Mycobacterium Tuberculosis enoyl-ACP Reductase Research Articles

Oxacyanopyridine-Benzofuran Hybrids: Synthesis, in silico Toxicity Assessment, in vitro Antimicrobial Activity and Dual Target Docking Studies

This study emphasizes the synthesis and characterization of a novel series of oxa cyano pyridine heterocyclic molecular hybrids (OCP 1-6), integrating pyridine and benzofuran motifs. Meticulously designed from 5-chlorosalicylaldehyde in a multistep process, the synthesized compounds were structurally confirmed through IR spectroscopy, 1H NMR spectroscopy, and mass spectrometry. Computational predictions highlighted diverse properties, including antitubercular and antibacterial attributes, bioactivity scores, toxicity profiles and potential molecular targets. In vitro assessments and Schrödinger docking simulations revealed binding affinities to enzymes viz. Mycobacterium tuberculosis enoyl-ACP reductase and Escherichia coli Topoisomerase IV. Compounds, particularly OCP 2, 3, 4 and 5, exhibited significant antitubercular and antibacterial activities in both in vitro assessments and docking simulations. This study underscores the substantial potential of the synthesized hybrids as promising candidates for further lead optimization, positioning them as valuable contributors to the development of pharmaceutical agents with heightened potency and enhanced safety profiles.

Synthesis, Antimicrobial Activity and Molecular Docking of Some Novel 2-Aryl-4H-[1,3]-thiazolo[4,5-b]indoles

In present study, six novel 2-aryl-4H-[1,3]-thiazolo[4,5-b]indoles (4a-f) were synthesized by integrating thiazole and indole motifs through a three-component one-pot condensation involving isatin, ammonium thiocyanate and arylaldehydes. Structural confirmation was achieved using IR, 1H NMR and mass spectrometric techniques. Molecular targets, bioactivity scores, drug-likeness, anti- tuberculosis and antibacterial properties and toxicity were all predicted using various computational techniques. To calculate binding affinities to Mycobacterium tuberculosis enoyl-ACP reductase and Escherichia coli Topoisomerase IV, in vitro bioactivity studies were performed and Schrödinger docking simulations were performed. Anti-tubercular efficacy was evaluated using the MABA method, while antibacterial effectiveness against both Gram-positive and Gram-negative bacteria was assessed through the agar plate method. The results highlight the potential of these heterocyclic molecular hybrids, positioning them as promising candidates for further lead optimization in the development of more potent and safer pharmaceutical agents.

Benzofuran-Isoxazole Hybrids: Synthesis, Antimicrobial Activity and Dual Target Docking Studies

In this study, five novel heterocyclic molecular hybrids (4a-e) were synthesized by combining benzofuran and isoxazole motifs. Thesecompounds were synthesized via a multistep process starting with 5-chlorosalicylaldehyde. The structures of the synthesized compounds were characterized by IR, 1H NMR and mass spectrometry. Computational tools were employed to predict properties, including antitubercular and antibacterial traits, drug-likeness, bioactivity scores, toxicity and potential molecular targets. In addition to these predictions, in vitro bioactivities were assessed and conducted Schrödinger docking simulations to determine binding affinities to Mycobacterium tuberculosis enoyl-ACP reductase and Escherichia coli topoisomerase IV. Antitubercular efficacy was determined via the MABA method, while antibacterial effectiveness was tested against both Gram-positive and Gram-negative bacteria. The obtained results highlighted the promise of these benzofuranyl-isoxazole hybrids, making them strong contenders for further lead optimization, with the goal of developing more potent and safer pharmaceutical agents.

