Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

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.     

Synthesis and biological evaluation of a C5-biphenyl thiolactomycin library

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 IC(50) value of 3 microM compared to the parent drug thiolactomycin (75 microM) against mtFabH. The biological analysis of this library reaffirms the requirement for a linear pi-rich system containing hydrogen bond accepting substituents attached to the para-position of the C5 biphenyl analogue to generate compounds with enhanced activity.     

Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the β-Ketoacyl-ACP Synthase mtFabH

Background

Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world''s population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration.

Methodology/Principal Findings

Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM''s novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H₃₇R_v and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H₃₇R_v with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC₅₀ of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism.

Conclusions/Significance

These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.     

Identification and substrate specificity of beta -ketoacyl (acyl carrier protein) synthase III (mtFabH) from Mycobacterium tuberculosis

The long-chain alpha-alkyl-beta-hydroxy fatty acids, termed mycolic acids, which are characteristic components of the mycobacterial cell wall are produced by successive rounds of elongation catalyzed by a multifunctional (type I) fatty acid synthase complex followed by a dissociated (type II) fatty acid synthase. In bacterial type II systems, the first initiation step in elongation is the condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) catalyzed by beta-ketoacyl-ACP III (FabH). An open reading frame in the Mycobacterium tuberculosis genome (Rv0533c), now termed mtfabH, was 37.3% identical to Escherichia coli ecFabH and contained the Cys-His-Asn catalytic triad signature. However, the purified recombinant mtFabH clearly preferred long-chain acyl-CoA substrates rather than acyl-ACP primers and did not utilize acetyl-CoA as a primer in comparison to ecFabH. In addition, purified mtFabH was sensitive to thiolactomycin and resistant to cerulenin in an in vitro assay. However, mtFabH overexpression in Mycobacterium bovis BCG did not confer thiolactomycin resistance, suggesting that mtFabH may not be the primary target of thiolactomycin inhibition in vivo and led to several changes in the lipid composition of the bacilli. The data presented is consistent with a role for mtFabH as the pivotal link between the type I and type II fatty acid elongation systems in M. tuberculosis. This study opens up new avenues for the development of selective and novel anti-mycobacterial agents targeted against mtFabH.     

Antitubercular agents. Part 2: new thiolactomycin analogues active against Mycobacterium tuberculosis

Structurally modified analogues of naturally occurring antibiotic thiolactomycin, substituted at 4-position of the thiolactone ring have been prepared and evaluated for their antitubercular activity. Some of the compounds have exhibited potential activity against Mycobacterium tuberculosis.     

Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin

The biochemical basis for the inhibition of fatty acid biosynthesis in Escherichia coli by the antibiotic thiolactomycin was investigated. A biochemical assay was developed to measure acetoacetyl-acyl carrier protein (ACP) synthase activity, a recently discovered third condensing enzyme from E. coli (Jackowski, S., and Rock, C.O. (1987) J. Biol. Chem. 262, 7927-7931). In contrast to the other two condensing enzymes in E. coli, acetoacetyl-ACP synthase (synthase III) condensed malonyl-ACP with acetyl-CoA, rather than with acetyl-ACP. The concentration dependence of thiolactomycin inhibition of fatty acid biosynthesis in vivo was the same as the inhibition of acetoacetyl-ACP synthase activity in vitro indicating that the two phenomena were related. A thiolactomycin-resistant mutant (strain CDM5) was isolated. The specific activity of acetoacetyl-ACP synthase in extracts from this mutant was 10-fold lower than in extracts from its thiolactomycin-sensitive parent resulting in a marked defect in the ability of strain CDM5 to incorporate acetyl-CoA into fatty acids in vitro. The residual acetoacetyl-ACP synthase activity in the resistant strain was refractory to thiolactomycin inhibition. In addition, acetyl-CoA:ACP transacylase activity in strain CDM5 was resistant to inactivation by thiolactomycin suggesting that the acetoacetyl-ACP synthase also catalyzes this transacylation reaction. These data point to acetoacetyl-ACP synthase as a target for thiolactomycin inhibition of bacterial fatty acid biosynthesis.     

Mechanism of action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of Escherichia coli

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.     

Crystal structure of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III

Mycolic acids (alpha-alkyl-beta-hydroxy long chain fatty acids) cover the surface of mycobacteria, and inhibition of their biosynthesis is an established mechanism of action for several key front-line anti-tuberculosis drugs. In mycobacteria, long chain acyl-CoA products (C(14)-C(26)) generated by a type I fatty-acid synthase can be used directly for the alpha-branch of mycolic acid or can be extended by a type II fatty-acid synthase to make the meromycolic acid (C(50)-C(56)))-derived component. An unusual Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as myristoyl-CoA (C(14)) and malonyl-ACP. This enzyme, presumed to play a key role in initiating meromycolic acid biosynthesis, was crystallized, and its structure was determined at 2.1-A resolution. The mtFabH homodimer is closely similar in topology and active-site structure to Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel leading from the enzyme surface to the buried active-site cysteine residue. Unlike ecFabH, mtFabH contains a second hydrophobic channel leading from the active site. In the ecFabH structure, this channel is blocked by a phenylalanine residue, which constrains specificity to acetyl-CoA, whereas in mtFabH, this residue is a threonine, which permits binding of longer acyl chains. This same channel in mtFabH is capped by an alpha-helix formed adjacent to a 4-amino acid sequence insertion, which limits bound acyl chain length to 16 carbons. These observations offer a molecular basis for understanding the unusual substrate specificity of mtFabH and its probable role in regulating the biosynthesis of the two different length acyl chains required for generation of mycolic acids. This mtFabH presents a new target for structure-based design of novel antimycobacterial agents.     

