Identification of new anti-mycobacterial agents based on quinoline-isatin hybrids targeting enoyl acyl carrier protein reductase (InhA).

Tuberculosis (TB) is a global issue that poses a significant economic burden as a result of the ongoing emergence of drug-resistant strains. The urgent requirement for the development of novel antitubercular drugs can be addressed by targeting specific enzymes. One such enzyme, Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein (enoyl-ACP) reductase (InhA), plays a crucial role in the survival of the MTB bacterium. In this research study, a series of hybrid compounds combining quinolone and isatin were synthesized and assessed for their effectiveness against MTB, as well as their ability to inhibit the activity of the InhA enzyme in this bacterium. Among the compounds tested, 7a and 5g exhibited the most potent inhibitory activity against MTB, with minimum inhibitory concentration (MIC) values of 55 and 62.5 µg/mL, respectively. These compounds were further evaluated for their inhibitory effects on InhA and demonstrated significant activity compared to the reference drug Isoniazid (INH), with IC50 values of 0.35 ± 0.01 and 1.56 ± 0.06 µM, respectively. Molecular docking studies investigated the interactions between compounds 7a and 5g and the target enzyme, revealing hydrophobic contacts with important amino acid residues in the active site. To further confirm the stability of the complexes formed by 5g and 7a with the target enzyme, molecular dynamic simulations were employed, which demonstrated that both compounds 7a and 5g undergo minor structural changes and remain nearly stable throughout the simulated process, as assessed through RMSD, RMSF, and Rg values.

Isatin-pyrimidine hybrid derivatives as enoyl acyl carrier protein reductase (InhA) inhibitors against Mycobacterium tuberculosis.

Tuberculosis is a worldwide problem that impose a burden on the economy due to continuous development of resistant strains. The development of new antitubercular drugs is a need and can be achieved through inhibition of druggable targets. Mycobacterium tuberculosis enoyl acyl carrier protein (ACP) reductase (InhA) is an important enzyme for Mycobacterium tuberculosis survival. In this study, we report the synthesis of isatin derivatives that could treat TB through inhibition of this enzyme. Compound 4l showed IC50 value (0.6 ± 0.94 µM) similar to isoniazid but is also effective against MDR and XDR Mycobacterium tuberculosis strains (MIC of 0.48 and 3.9 µg/mL, respectively). Molecular docking studies suggest that this compound binds through the use of relatively unexplored hydrophobic pocket in the active site. Molecular dynamics was used to investigate and support the stability of 4l complex with the target enzyme. This study paves the way for the design and synthesis of novel antitubercular drugs.

Inhibition of Mycobacterium tuberculosis InhA (Enoyl-acyl carrier protein reductase) by synthetic Chalcones: a molecular modelling analysis and in-vitro evidence.

Tuberculosis (TB) is a serious infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The World Health Organization (WHO) estimates that 1.8 million people die each year from TB, with 10 million new cases being registered each year. In this study, 50 Chalcones were developed, five of which were synthesized, and their inhibitory effects against Mtb were studied. The discovery of new powerful inhibitors with IC50 values in the sub-micro molar range resulted from the development of structure-activity relationships (SAR). The goal of the molecular modelling studies was to uncover the most important structural criteria underpinning the binding affinity and selectivity of this class of inhibitors as possible anti-TB drugs. Because of their great efficacy and selectivity, our developed nitro and benzyloxy substituted Chalcones compounds appear to be promising anti-TB therapies.Communicated by Ramaswamy H. Sarma.

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3.

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence-mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC-DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC-POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.

Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity.

Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.

Development of a scintillation proximity assay for beta-ketoacyl-acyl carrier protein synthase III.

Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonyl-acyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP. This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K(m) of 7.1 microM was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K(m) (6 microM) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC(50) values of 15.2 microM (SPA) and 24.8 microM were obtained with iodoacetamide and the S. glaucescens FabH. Comparable IC(50) values of 160 microM (SPA) and 125 microM were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics.

Kinetic analysis of the actinorhodin aromatic polyketide synthase.

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

Fatty acid synthesis of an eicosapentaenoic acid-producing bacterium: de novo synthesis, chain elongation, and desaturation systems.

The fatty acid synthesis systems of a Shewanella sp., strain SCRC-2738, that produces a large amount of eicosapentaenoic acid were investigated. Two kinds of fatty acid synthesis system, de novo synthesis and chain elongation ones, were detected in the cytosol. The de novo synthesis system required an acyl carrier protein, and produced palmitoyl- and palmitoleoyl-acyl carrier proteins as final products. The chain elongation system also required an acyl carrier protein, and produced an acyl-acyl carrier protein as a product, using palmitoyl-, palmitoleoyl-, stearoyl-, and oleoyl-CoAs as primers but not eicosanoyl- or eicosenoyl-CoA. There were an anaerobic pathway and an aerobic desaturation one for the production of unsaturated fatty acids. Eicosapentaenoic acid seemed to be produced through the aerobic desaturation pathway and not through the anaerobic one, since the latter pathway produced n-7 type monoenoic fatty acids, which are different from eicosapentaenoic acid in the position of the double bond. The desaturase utilized an acyl-acyl carrier protein as a substrate, and this activity increased in the presence of ferredoxin and ferredoxin NADP+ reductase. Thus, Shewanella sp., strain SCRC-2738, has novel characteristics as to both fatty acid chain elongation and desaturation systems.

The unmodified (apo) form of Escherichia coli acyl carrier protein is a potent inhibitor of cell growth.

