Beensupporting

confidence: 91%

Section: Methodsmentioning

confidence: 97%

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Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

Warrier

Tropis

Werngren

et al. 2012

Antimicrob Agents Chemother

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“…Fragment-based screening has also been utilized in identifying new leads for Ag85 inhibition [146, 147]. Fragments 109 - 112 (Figure 22) have MIC values of 1.3 mM, 250 μM, 100 μM and 50 μM, respectively, against whole cell M. tb H37Rv [147].…”

Section: New Inhibitors Of the Mycolic Acid Biosynthetic Pathwaymentioning

confidence: 99%

New Approaches to Target the Mycolic Acid Biosynthesis Pathway for the Development of Tuberculosis Therapeutics

North¹,

Jackson²,

Lee³

2013

CPD

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Mycolic acids are the major lipid component of the unique mycobacterial cell wall responsible for the protection of the tuberculosis bacilli from many outside threats. Mycolic acids are synthesized in the cytoplasm and transported to the outer membrane as trehalose-containing glycolipids before being esterified to the arabinogalactan portion of the cell wall and outer membrane glycolipids. The large size of these unique fatty acids is a result of a huge metabolic investment that has been evolutionarily conserved, indicating the importance of these lipids to the mycobacterial cellular survival. There are many key enzymes involved in the mycolic acid biosynthetic pathway, including fatty acid synthesis (KasA, KasB, MabA, InhA, HadABC), mycolic acid modifying enzymes (SAM-dependent methyltransferases, aNAT), fatty acid activating and condensing enzymes (FadD32, Acc, Pks13), transporters (MmpL3) and tranferases (Antigen 85A-C) all of which are excellent potential drug targets. Not surprisingly, in recent years many new compounds have been reported to inhibit specific portions of this pathway, discovered through both phenotypic screening and target enzyme screening. In this review, we analyze the new and emerging inhibitors of this pathway discovered in the post-genomic era of tuberculosis drug discovery, several of which show great promise as selective tuberculosis therapeutics.

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“…Moreover, I3-AG85 was active against a panel of MDR/XDR strains although it exhibited an MIC of 100 µM (25). By combining fragment-based drug discovery with early whole cell antibacterial screening, tetrahydro-1-benzothiophene analogs were discovered as potent Ag85C inhibitory molecules against drugsusceptible and drug-resistant M. tuberculosis strains (26). The selenazole compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) was found to inhibit the activity of Ag85C through an original mechanism by reacting with the conserved Cys209 residue located near the active site of the enzyme but not involved in the catalytic activity (27,28).…”

Section: Introductionmentioning

confidence: 99%

Cyclipostins and cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo

Viljoen

Richard

Nguyen

et al. 2018

Journal of Biological Chemistry

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An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of Cyclipostins and Cyclophostin (CyC) analogs exhibiting potent activity against Mycobacterium tuberculosis both in vitro and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex (i.e. Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate (TDM) and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC7β, CyC8β, and CyC17 bind covalently to the catalytic Ser124 residue in Ag85C, inhibit mycolyltransferase activity, i.e. the transfer of a fatty acid molecule onto trehalose, and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC8β disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of M. tuberculosis cultures revealed that the CyC compounds impair both TDM synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex in vitro and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against M. tuberculosis. http://www.jbc.org/cgi

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Novel Small Molecule Inhibitors of MDR Mycobacterium tuberculosis by NMR Fragment Screening of Antigen 85C

Cited by 32 publications

References 25 publications

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

New Approaches to Target the Mycolic Acid Biosynthesis Pathway for the Development of Tuberculosis Therapeutics

Cyclipostins and cyclophostin analogs inhibit the antigen 85C from Mycobacterium tuberculosis both in vitro and in vivo

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