A Mechanism of Drug Action Revealed by Structural Studies of Enoyl Reductase

Baldock, Clair; Rafferty, John B.; Sedelnikova, Svetlana E.; Baker, Patrick J.; Stuitje, Antoine R.; Slabas, Antoni R.; Hawkes, Timothy R.; Rice, David W.

doi:10.1126/science.274.5295.2107

Cited by 236 publications

(230 citation statements)

References 22 publications

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“…Similarly, DZB was also able to inhibit InhA activity, although to a lesser extent than INH. The IC 50 was greater than 1000 M, a value consistent with the high MIC of this compound against M. smegmatis. TRC, a widely used biocide (58), was found to be very active against InhA with an IC 50 Ͻ1 M, which is in agreement with previous findings demonstrating that TRC inhibits mycobacterial InhA, both in vitro and in vivo (59,60).…”

Section: Induction Of the Kasa-containing Complex By Inha-inhibitory mentioning

confidence: 48%

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

Journal of Biological Chemistry

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(6) further demonstrated that the inhibition of mycolic acid biosynthesis mediated by INH leads to the accumulation of a saturated C 26 fatty acid within 1 h of INH treatment. By using scanning electron microscopy, it was established that M. tuberculosis treated with INH undergoes a defined order of molecular events that leads to lysis 24 h after initiation of treatment, thus after approximately one cell generation (7). The events that lead from the inhibition of mycolic acid biosynthesis and the accumulation of a saturated C 26 fatty acid to cell lysis are still poorly understood. Moreover, there exists controversy as to which enzymes of the mycolic acid biosynthetic pathway are the actual targets of INH.The mode of action of INH appears to be rather complex and requires activation by the mycobacterial catalase-peroxidase KatG (8,9). Activation of the pro-drug leads to reactive radicals that exert a toxic effect on the tubercle bacillus (9 -11). Presumably activated INH inhibits one or more targets of the mycolic acid biosynthetic pathway which eventually leads to cell death. At least two enzymes have been identified as potential targets of activated INH, the enoyl-ACP reductase InhA (12) and the ␤-ketoacyl-ACP synthase KasA (13). The inhA gene was first identified by conferring co-resistance to INH and ethionamide (ETH) after overexpression in mycobacteria (12). In addition, mutations in inhA confer resistance to both INH and ETH in drug-resistant M. tuberculosis isolates (14 -17).

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Section: Induction Of the Kasa-containing Complex By Inha-inhibitory mentioning

confidence: 48%

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Kremer

Dover

Morbidoni

et al. 2003

Journal of Biological Chemistry

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“…Dried samples were resuspended in 20 μL hexanes, spotted on a silica gel TLC plate, and chromatographed in a 70∶30∶2 (v∕v) mixture of hexanes∶ether∶ acetic acid. Radioactivity was quantified on a Packard Phosphorimager, using [1][2][3][4][5][6][7][8][9][10][11][12][13][14] ACP Analysis via Nondenaturing PAGE. An earlier protocol was used to separate individual acyl-ACP species in the reaction mixture (15).…”

Section: Methodsmentioning

confidence: 99%

“…Two cofactors are utilized in the process. FabG uses NADPH (10,11), whereas FabI preferentially utilizes NADH (12,13). To explore the dependence of the reconstituted FAS on the two acyl-CoA substrates and the two cofactors, we titrated these reactants individually into the system.…”

Section: Messenger Rna and Protein Concentrations Of Fatty Acid Synthasementioning

