cerulenin and thiolactomycin

As a result of increasing drug resistance in pathogenic bacteria, there is a critical need for novel broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria is an essential process for cell survival, the enzymes involved in the FAS pathway have emerged as promising targets for antimicrobial agents. Several lines of evidence have indicated that bacterial condensing enzymes are central to the initiation and elongation steps in bacterial fatty acid synthesis and play a pivotal role in the regulation of the entire fatty acid synthesis pathway. beta-ketoacyl-acyl carrier protein (ACP) synthases (KAS) from various bacterial species have been cloned, expressed and purified in large quantities for detailed enzymological, structural and screening studies. Availability of purified KAS from a variety of bacteria, along with a combination of techniques, including combinatorial chemistry, high-throughput screening, and rational drug design based on crystal structures, will undoubtedly aid in the discovery and development of much needed potent and broad-spectrum antibacterial agents. In this review we summarize the biochemical, biophysical and inhibition properties of beta-ketoacyl-ACP synthases from a variety of bacterial species.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Anti-Bacterial Agents; Bacterial Proteins; Catalytic Domain; Cerulenin; Enzyme Inhibitors; Indoles; Thiophenes

Escherichia coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), traveler's diarrhea and other clinical infections such as neonatal meningitis and pneumonia. The fatty acid biosynthesis is essential for the bacterial viability and growth. There are three types of β-ketoacyl acyl carrier protein synthase (KAS) which are important for overcoming the bacterial resistance problem. β-ketoacyl acyl carrier protein synthase I (KAS I) is member of the condensing enzyme family, which is a key catalyst in bacterial fatty acid biosynthesis, and thus an attractive target for novel antibioticsis related to the elongation of unsaturated fatty acids in bacterial fatty acid synthesis and can be a good therapeutic target of designing novel antibiotics. In this report, we performed docking study of E. coli (KAS I) and 50 flavonoids. Out of these 50 flavonoids, there are two compounds, genistein and isorhamnetin, that showed the superior binding energy while fully satisfying the conditions of drug likeliness. The predicted binding energy of genistein and isorhamnetin toward KAS I are -135.76kcal/mol and -132.42kcal/mol, respectively. These energies favorably compare to the biding energy of known drugs thiolactomicin and cerulenin that are -90.26kcal/mol and -99.64kcal/mol, respectively. The method used was docking with the selected E. coli (KAS I-PDB ID-1FJ4) using iGemdock. This was also found to obey the Lipinski's guidelines of five and to show the drug likeliness and bioavailability.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Amino Acid Motifs; Anti-Bacterial Agents; Cerulenin; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Genistein; High-Throughput Screening Assays; Isoenzymes; Molecular Docking Simulation; Molecular Sequence Data; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Quercetin; Structure-Activity Relationship; Thermodynamics; Thiophenes; User-Computer Interface

Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413-K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.

Topics: Adamantane; Aminobenzoates; Anilides; Cerulenin; Fatty Acid Synthases; Models, Molecular; Protein Structure, Secondary; Protein Structure, Tertiary; Saccharomyces cerevisiae; Substrate Specificity; Thiophenes

Fatty acids are essential for bacterial growth and viability, with the type II fatty acid synthesis (FAS II) pathway being a potential antibacterial target. A new, selective, and highly sensitive whole cell-based antisense strategy has been designed to screen for natural product inhibitors of FabH/F of the FAS II pathway using a high-throughput two-plate agar-based differential sensitivity assay (FabF(2)p). An antisense assay along with the FASII enzyme prepared from Staphylococcus aureus was used for bioactivity-guided fractionation, leading to the isolation of phomallenic acids A-C (1-3) from a leaf litter fungus identified as Phoma sp. Compounds 1-3 exhibited minimum detection concentrations (MDC) of 0.63, 0.31, and 0.15 microg/mL in the FabF(2P) assay, IC(50) values of 22, 3.4, and 0.77 microg/mL in the FASII enzyme assay, and minimum inhibitory concentrations (MIC) of 250, 7.8, and 3.9 microg/mL, respectively, against wild-type S. aureus. Phomallenic acid C (3), the analogue with the longest chain, exhibited the best overall activity within the phomallenic acids obtained and was superior to cerulenin and thiolactomycin, the two most studied and commonly used FabF inhibitors.

