The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Gurdyal S Besra - One of the best experts on this subject based on the ideXlab platform.
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and Development,
2013Co-Authors: James D. Douglas, Gurdyal S Besra, David E Minnikin, Ian B Campbell, Caroline Morehouse, Benjawan Phetsukiri, Suzanne J., Centre Gunnels WoodAbstract:Analogues of the antibiotic Thiolactomycin (TLM) have been synthesized and have been shown to have enhanced activity against whole cells of Mycobacterium tuberculosis H37Rv and against mycolic acid biosynthesis in cell extracts of Mycobacterium smegmatis. TLM has a methyl-branched butadienyl side chain attached at position 5 on a ‘thiolactone ’ ring, namely 4-hydroxy-3,5dimethyl-5H-thiophen-2-one. Various combinations of strong bases were explored to create a reactive anion at position 5 on the thiolactone ring which could react with halides to produce 5-substituted derivatives; the best reagent was two equivalents of lithium-bis-(trimethylsilyl)amide in tetrahydrofuran. The analogue with a 5-tetrahydrogeranyl substituent showed the best biological activity with an MIC 90 for M. tuberculosis of 29 µM and 92% mycolate inhibition in extracts of M. smegmatis, as compared to 125 µM and 54%, respectively, for TLM; other related
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The chemical structures of Thiolactomycin 1, its analogue 4, and inhibitors 2 and 3.
2013Co-Authors: Qosay Al-balas, Gurdyal S Besra, Nahoum G. Anthony, Bilal Al-jaidi, Amani Alnimr, Grainne Abbott, Alistair K. Brown, Rebecca C. Taylor, Timothy D. Mchugh, Stephen H. GillespieAbstract:The chemical structures of Thiolactomycin 1, its analogue 4, and inhibitors 2 and 3.
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Synthesis and biological evaluation of a C5-biphenyl Thiolactomycin library.
Bioorganic & medicinal chemistry letters, 2007Co-Authors: Veemal Bhowruth, Alistair K. Brown, Suzanne J Senior, John S. Snaith, Gurdyal S BesraAbstract:Abstract Fifteen novel C5 analogues of Thiolactomycin (13 biphenyl analogues and two biphenyl mimics) have been synthesised and assessed for their in vitro mtFabH and whole cell Mycobacterium bovis BCG activity, respectively. Analysis of the 15 compounds revealed that six possessed enhanced in vitro activity in a direct mtFabH assay. Encouragingly analogues 11, 12 and 13 gave a significant enhancement in in vitro activity against mtFabH. Analogue 13 (5-(4-methoxycarbonyl-biphenyl-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one) gave an IC50 value of 3 μM compared to the parent drug Thiolactomycin (75 μM) against mtFabH. The biological analysis of this library reaffirms the requirement for a linear π-rich system containing hydrogen bond accepting substituents attached to the para-position of the C5 biphenyl analogue to generate compounds with enhanced activity.
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Acetylene-based analogues of Thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme.
Bioorganic & medicinal chemistry letters, 2004Co-Authors: Suzanne J Senior, David E Minnikin, Petr A Illarionov, Sudagar S Gurcha, Ian B Campbell, Merrill L Schaeffer, Gurdyal S BesraAbstract:Analogues of the natural antibiotic Thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis β-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to Thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic Thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.
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Biphenyl-based analogues of Thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme.
Bioorganic & medicinal chemistry letters, 2003Co-Authors: Suzanne J Senior, David E Minnikin, Petr A Illarionov, Sudagar S Gurcha, Ian B Campbell, Merrill L Schaeffer, Gurdyal S BesraAbstract:Analogues of the antibiotic Thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-(4'-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to Thiolactomycin.
Charles O. Rock - One of the best experts on this subject based on the ideXlab platform.