confidence: 99%

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Yu¹,

Liu

Zhu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

152

208

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Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM∕ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.D ue to their high energy density and low water solubility, fatty acids are arguably the most appropriate biofuel precursors. Therefore, fatty acid synthases (FASs) have emerged as attractive engineering targets in society's recent quest for transportation fuels from renewable sources. Among different FASs, the Escherichia coli synthase is perhaps most well understood (1). However, notwithstanding extensive analysis of fatty acid biosynthesis and its regulation in E. coli (2-5), little is known about its steady-state kinetic properties. We therefore sought to undertake systematic kinetic analysis of the fully reconstituted E. coli FAS. It was anticipated that such analysis would provide a fundamentally new basis for assessing the scope and limitations of producing fatty acids using E. coli as a biocatalyst.Fatty acid biosynthesis in E. coli is catalyzed by an enzyme system consisting of nine distinct proteins-FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and ACP (Fig. 1). Together, they convert one equivalent of acetyl-CoA and 6-8 equivalents of malonyl-CoA into C 14 -C 18 acyl-ACP species. One or two reducing equivalents of NAD(P)H are utilized in each round of chain elongation. Whereas most of the resulting fatty acyl chains are directly harnessed for phospholipid biosynthesis, the cytoplasmic mutant of the periplasmic thioesterase, TesA, is capable of releasing free fatty acids via hydrolysis of acyl-...

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“…[12][13][14][15][16][17][18][19][20] In this study we describe two classes of molecules in which alterations have been made to the diphenyl ether 'B' ring. In one series of compounds we have replaced the B ring with isosteric heterocycles that incorporate nitrogen atoms within the ring, thereby causing little steric perturbation to the overall structure of the molecule (Scheme 2).…”

mentioning

confidence: 99%

Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA inhibitors

Ende

Knudson

Liu

et al. 2008

Bioorganic & Medicinal Chemistry Letters

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In earlier work structure-based design studies resulted in the discovery of alkyl substituted diphenyl ether inhibitors of InhA, the enoyl reductase from Mycobacterium tuberculosis. Compounds such as 5-hexyl-2-phenoxyphenol 19 are nM inhibitors of InhA and inhibit the growth of both sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis with MIC 90 values of 1-2 µg/mL. However, despite their promising in vitro activity, these compounds have ClogP values of over 5. In efforts to reduce the lipophilicity of the compounds, and enhance potentially enhance compound bioavailability, a series of B ring analogues of 19 were synthesized that contained either heterocylic nitrogen rings or phenyl rings having amino, nitro, amide or piperazine functions. Compounds 3c, 3e and 14a show comparable MIC 90 values to that of 19, but have improved ClogP values. KeywordsTuberculosis; Diaryl ether; enoyl reductase; mycolic acids; InhA; isoniazid Tuberculosis (TB) is responsible for more than 1.6 million deaths per annum with 8.8 million new cases being reported each year. These numbers make TB one of the leading infectious causes of death, eclipsed only by AIDS. In addition, according to the World Health Organization, the number of multi-drug-resistant and extensively drug-resistant TB cases is growing with almost a half million new cases being reported each year. Therefore, there is an urgent need to develop novel TB chemotherapeutic agents. 1 *Corresponding authors, PJT: Tel.: (631) 632 7907; Email: peter.tonge@sunysb.edu, RAS: Tel.: (970) 491 1925; Email: richard.slayden@colostate.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In this study we describe two classes of molecules in which alterations have been made to the diphenyl ether 'B' ring. In one series of compounds we have replaced the B ring with isosteric heterocycles that incorporate nitrogen atoms within the ring, thereby causing little steric perturbation to the overall structure of the molecule (Scheme 2). The second series of compounds have nitro, amino, amide and piperazino functionalities incorporated at the ortho, meta, or para positions of the B ring (Scheme 3 and Scheme 4). This second series of compounds was synthesized not only to improve solubility but also to systematically identify positions on the B ring which could be substituted without diminishing biological activity. NIH Public AccessThe synthesis of the heterocyclic diaryl ether compounds was initiated either by nucleophilic aromatic substitution or by Buchwald-Hartwig cross-coupling of the appropriate nitrogen heterocycle with 4-b...

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A Mechanism of Drug Action Revealed by Structural Studies of Enoyl Reductase

Cited by 236 publications

References 22 publications

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

Inhibition of InhA Activity, but Not KasA Activity, Induces Formation of a KasA-containing Complex in Mycobacteria

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA inhibitors

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