NADH-dependent Enoyl-acyl Carrier Protein Reductase Research Articles

Design and synthesis of Thieno[3, 2-b]pyridinone derivatives exhibiting potent activities against Mycobacterium tuberculosis in vivo by targeting Enoyl-ACP reductase

In this study, a series of novel thieno [3, 2-b]pyridinone derivatives were designed and synthesized using a scaffold hopping strategy. Six compounds showed potent anti-mycobacterial activity (minimum inhibitory concentration (MIC) ≤ 1 μg/mL) against Mycobacterium tuberculosis (Mtb) UAlRa. Compound 6c displayed good activity against Mtb UAlRv (MIC = 0.5–1 μg/mL). Compounds 6c and 6i also showed activity against Mtb UAlRa in macrophages and exhibited low cytotoxicity against LO-2 cells. The selected compounds displayed a narrow antibacterial spectrum, with no activity against representative Gram-positive, Gram-negative bacteria, as well as fungi. Furthermore, compound 6c demonstrated favorable oral pharmacokinetic properties with a T1/2 value of 47.99 h and exhibited good in vivo activity in an acute mouse model of tuberculosis (TB). The target of compound 6c was identified as a NADH-dependent enoyl-acyl carrier protein reductase (InhA) by genome sequencing of spontaneously compound 6c-resistant Mtb mutants, indicating that compound 6c may not require activation and can directly target InhA. In vitro antimicrobial assays against a recombinant M. smegmatis overexpressing the Mtb-InhA, along with InhA inhibition assays, confirmed that InhA is the target of thieno [3, 2-b]pyridinone derivatives. Overall, this study identified thieno [3, 2-b]pyridinone scaffold as a novel chemotype that is promising for the development of anti-TB agents.

Design and In-silico Screening of Short Antimicrobial Peptides (AMPs) as Anti-Tubercular Agents Targeting INHA

Introduction: Tuberculosis has been studied as a potentially serious bacterial infection affecting the lung caused by Mycobacterium tuberculosis. In addition to its severe impact on health, resistance to existing drugs has also been seen as a rising concern in the space of medicinal solutions. Therapeutic peptides have the potential to complement existing drug designs to provide effective outcomes against Mycobacterium tuberculosis-resistant strains. Methods: This study illustrated a computational approach to design and test peptides against NADHdependent enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis. A human antimicrobial peptide LL-37 was used as a template, and a further 6 peptides were designed, and their binding and interactions against NADH-dependent enoyl-acyl carrier protein reductase were examined. Further, toxicity, immunogenicity, and a broad spectrum of physicochemical properties were calculated to evaluate the therapeutic and safety profile of these peptides. Results: These peptides were structurally modelled and docked with the protein to determine their binding poses and affinity. The molecular interaction of LL-37 with protein was treated as a reference to evaluate the effectiveness of designed peptides. Solvent accessible surface area (SASA) and ΔG binding free energy of docked complexes assisted in the ranking of these peptides. Eventually, peptides P1: LLGDFFRKSKEK, P3: LLFGDRFLLKEK and P7: LLGDFFRLLKEK were selected for 100 ns molecular dynamic simulation as they showed predicted dissociation constants of 8.7×10-4 M, 3.3×10-4 M and 1.2×10-4 M, respectively. These peptides showed direct hydrogen bond formation with ILE21 and LYS165, which are critical active site residues of the protein. The structural variation pattern collected from the MD simulation suggested a strong and stable binding of P3 and P1 with the protein with RMSD 4-5 Å with the starting conformation under the non-fluctuating state. These two peptides showed relatively similar binding results compared with the control peptide LL-37. Comprehensive structural analysis was performed for the middle structures of the most populated cluster generated from 100 ns MD simulation trajectory. Conclusion: Later, MMPBSA binding energies of these structures were computed, where the average binding free energies of P1, P3, and P7 peptides were -146.93 kcal/mole, -161.16 kcal/mole, and - 151.44 kcal/mole, respectively. These energies suggested that P3 is strongly bound to the active site of NADH-dependent enoyl-acyl carrier protein reductase. Overall, this study proposed the application of these peptides as a possible therapeutic solution to inhibit the growth of Mycobacterium tuberculosis.

Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity, SAR, and preclinical evaluation

Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals, satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-aminoquinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-resistant tuberculosis treatments.

Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

Identification of Mycobacterium tuberculosis enoyl-acyl carrier protein reductase inhibitors: A combined in-silico and in-vitro analysis

Mycobacterium tuberculosis (Mtb), had developed evolutionary changes in its genome to adapt for survival and thereby generated multi-drug resistant strains. However, novel drug targets that remained unchanged for their biochemical role has impressed the research community to target such proteins. The comprehensive analysis of multiple protein targets has influenced us to make a consensus structural rule exploited by pharmacophore and other allied techniques from a large repository of protein structures. In this pursuit, we made a retrospective analysis of pharmacophores mapped from the tuberculosis structural proteome and identified unique patterns that can be employed for the novel molecules design. The current work on NADH–dependent enoyl–acyl carrier protein reductase (InhA) has yielded top scored pharmacophore models which were searched over SPECS natural product database to prioritize the molecules that can be targeted against Mtb. With efforts on rigorous validation and expertise, we have identified such pharmacophoric patterns from natural compounds that can be used as initial hits. Subsequently, these hits were subjected to in-vitro antitubercular evaluation to ensure the inhibitory activity against the mycobacterium culture growth (MtbH37Rv). Furthermore, docking simulations were carried out to provide an insight on the possible modes of interaction between the experimentally explored compounds and InhA.