Topics: Acetyltransferases; Alkadienes; Anti-Bacterial Agents; Ascomycota; Fatty Acid Synthase, Type II; Fatty Acid Synthases; Fatty Acids; France; Methicillin Resistance; Microbial Sensitivity Tests; Molecular Structure; Multienzyme Complexes; Staphylococcus aureus; Thiophenes

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.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Binding Sites; Cerulenin; Enzyme Inhibitors; Isoenzymes; Structure-Activity Relationship; Thiophenes

Mycolic acids are vital components of the Mycobacterium tuberculosis cell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performs de novo fatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two beta-ketoacyl-ACP synthase (KAS) enzymes of the M. tuberculosis FASII system. KasA and KasB were expressed in E. coli and purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use of M. tuberculosis AcpM, suggesting that structural differences between AcpM and E. coli ACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Acetyltransferases; Anti-Bacterial Agents; Antifungal Agents; Bacterial Proteins; Carbon; Cerulenin; Chromatography, Affinity; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Escherichia coli; Fatty Acid Synthase, Type II; Fatty Acids; Gene Deletion; Inhibitory Concentration 50; Kinetics; Models, Chemical; Multienzyme Complexes; Mycobacterium tuberculosis; Mycolic Acids; Phylogeny; Protein Binding; Substrate Specificity; Thiophenes; Time Factors

We report here the molecular analysis of a Type I fatty acid synthase in the apicomplexan Cryptosporidium parvum (CpFAS1). The CpFAS1 gene encodes a multifunctional polypeptide of 8243 amino acids that contains 21 enzymatic domains. This CpFAS1 structure is distinct from that of mammalian Type I FAS, which contains only seven enzymatic domains. The CpFAS1 domains are organized into: (i) a starter unit consisting of a fatty acid ligase and an acyl carrier protein; (ii) three modules, each containing a complete set of six enzymes (acyl transferase, ketoacyl synthase, ketoacyl reductase, dehydrase, enoyl reductase, and acyl carrier protein) for the elongation of fatty acid C2-units; and (iii) a terminating domain whose function is as yet unknown. The CpFAS1 gene is expressed throughout the life cycle of C. parvum, since its transcripts and protein were detected by RT-PCR and immunofluorescent localization, respectively. This cytosolic Type I CpFAS1 differs from the organellar Type II FAS enzymes identified from Toxoplasma gondii and Plasmodium falciparum which are targetted to the apicoplast, and are sensitive to inhibition by thiolactomycin. That the discovery of CpFAS1 may provide a new biosynthetic pathway for drug development against cryptosporidiosis, is indicated by the efficacy of the FAS inhibitor cerulenin on the growth of C. parvum in vitro.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Cerulenin; Cryptosporidium parvum; Fatty Acid Synthases; Fluorescent Antibody Technique; Genes, Protozoan; Humans; Molecular Sequence Data; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Thiophenes

African trypanosomes, the cause of sleeping sickness, need massive amounts of myristate to remodel glycosyl phosphatidylinositol (GPI) anchors on their surface glycoproteins. However, it has been believed that the parasite is unable to synthesize any fatty acids, and myristate is not abundant in the hosts' bloodstreams. Thus, it has been unclear how trypanosomes meet their myristate requirement. Here we found that they could indeed synthesize fatty acids. The synthetic pathway was unique in that the major product, myristate, was preferentially incorporated into GPIs and not into other lipids. The antibiotic thiolactomycin inhibited myristate synthesis and killed the parasite, making this pathway a potential chemotherapeutic target.

Topics: Acyl Coenzyme A; Animals; Anti-Bacterial Agents; Caprylates; Cell-Free System; Cerulenin; Decanoic Acids; Fatty Acid Synthases; Fatty Acids; Glycosylphosphatidylinositols; Laurates; Myristates; Subcellular Fractions; Thiophenes; Trypanosoma brucei brucei

Many strains of Pseudomonas aeruginosa are resistant to the antibiotics cerulenin and thiolactomycin, potent inhibitors of bacterial fatty acid biosynthesis. A novel yeast Flp recombinase-based technique was used to isolate an unmarked mexAB-oprM deletion encoding an efflux system mediating resistance to multiple antibiotics in P. aeruginosa. The experiments showed that the MexAB-OprM system is responsible for the intrinsic resistance of this bacterium to cerulenin and thiolactomycin. Whereas thiolactomycin was not a substrate of the MexCD-OprJ pump expressed in a delta(mexAB-oprM) nfxB mutant, cerulenin was efficiently effluxed by the MexCD-OprJ system. It was also found that the MexAB-OprM system is capable of efflux of irgasan, a broad-spectrum antimicrobial compound used in media selective for Pseudomonas.