Synthesis and biological activity of enantiomeric pairs of 5-vinylthiolactomycin congeners

Twelve enantiomeric pairs of 5-vinylthiolactomycin congeners were synthesized by employing our efficient synthetic route previously explored for the synthesis of enantiomeric pairs of thiolactomycin and its 3-demethyl derivative. From the biological activity assay carried out using the obtained congeners along with enantiomeric pairs of thiolactomycin and its 3-demethyl derivative previously prepared, it appeared evident that in vitro antibacterial and mammalian type I FAS inhibitory activity of thiolactomycin congeners can be cleanly separated by changing not only the structure but also the absolute configuration of the side chain at the C(5)-position. These studies led us to explore (S)-3-demethyl-5-(pent-1-enyl)thiolactomycin derivative [(S)-4-hydroxy-5-methyl-5-(pent-1-enyl)-5H-thiophen-2-one] which exhibits type I FAS inhibitory activity equal to that of C75, the potent inhibitor so far reported, with complete loss of in vitro antibacterial activity.     

Effect of thiolactomycin on the individual enzymes of the fatty acid synthase system in Escherichia coli

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.     

Mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities

Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against Staphylococcus aureus, Mycobacterium tuberculosis, Francisella tularensis and Escherichia coli. While the new derivatives still bound the gyrase B protein potently (0.07-1.8 μM, IC(50)), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 μg/ml.     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability

Despite the existence of efficient chemotherapy, tuberculosis remains a leading cause of mortality worldwide. New drugs are urgently needed to reduce the potential impact of the emergence of multidrug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb). The front-line antibiotic isoniazid (INH), and several other drugs, target the biosynthesis of mycolic acids and especially the Fatty Acid Synthase-II (FAS-II) elongation system. This biosynthetic pathway is essential and specific for mycobacteria and still represents a valuable system for the search of new anti-tuberculous agents. Several data, in the literature, suggest the existence of protein-protein interactions within the FAS-II system. These interactions themselves might serve as targets for a new generation of drugs directed against Mtb. By using an extensive in vivo yeast two-hybrid approach and in vitro co-immunoprecipitation, we have demonstrated the existence of both homotypic and heterotypic interactions between the known components of FAS-II. The condensing enzymes KasA, KasB and mtFabH interact with each other and with the reductases MabA and InhA. Furthermore, we have designed and constructed point mutations of the FAS-II reductase MabA, able to disrupt its homotypic interactions and perturb the interaction pattern of this protein within FAS-II. Finally, we showed by a transdominant genetic approach that these mutants are dominant negative in both non-pathogenic and pathogenic mycobacteria. These data allowed us to draw a dynamic model of the organization of FAS-II. They also represent an important step towards the design of a new generation of anti-tuberculous agents, as being inhibitors of essential protein-protein interactions.     

The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions

Tuberculosis kills about two million people every year and remains one of the leading causes of mortality worldwide. As a result of the increasing antibiotic resistance of Mycobacterium tuberculosis (Mtb) strains, there is an urgent need for new antitubercular drugs. Several efficient antibiotics, including isoniazid, specifically target the fatty acid synthase-II (FAS-II) complex of mycolic acid biosynthesis. We have previously shown that there are protein-protein interactions between the components of FAS-II that are essential for mycobacterial survival. We have now looked at the potential partners of FAS-II, mtFabD, the methyltransferases MmaAs, and Pks13. A combination of yeast two-hybrid and co-immunoprecipitation experiments showed that mtFabD interacts with each beta-ketoacyl-synthase (KasA, KasB and mtFabH) and with the core of FAS-II (InhA and MabA). The methyltransferases have a greater affinity for KasA and KasB than for mtFabH, suggesting that modifications on the meromycolic chains may occur during their elongation. Finally, Pks13, which catalyzes the final Claisen condensation of mycolic acids, interacts specifically with KasB. These data allowed us to determine the architecture of the multiple specialized FAS-II complexes, giving us insights into the organization of the complete mycolic acids biosynthesis. Our studies suggest a new and crucial interaction (KasB-Pks13) as a putative target for peptidomimetic antibiotics.     

Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-(14)C]cholesterol or [1,7,15,22,26-(13)C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2'-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2'-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.     

Synthesis and structure-activity relationships of thiotetronic acid analogues of thiolactomycin

3-Acetyl analogues of thiolactomycin, a thiotetronic acid natural product, were synthesized and profiled against livestock pathogens. Some analogues showed improved activity over thiolactomycin against Staphylococcus aureus and comparable activity against Pasteurella multocida. Several semisynthetically modified analogues of thiolactomycin showed no improvement in activity over thiolactomycin.     

Synthesis and biological evaluation of thiazolidine-2-one 1,1-dioxide as inhibitors of Escherichia coli beta-ketoacyl-ACP-synthase III (FabH)

A series of cyclic sulfones has been synthesized and their activity against beta-ketoacyl-ACP-synthase III (FabH) has been investigated. The compounds are selectively active against Escherichia coli FabH (ecFabH), but not Mycobacterium tuberculosis FabH (mtFabH) or Plasmodium falciparum KASIII (PfKASIII). The activity against ecFabH ranges from 0.9 to >100microM and follows a consistent general SAR trend. Many of the compounds were shown to have antimalarial activity against chloroquine (CQ)-sensitive (D6) P. falciparum (IC(50)=5.3microM for the most potent inhibitor) and some were active against E. coli (MIC=6.6microg/ml for the most potent inhibitor).     

Purification and biochemical characterization of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthases KasA and KasB

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.     

X-ray crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase II (mtKasB)

Mycolic acids are long chain alpha-alkyl branched, beta-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C(56)) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis beta-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis beta-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 A resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds, which inhibit mycobacterial KAS enzymes more effectively.     

β-Ketoacyl-acyl carrier protein synthase III from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): properties,inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.