Synthesis, Antimicrobial Activity and Dual Target Docking Studies of Novel 1-(5-Chloro-1-benzofuran-2-yl)-3-substituted Phenyl Prop-2-en-1-ones

This study involved synthesizing five novel derivatives of 1-(5-chloro-1-benzofuran-2-yl)-3-substituted phenyl prop-2-en-1-ones throughthe Claisen-Schmidt condensation reaction, using 5-chloro-2-acetyl benzofuran and aromatic aldehydes in the presence of base catalyst. The chemical structures of these compounds were confirmed by using IR spectroscopy, 1H NMR spectroscopy and mass spectrometry. Schrödinger docking simulations were employed to ascertain the binding affinity of the synthesized compounds to Mycobacterium tuberculosis enoyl-ACP reductase and Escherichia coli Topoisomerase IV. Subsequently, the anti-tubercular and in vitro antibacterial activities of the synthesized compounds were also investigated. Anti-tubercular efficacy was determined using the MABA method, while the antibacterial effectiveness was assessed against both Gram-positive and Gram-negative bacterial strains through the agar cup plate. The findings from these assays provide insights into the potential of these compounds as agents possessing anti-tubercular and antibacterial characteristics, thereby offering promising prospects for further investigation in the field of drug development.

Evaluating the Impact of the Tyr158 pKa on the Mechanism and Inhibition of InhA, the Enoyl-ACP Reductase from Mycobacterium tuberculosis.

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F2Y) has no effect on kcatapp/KMapp nor on the binding of inhibitors to the open form of the enzyme (Kiapp), whereas both kcatapp/KMapp and Kiapp are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F3Y158 InhA). 19F NMR spectroscopy suggests that 2,3,5-F3Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, Ki*app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F2Y158 and 2,3,5-F3Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced ∼four-fold for 2,3,5-F3Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.

Synthesis, Biological Evaluation and In-silico Studies of 3-((4-(6-(Substitutedphenyl)-2-Mercaptopyrimidin-4-Yl) Phenyl) Imino)- 1-((Dimethylamino) Methyl) Indolin-2-One

A novel isatin and pyrimidine prototype derivatives 3-((4-(6-(substitutedphenyl)-2-mercaptopyrimidin-4-yl)phenyl)imino)- 1-((dimethylamino)methyl)indolin-2-one were synthesized and designed from the intermediate chalcones. The produced components were characterized, anti-tubercular and antimicrobial screening was done by agar dilution and agar-cup plate methods, respectively. From the studies, it was revealed that derivatives 5e, 5h and 5i showed increased potency. Further in-silico studies were carried out by molecular docking with the interaction of target components 5a-5j within the Mycobacterium tuberculosis enoyl-acp reductase enzyme (1ZID), drug likeness, and pharmacokinetic parameters were measured by osiris property explorer and molinspiration online toolkit respectively.

Crystal structures, DFT calculations and Hirshfeld surface analysis of two (E)-3-(aryl)-1-(naphthalen-1-yl)prop-2-en-1-one chalcone derivatives, potential Mycobacterium tuberculosis Enoyl ACP reductase (InhA) inhibitors and optical materials: conformational differences within the prop-2-en-1-one unit