Topics: Anti-Bacterial Agents; Cerulenin; Chromosomes, Bacterial; Drug Resistance, Microbial; Fatty Acids; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Thiophenes

Topics: Cerulenin; Fatty Acid Synthases; Fatty Acids; Pisum sativum; Structure-Activity Relationship; Thiophenes

beta-ketoacyl-ACP synthetase III (KAS III) has been purified from avocado using a six-step purification procedure. The enzyme, which is cerulenin-insensitive and thiolactomycin-sensitive, was assayed using a partial component reaction: acetyl CoA:ACP transacylase (ACAT) activity. KAS III activity is distinguished from ACAT activity on the basis that the former is highly stimulated by the addition of malonyl CoA in the presence of malonyl-CoA:ACP transacylase, and the latter is not. KAS III and ACAT activity have been separated from each other thus providing the first evidence that these two discrete activities exist in higher plants. Both of these enzymes have been implicated in the initial reactions of fatty acid synthesis. KAS III was purified 134-fold using a combination of PEG precipitation, Fast Q, ammonium sulphate precipitation, Phenyl Sepharose and ACP-affinity chromatography. The enzyme requires Triton X-100 for solubility and is highly salt sensitive. The subunit molecular mass of 37 kDa has been identified by SDS-PAGE. The results of gel filtration analysis are consistent with the native enzyme being homodimeric. The native molecular mass of KAS III is 69 kDa and that of ACAT 18.5 kDa. The enzyme has a pH optimum of 7.0-7.5, which is similar to the pH optimum of the ACAT reaction. The Km for acetyl CoA is 12.5 microM and the Km for malonyl-ACP is 14 microM. Both KAS III and ACAT are sensitive to thiolactomycin inhibition. The results are discussed with respect to the potential role of acetyl CoA:ACP transacylase in plants.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Acetyl Coenzyme A; Acetyltransferases; Acyl-Carrier Protein S-Acetyltransferase; Buffers; Cerulenin; Fruit; Hydrogen-Ion Concentration; Kinetics; Malonyl Coenzyme A; Molecular Weight; Thiophenes

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.

Topics: Biological Transport; Cerulenin; Chromosome Mapping; Drug Resistance, Microbial; Escherichia coli; Fatty Acid Synthases; Genes, Bacterial; Malonyl Coenzyme A; Operon; Thiophenes

Thiolactomycin, an antibiotic with the structure of (4S)-(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-++ +thiolide, selectively inhibits type II fatty acid synthases. The mode of the thiolactomycin action on the fatty acid synthase system of Escherichia coli was investigated. Of the six individual enzymes of the fatty acid synthase system, [acyl-carrier-protein] (ACP) acetyltransferase and 3-oxoacyl-ACP synthase were inhibited by thiolactomycin. On the other hand, the other enzymes were not affected by this antibiotic. The thiolactomycin inhibition of the fatty acid synthase system was reversible. As to ACP acetyltransferase, the inhibition was competitive with respect to ACP and uncompetitive with respect to acetyl-CoA. As to 3-oxoacyl-ACP synthase, the inhibition was competitive with respect to malonyl-ACP and noncompetitive with respect to acetyl-ACP. The thiolactomycin action on the fatty acid synthase system was compared with that of cerulenin.

Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Acetyltransferases; Acyl-Carrier Protein S-Acetyltransferase; Anti-Bacterial Agents; Cerulenin; Escherichia coli; Fatty Acid Synthases; Lactones; Thiophenes

Thiolactomycin, an antibiotic with the structure of (4S)-(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide, inhibits the incorporation of [14C]acetate into cellular fatty acids of Escherichia coli. This antibiotic inhibits the fatty acid synthetase system of E. coli. However, the fatty acid synthetases from Saccharomyces cerevisiae, Candida albicans and rat liver are insensitive to thiolactomycin. This effect may account for the antibacterial activity of thiolactomycin and for its low toxicity in animals.

Topics: Acetates; Animals; Anti-Bacterial Agents; Candida albicans; Cerulenin; Escherichia coli; Fatty Acid Synthases; Fatty Acids; Kinetics; Lactones; Liver; Rats; Saccharomyces cerevisiae; Species Specificity; Structure-Activity Relationship; Thiophenes