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The reductase steps of the type II fatty acid synthase as antimicrobial targets
Lipids, 2004Co-Authors: Yong-mei Zhang, Charles O. RockAbstract:The increasing of multidrug resistance of clinically important pathogens calls for the development of novel antibiotics with unexploited cellular targets. FA biosynthesis in bacteria is catalyzed by a group of highly conserved proteins known as the type II FA synthase (FAS II) system. Bacteria FAS II organization is distinct from its mammalian counterpart; thus the FAS II pathway offers several unique steps for selective inhibition by antibacterial agents. Some known antibiotics that target the FAS II system include triclosan, isoniazid, and Thiolactomycin. Recent years have seen remarkable progress in the understanding of the genetics, biochemistry, and regulation of the FAS II system with the availability of the complete geome, sequence for many bacteria. Crystal structures of the FAS II pathway enzymes have been determined for not only the Escherichia coli model system but also other gram-netative and gram-positive pathogens. The protein structures have greatly facilitated structure-based design of novel inhibitors and the improvement of existing antibacterial agents. This review discusses new developments in the discovery of inhibitors that specifically target the two reductase steps of the FAS II system, β-ketoacyl-acyl carrier potein (ACP) reductase and enoyl-ACP reductase.
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Fatty acid biosynthesis as a target for novel antibacterials.
Current opinion in investigational drugs (London England : 2000), 2004Co-Authors: Richard J. Heath, Charles O. RockAbstract:The bacterial fatty acid synthesis pathway has significant potential as a target for the development of novel antibacterials. The pathway has been extensively studied in Escherichia coli, the crystal structures of the compounds involved are known and homologous genes are readily identified in the genomes of important pathogens. The, currently used drugs triclosan and isoniazid are known to target one step in the pathway. Other experimental compounds such as Thiolactomycin and cerulenin effectively inhibit other steps. These known pathway inhibitors are reviewed and the areas for potential future developments are explored.
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The Claisen condensation in biology
Natural product reports, 2002Co-Authors: Richard J. Heath, Charles O. RockAbstract:Covering: 1997–2002 The mechanism for carbon–carbon bond formation used in the biosynthesis of natural products such as fatty acids and polyketides is a decarboxylating Claisen condensation. The enzymes that catalyze this reaction in various bacterial systems, collectively referred to as condensing enzymes, have been intensively studied in the past several decades, and members of the family have been crystallized. The condensing enzymes share a common 3-dimensional fold, first described for the biosynthetic thiolase I that catalyzes a non-decarboxylating Claisen condensation, although they share little similarity at the amino acid level. Their active sites, however, possess significant similarities. The initiation condensing enzymes use CoA primers and possess a catalytic triad of Cys, His, Asn; and the elongating condensing enzymes that exclusively use ACP thioesters have a triad of Cys, His, His. These active site differences affect the sensitivity of the respective enzymes to the antibiotics Thiolactomycin and cerulenin. Different reaction mechanisms have been proposed for the condensing enzymes. This review covers the recent structural and mechanistic data to see if a unifying hypothesis for the reaction mechanism catalyzed by this important family of enzymes can be established.
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inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics
Applied Microbiology and Biotechnology, 2002Co-Authors: Richard J. Heath, Stephen W White, Charles O. RockAbstract:Fatty acid biosynthesis is an emerging target for the development of novel antibacterial chemotherapeutics. The dissociated bacterial system is substantially different from the large, multifunctional protein of mammals, and many possibilities exist for type-selective drugs. Several compounds, both synthetic and natural, target bacterial fatty acid synthesis. Three compounds target the FabI enoyl-ACP reductase step; isoniazid, a clinically used antituberculosis drug, triclosan, a widely used consumer antimicrobial, and diazaborines. In addition, cerulenin and Thiolactomycin, two fungal products, inhibit the FabH, FabB and FabF condensation enzymes. Finally, the synthetic reaction intermediates BP1 and decynoyl-N-acetyl cysteamine inhibit the acetyl-CoA carboxylase and dehydratase isomerase steps, respectively. The mechanisms of action of these compounds, as well as the potential development of new drugs targeted against this pathway, are discussed.