A combined structural and computational study has been conducted on two chalcone compounds, namely (E)-3-(4-bromophenyl)-1-(4,8-dimethoxynaphthalen-1-yl)prop-2-en-1-one (1), and (E)-3-(3,4-dichlorophenyl)-1-(4,8-dimethoxynaphthalen-1-yl)prop-2-en-1-one (2). These compounds are members of a series of compounds identified from a molecular modelling study as potentially active Mycobacterium tuberculosis Enoyl ACP reductase (InhA) inhibitors. In addition, the computational study indicated that the compounds have potential as optical materials.The combined study revealed that both molecules in the solid state displayed similar overall molecular conformations between a ‘‘T’’ and a ‘‘Y’’ shape, but with different arrangements in the bridging prop-2-en-1-one unit: an anti-periplanar in 1 and a syn-periplanar in 2. The experimental and calculated bond lengths and angles for anti-periplanar1 and syn-periplanar2 were generally in good agreement.Theoretical calculations at the B3LYP/DGTZVP level in the gas phase, DMSO and water media indicated that for both compounds, the syn-periplanar form was the energy local minimum while the anti-periplanar form was the energy global minimum. However, the energy differences are very small, with slight variations when using the different basis sets (0.80 and 0.84 kcal/mol using the B3LYP/DGTVP theory level in gas phase, 0.28 and 0.32 in DMSO and 0.27 and 0.31 kcal/mol in water). The differences in the solid state arrangements within the bridging prop-2-en-1-one unit in 1 and 2 arise from variations in the intermolecular interactions, with halogen bonds being considered to be an important factor in stabilizing a syn-periplanar arrangement in compound 2. PLATON and Hirshfeld surface analyses confirmed the important intermolecular interactions in 1 and 2.Calculations were also carried out on the electronic and vibrational spectra, dipole moments and charge distributions of 1 and 2. The HOMO was located in the dimethoxynaphthalenyl region of the molecule with mild donor character, while the LUMO was located in the propenyl and phenyl regions of each molecule. The energy gaps between these orbitals are 3.35 eV for 1 3.25 eV for 2. The delocalization present in compounds 1 and 2 facilitates the HOMO-LUMO charge transfer process.The total electronic molecular dipolar moments (μTotal) were calculated to be 4.1511 and 5.9316 D for 1 and 2, respectively. The values of μTotal found for 1 and 2 are both larger than reported values for similar compounds, indicating that the compounds studied here should exhibit a good non-linear optical response.

ChemInform Abstract: Recent Advances and Structural Features of Enoyl‐ACP Reductase Inhibitors of Mycobacterium tuberculosis

AbstractReview: 40 refs.

Recent Advances and Structural Features of Enoyl-ACP Reductase Inhibitors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis enoyl-ACP reductase (InhA) has been validated as a promising target for antitubercular agents. Isoniazid (INH), the most prescribed drug to treat tuberculosis (TB), inhibits a NADH-dependent InhA that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. It is a pro-drug that needs activation to form the inhibitory INH-NAD adduct by KatG coding for catalase-peroxidase. The INH resistance of M. tuberculosis is caused by mutations in KatG, which may lead to multidrug-resistant TB (MDR-TB). Hence, there is a need for new drugs that can combat MDR-TB. The rationale for the development of new drugs to combat MDR-TB strains is the design of InhA inhibitors that can bypass bioactivation by KatG. In the present review, special attention was paid to discuss the chemical nature and recent developments of direct InhA inhibitors. The InhA inhibitors reported here have significant inhibitory effects against Mtb InhA. The diphenyl ether derivatives have shown slow onset, a tight-binding mechanism, and high affinity at the InhA active site. However, some of the diphenyl ethers have significant in vitro efficacy, which fails to transform into in vivo efficacy. Among the InhA inhibitors, 4-hydroxy-2-pyridones have emerged as a new chemical class with significant InhA inhibitory activity and better pharmacokinetic parameters when compared to diphenyl ethers.

Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

Design, Synthesis, Toxicity Estimation and Molecular Docking Studies of N-(furan-2-yl)-1-(5-substituted) phenyl-1,3,4-oxadiazol-2-yl) methanimine as Antitubercular Agents.

A series of novel N-(furan-2-yl)-1-(5-substituted) phenyl-1,3,4-oxadiazol-2-yl) methanimines (Fa-e) were synthesized and evaluated for antitubercular activity against Mycobacterium tuberculosis (H37Rv) strain by using alamar blue assay. The synthesized compounds were characterized based on IR, (1)HMR and mass spectral analysis. The toxicity profile was predicted by organic chemistry portal, a web based application for predicting in silico absorption, distribution, metabolism, excretion and toxicity, and the novel derivatives under study did not show any toxicity issues. The mechanism of action of the titled derivatives was predicted by docking on the Mycobacterium tuberculosis Enoyl-ACP reductase enzyme. The docking study concluded that Fb and Fa possessed good binding energy indicating more prominent interaction towards the active sites NAD and TYR 158. The antitubercular studies showed that the both Fa and Fb possessed significant activity with the MIC as low as 3.125 μg/ml.