Acyl carrier protein (ACP) is the carrier of fatty acids during their synthesis and utilization. ACPs (or ACP-like protein domains) have been found throughout biology and share significant amino acid sequence similarities. All ACPs undergo a post-translational modification in which 4'-phosphopantetheine is transferred from CoA to a specific serine of apo-ACP. This modification is essential for activity because fatty acids are bound in thioester linkage to the sulfhydryl of the prosthetic group. Overproduction of Escherichia coli ACP from multicopy plasmids strongly inhibits growth of E. coli. We report that upon overexpression of ACP in E. coli post-translational modification is inefficient and the apo protein accumulates and blocks cell growth by inhibition of lipid metabolism. Moreover, a mutant form of ACP that is unable to undergo post-translational modification is a potent inhibitor of growth. Finally, we observed that an increase in the efficiency of modification of overexpressed ACP results in decreased toxicity. The accumulated apo-ACP acts as a potent in vitro inhibitor of the sn-glycerol-3-phosphate acyltransferase resulting in an inability to transfer the completed fatty acid to sn-glycerol 3-phosphate. The degree of inhibition depended upon the species of donor acyl chain. Utilization of cis-vaccenoyl-ACP by the sn-glycerol-3-phosphate acyltransferase was inhibited to a much greater extent by apo-ACP than was utilization of palmitoyl-ACP. 1-Acyl glycerol-3-phosphate acyltransferase was also inhibited in vitro by apo-ACP, although not at physiologically relevant concentrations. These in vitro data are supported by in vivo labeling data, which showed a large decrease in cis-vaccenate incorporation into phospholipid during overproduction of ACP, but no decrease in the rate of synthesis of long chain acyl-ACPs. These data indicate that acylation of sn-glycerol 3-phosphate is the major site of inhibition by apo-ACP.

Overproduction of the acyl carrier protein component of a type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens.

The development of microorganisms with improved antibiotic production is an important goal in the commercialization of new pharmaceuticals or in lowering the cost of established drugs. We report a way to achieve this for biosynthetic intermediates of an antibiotic made by the polyketide pathway whose earliest steps involve a Type II multienzyme complex. Introduction of the tcmKLM beta-ketoacyl: ACP synthase and acyl carrier protein (ACP) genes or just the tcmM ACP gene into the tetracenomycin (Tcm) C-producing Streptomyces glaucescens wild-type strain, or its tcmN or tcmO blocked mutants, on high copy vectors under the control of strong promoters caused a 2 to 30-fold overproduction of Tcm D3 and some other biosynthetic intermediates (or shunt products) and a 25 to 30% increase in Tcm C production relative to the control strains carrying the plasmid vector only. However, Tcm C production was not greater than that obtained with the vector-free wild-type strain. The unexpected effect of increased ACP on Tcm D3 production suggests that the level of this protein can influence either the activity or level of the three other components of the Tcm polyketide synthase.

Coenzyme A biosynthesis in plants: partial purification and characterization of pantothenate kinase from spinach.

A study of the biosynthesis of coenzyme A (CoA), a critical cofactor in the metabolism of lipids and other molecules in higher plants, was initiated. Pantothenate kinase was partially purified from spinach leaves. This enzyme was predominantly localized in the chloroplast with very little activity observed in the mitochondria or cytosol. DEAE-agarose chromatography resolved two pantothenate kinase activity peaks which differed in their requirement for reductant, stability upon boiling, and reactivity in the presence of spinach holo-acyl carrier protein (ACP) I. One active peak of this enzyme was further purified on Cibacron blue 3GA to yield a preparation containing pantothenate kinase enriched to 20% of the total protein within the fraction. Pantothenate kinase was inhibited by malonyl-CoA, but not by CoASH or acetyl-CoA, and the activity was stabilized by the phosphatase inhibitors sodium molybdate, sodium tungstate, and the phosphatase substrate glycerol 2-phosphate, but was inhibited by sodium fluoride. Further experiments demonstrated a linear increase in pantothenate kinase activity during spinach seed germination, consistent with a role for this enzyme in the developmental utilization of seed triacylglycerol.

Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation.

Haemolysin secreted by pathogenic Escherichia coli binds to mammalian cell membranes, disrupting cellular activities and lysing cells by pore-formation. It is synthesized as nontoxic prohaemolysin (proHlyA), which is activated intracellularly by a mechanism dependent on the cosynthesized HlyC. Haemolysin is one of a family of membrane-targeted toxins, including the leukotoxins of Pasteurella and Actinobacillus and the bifunctional adenylate cyclase haemolysin of Bordetella pertussis, which require this protoxin activation 1-5. HlyC alone cannot activate proHlyA, but requires a cytosolic activating factor6. Here we report the cytosolic activating factor is identical to the acyl carrier protein and that activation to mature toxin is achieved by the transfer of a fatty acyl group from acyl carrier protein to proHlyA. Only acyl carrier protein, not acyl-CoA, can promote HlyC-directed proHlyA acylation, but a range of acyl groups are effective.

Biosynthesis of very long chain fatty acids in microsomes from epidermal cells of Allium porrum L.

The elongation system present in leek epidermal cells functions to synthesize very long chain fatty acids which, in turn, are the precursors to alkanes. The elongation system is microsomal, employs only saturated acyl components of the endogenous lipid pool as acceptors, utilizes malonyl-CoA as the C2 donor, has an absolute requirement for ATP, and is markedly inhibited by acetyl-ACP. Only saturated acyl-CoAs are readily elongated to very long chain fatty acids by malonyl-CoA in the absence of ATP. ACP is not required by the microsomal system.