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inhibition of beta ketoacyl acyl carrier protein synthases by Thiolactomycin and cerulenin structure and mechanism
Journal of Biological Chemistry, 2001Co-Authors: Allen C. Price, Richard J. Heath, Stephen W White, Keum-hwa Choi, Charles O. RockAbstract:The beta-ketoacyl-acyl carrier protein (ACP) synthases are key regulators of type II fatty acid synthesis and are the targets for two natural products, Thiolactomycin (TLM) and cerulenin. The high resolution structures of the FabB-TLM and FabB-cerulenin binary complexes were determined. TLM mimics malonyl-ACP in the FabB active site. It forms strong hydrogen bond interactions with the two catalytic histidines, and the unsaturated alkyl side chain interaction with a small hydrophobic pocket is stabilized by pi stacking interactions. Cerulenin binding mimics the condensation transition state. The subtle differences between the FabB-cerulenin and FabF-cerulenin (Moche, M., Schneider, G., Edwards, P., Dehesh, K., and Lindqvist, Y. (1999) J. Biol. Chem. 244, 6031-6034) structures explain the differences in the sensitivity of the two enzymes to the antibiotic and may reflect the distinct substrate specificities that differentiate the two enzymes. The FabB[H333N] protein was prepared to convert the FabB His-His-Cys active site triad into the FabH His-Asn-Cys configuration to test the importance of the two His residues in TLM and cerulenin binding. FabB[H333N] was significantly more resistant to both antibiotics than FabB and had an affinity for TLM an order of magnitude less than the wild-type enzyme, illustrating that the two-histidine active site architecture is critical to protein-antibiotic interaction. These data provide a structural framework for understanding antibiotic sensitivity within this group of enzymes.
Bradley S. Moore - One of the best experts on this subject based on the ideXlab platform.
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Engineering Salinispora tropica for heterologous expression of natural product biosynthetic gene clusters.
Applied Microbiology and Biotechnology, 2018Co-Authors: Jia Jia Zhang, Bradley S. Moore, Xiaoyu TangAbstract:The marine actinomycete genus Salinispora is a remarkably prolific source of structurally diverse and biologically active secondary metabolites. Herein, we select the model organism Salinispora tropica CNB-440 for development as a heterologous host for the expression of biosynthetic gene clusters (BGCs) to complement well-established Streptomyces host strains. In order to create an integratable host with a clean background of secondary metabolism, we replaced three genes (salA-C) essential for salinosporamide biosynthesis with a cassette containing the Streptomyces coelicolor ΦC31 phage attachment site attB to generate the mutant S. tropica CNB-4401 via double-crossover recombination. This mutagenesis not only knocks-in the attachment site attB in the genome of S. tropica CNB-440 but also abolishes production of the salinosporamides, thereby simplifying the strain's chemical background. We validated this new heterologous host with the successful integration and expression of the Thiolactomycin BGC that we recently identified in several S. pacifica strains. When compared to the extensively engineered superhost S. coelicolor M1152, the production of Thiolactomycins from S. tropica CNB-4401 was approximately 3-fold higher. To the best of our knowledge, this is the first example of using a marine actinomycete as a heterologous host for natural product BGC expression. The established heterologous host may provide a useful platform to accelerate the discovery of novel natural products and engineer biosynthetic pathways.
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Enzymatic C-H Oxidation-Amidation Cascade in the Production of Natural and Unnatural Thiotetronate Antibiotics with Potentiated Bioactivity.
Angewandte Chemie (International ed. in English), 2017Co-Authors: Xiaoyu Tang, Takayoshi Awakawa, Bradley S. MooreAbstract:The selective activation of unreactive hydrocarbons by biosynthetic enzymes has inspired new synthetic methods in C-H bond activation. Herein, we report the unprecedented two-step biosynthetic conversion of thiotetromycin to thiotetroamide C involving the tandem oxidation and amidation of an unreactive ethyl group. We detail the genetic and biochemical basis for the terminal amidation in thiotetroamide C biosynthesis, which involves a uniquely adapted cytochrome P450-amidotransferase enzyme pair and highlights the first oxidation-amidation enzymatic cascade reaction leading to the selective formation of a primary amide group from a chemically inert alkyl group. Motivated by the ten-fold increase in antibiotic potency of thiotetroamide C ascribed to the acetamide group and the unusual enzymology involved, we enzymatically interrogated diverse Thiolactomycin analogues and prepared an unnatural thiotetroamide C analogue with potentiated bioactivity compared to the parent molecule.
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minimization of the Thiolactomycin biosynthetic pathway reveals that the cytochrome p450 enzyme tlmf is required for five membered thiolactone ring formation
ChemBioChem, 2017Co-Authors: Xiaoyu Tang, Bradley S. Moore, Jie LiAbstract:: Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered δ-Thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.
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Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation
Chembiochem : a European journal of chemical biology, 2017Co-Authors: Xiaoyu Tang, Bradley S. MooreAbstract:Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics that inhibit bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years ago, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results revealed that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in sulfur insertion and C-S bond formation chemistry but rather in the construction of the five-membered thiolactone ring as, upon its deletion, we observed the alternative production of the six-membered δ-Thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.