Synthesis and biological evaluation of 1,3,4-trisubstituted pyrazole analogues as anti-mycobacterial agents

A series of 1,3,4-trisubstituted pyrazole derivatives (3a–f), (4a–r), (5a–f) and (6a–f) have been synthesized and evaluated for their Mycobacterium tuberculosis (MTB) (H37Rv) inhibitory activity. The structures of newly synthesized compounds were characterized by IR, 1H NMR, 13C NMR and mass spectral analysis. Among the thirty six compounds screened for in vitro anti-mycobacterial activity against MTB, three compounds 3b, 3e and 3f demonstrated significant growth inhibitory activity with minimum inhibitory concentration of 0.78 µg/ml. The selected compounds were further tested for cytotoxic activity against normal human dermal fibroblast cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and exhibited no significant cytotoxicity. Molecular docking simulation studies were carried out in order to better understand the hypothetical binding interaction to the MTB NADH-dependent enoyl–acyl carrier protein reductase.

Triclosan Resistance in Francisella tularensis: A Site-Directed Mutagenesis Study of the FabI Enzyme

An NADH-dependent enoyl-acyl carrier protein reductase, FabI, catalyzes the final step of bacterial fatty acid biosynthesis, reducing the double bond of trans-2-enoyl-ACP to a single bond forming acyl-ACP. Given its importance in bacterial fatty acid synthesis, FabI has become a recognized drug target. Triclosan, a diphenyl ether, targets the FabI, inhibits its activity, and stops bacterial growth. However, as a consequence of triclosan's popularity, and thus its overuse, bacterial resistance to triclosan has been reported. The mutation G93V in Escherichia coli (E. coli) FabI allows E. coli to resist the action of triclosan. We have identified the equivalent residue of G93 in Francisella tularensis FabI (ftFabI) as A92, and prepared a mutant A92V. E. coli cells, transformed with a plasmid containing the ftFabI-A92V gene, were grown, and the gene was overexpressed. From two growths (6 G of cells), 62 mG of protein, with a histidine tag, at a purity of 85% were obtained. Enzymatic activity was assayed by monitoring the absorbance of NADH at 340 nm. In the presence of triclosan, the wild-type protein was almost completely inhibited after NADH was converted to NAD$^+$ in the enzymatic reaction; however the A92V mutant exhibited similar activity with and without triclosan, demonstrating that triclosan resistance may also develop in Francisella tularensis.

Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare.

Dilemmas with ethionamide susceptibility testing of Mycobacterium tuberculosis: A microbiologist & physician's nightmare.

Piperazine derivatives: synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase and SAR studies.

The Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) catalyzes hydride transfer to long-chain enoyl thioester substrates. MtInhA is a member of the mycobacterial type II dissociated fatty acid biosynthesis system, and is the bona fide target for isoniazid, the most prescribed drug for tuberculosis treatment. Here, a series of piperazine derivatives was synthesized and screened as MtInhA inhibitors, which resulted in the identification of compounds with IC50 values in the submicromolar range. A structure-activity relationship (SAR) evaluation indicated the importance of the chemical environment surrounding the carbonyl group for inhibition. In addition, the structure of one selected compound was supported by crystallographic studies, and experimental geometrical values were compared with semi-empirical quantum chemical calculations. Furthermore, the mode of inhibition and inhibitory dissociation constants were determined for the nine most active compounds. These findings suggest that these 9H-fluoren-9-yl-piperazine-containing compounds interact with MtInhA at the enoyl thioester (2-trans-dodecenoyl-CoA) substrate binding site.

Cross-docking study on InhA inhibitors: a combination of Autodock Vina and PM6-DH2 simulations to retrieve bio-active conformations

InhA, the NADH-dependent enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis (Mtb) is the proposed main target of the first-line antituberculosis drug isoniazid (INH). INH activity is dependent on activation by the catalase peroxidase KatG, a Mtb enzyme whose mutations are linked to clinical resistance to INH. Other inhibitors of InhA that do not require any preliminary activation are known. The design of such direct potent inhibitors represents a promising approach to circumvent this resistance mechanism. An ensemble-docking process with four known InhA X-ray crystal structures and employing the Autodock Vina software was performed. Five InhA inhibitors whose bioactive conformations are known were sequentially docked in the substrate cavity of each protein. The efficiency of the docking was assessed and validated by comparing the calculated conformations to the crystallographic structures. For a same inhibitor, the docking results differed from one InhA conformation to another; however, docking poses that matched correctly or were very close to the expected bioactive conformations could be identified. The expected conformations were not systematically well ranked by the Autodock Vina scoring function. A post-docking optimization was carried out on all the docked conformations with the AMMP force field implemented on the VEGAZZ software, followed by a single point calculation of the interaction energy, using the MOPAC PM6-DH2 semi-empirical quantum chemistry method. The conformations were subsequently submitted to a PM6-DH2 optimization in partially flexible cavities. The resulting interaction energies combined with the multiple receptor conformations approach allowed us to retrieve the bioactive conformation of each ligand.

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2- trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH–NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH–NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

In vitro inhibition of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA by isoniazid.

The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent beta-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between Mn(III)-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP(+). The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the beta-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

CDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.