Kevin A. Reynolds - One of the best experts on this subject based on the ideXlab platform.
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Biosynthetic origins of the natural product, Thiolactomycin: a unique and selective inhibitor of type II dissociated fatty acid synthases.
Journal of the American Chemical Society, 2003Co-Authors: Maria S. Brown, Hamish A I Mcarthur, Konstantin Akopiants, Diane M Resceck, Ellen L. Mccormick, Kevin A. ReynoldsAbstract:Thiolactomycin (TLM), a natural product produced by both Nocardia and Streptomyces spp., is a potent and highly selective inhibitor of the type II dissociated fatty acid synthases of plants and bacteria. The unique mode of action of TLM and its low toxicity make it an attractive compound for development of new antimicrobial agents. In this study, incorporation studies with 13C-labeled precursors demonstrate that TLM is derived from one acetate-derived starter unit and three methylmalonate-derived extender units. The unusual thiolactone represented by TLM represents a novel class of polyketide-derived antibiotics in which an unusual cyclization process, which terminates the biosynthetic pathway, involves incorporation of a sulfur atom from l-cysteine. Manipulation of this pathway through techniques such a combinatorial biosynthesis and mutasynthesis may provide a new route for economically viable production of useful TLM analogues.
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Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis
Journal of bacteriology, 1998Co-Authors: Lei Han, Sandra Lobo, Kevin A. ReynoldsAbstract:The Streptomyces glaucescens fabH gene, encoding beta-ketoacyl-acyl carrier protein (beta-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an Mr of 37,000, while gel filtration analysis determined a native Mr of 72,000 +/- 3,000 (mean +/- standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to Thiolactomycin inhibition. The KAS activity of the protein demonstrated a Km value of 3.66 microM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 microM for acetyl-CoA, 0.71 microM for butyryl-CoA, and 0.41 microM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using Thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.
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In vivo and in vitro effects of Thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.
Journal of Bacteriology, 1997Co-Authors: Kimberlee K. Wallace, Hamish A I Mcarthur, S Lobo, Kevin A. ReynoldsAbstract:A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by Thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of Thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of Thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of Thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to Thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.
Peter J. Tonge - One of the best experts on this subject based on the ideXlab platform.
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Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity.
Journal of medicinal chemistry, 2016Co-Authors: Gopal R. Bommineni, R A Slayden, Kanishk Kapilashrami, Jason E. Cummings, Susan E. Knudson, Stephen G. Walker, Peter J. TongeAbstract:β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including Thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-Thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection.
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Thiolactomycin-Based Inhibitors of Bacterial β‑Ketoacyl-ACP Synthases with in Vivo Activity
2016Co-Authors: Gopal R. Bommineni, R A Slayden, Kanishk Kapilashrami, Jason E. Cummings, Stephen G. Walker, Susan E. Knudson, Peter J. TongeAbstract:β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including Thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-Thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection
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structural basis for the recognition of mycolic acid precursors by kasa a condensing enzyme and drug target from mycobacterium tuberculosis
Journal of Biological Chemistry, 2013Co-Authors: Johannes Schiebel, Gopal R. Bommineni, Kanishk Kapilashrami, Peter J. Tonge, Agnes Fekete, C M Schaefer, Martin J Mueller, Caroline KiskerAbstract:Abstract The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several Thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.
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Thiolactomycin based β ketoacyl acpm synthase a kasa inhibitors fragment based inhibitor discovery using transient one dimensional nuclear overhauser effect nmr spectroscopy
Journal of Biological Chemistry, 2013Co-Authors: Kanishk Kapilashrami, Pilho Kim, Gopal R. Bommineni, Carl A. Machutta, Cheng Tsung Lai, Carlos Simmerling, Francis Picart, Peter J. TongeAbstract:Thiolactomycin (TLM) is a natural product inhibitor of KasA, the β-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy.
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Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin
The Journal of biological chemistry, 2009Co-Authors: Carl A. Machutta, Gopal R. Bommineni, Kanishk Kapilashrami, Sylvia R. Luckner, Caroline Kisker, Carlos Simmerling, Bela Ruzsicska, Peter J. TongeAbstract:Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices α5 and α6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100–200 μg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.