Rational Modulation of the Induced-Fit Conformational Change for Slow-Onset Inhibition in Mycobacterium tuberculosis InhA.

Slow-onset enzyme inhibitors are the subject of considerable interest as an approach to increasing the potency of pharmaceutical compounds by extending the residence time of the inhibitor on the target (the lifetime of the drug-receptor complex). However, rational modulation of residence time presents significant challenges because it requires additional mechanistic insight, such as the nature of the transition state for postbinding isomerization. Our previous work, based on X-ray crystallography, enzyme kinetics, and molecular dynamics simulation, suggested that the slow step in inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA involves a change in the conformation of the substrate binding loop from an open state in the initial enzyme-inhibitor complex to a closed state in the final enzyme-inhibitor complex. Here, we use multidimensional free energy landscapes for loop isomerization to obtain a computational model for the transition state. The results suggest that slow-onset inhibitors crowd key side chains on helices that slide past each other during isomerization, resulting in a steric clash. The landscapes become significantly flatter when residues involved in the steric clash are replaced with alanine. Importantly, this lower barrier can be increased by rational inhibitor redesign to restore the steric clash. Crystallographic studies and enzyme kinetics confirm the predicted effects on loop structure and flexibility, as well as inhibitor residence time. These loss and regain of function studies validate our mechanistic hypothesis for interactions controlling substrate binding loop isomerization, providing a platform for the future design of inhibitors with longer residence times and better in vivo potency. Similar opportunities for slow-onset inhibition via the same mechanism are identified in other pathogens.

Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase.

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA.

Slow-onset enzyme inhibitors are of great interest for drug discovery programs since the slow dissociation of the inhibitor from the drug–target complex results in sustained target occupancy leading to improved pharmacodynamics. However, the structural basis for slow-onset inhibition is often not fully understood, hindering the development of structure-kinetic relationships and the rational optimization of drug-target residence time. Previously we demonstrated that slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA correlated with motions of a substrate-binding loop (SBL) near the active site. In the present work, X-ray crystallography and molecular dynamics simulations have been used to map the structural and energetic changes of the SBL that occur upon enzyme inhibition. Helix-6 within the SBL adopts an open conformation when the inhibitor structure or binding kinetics is substrate-like. In contrast, slow-onset inhibition results in large-scale local refolding in which helix-6 adopts a closed conformation not normally populated during substrate turnover. The open and closed conformations of helix-6 are hypothesized to represent the EI and EI* states on the two-step induced-fit reaction coordinate for enzyme inhibition. These two states were used as the end points for nudged elastic band molecular dynamics simulations resulting in two-dimensional potential energy profiles that reveal the barrier between EI and EI*, thus rationalizing the binding kinetics observed with different inhibitors. Our findings indicate that the structural basis for slow-onset kinetics can be understood once the structures of both EI and EI* have been identified, thus providing a starting point for the rational control of enzyme–inhibitor binding kinetics.

Binding of the tautomeric forms of isoniazid-NAD adducts to the active site of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA): A theoretical approach

The front-line antituberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis enoyl ACP-reductase from Mycobacterium tuberculosis, via formation of covalent adducts with NAD (INH-NAD adducts). While ring tautomers were found the main species formed in solution, only the 4 S chain INH-NAD tautomer was evidenced in the crystallized InhA:INH-NAD complex. In this study we attempted to explore the modes of interaction and energy binding of the different isomers placed in the active site of InhA with the help of various molecular modelling techniques. Ligand and enzyme models were generated with the help of the Vega ZZ program package. Resulting ligands were then docked into the InhA active site individually using computational automated docking package AUTODOCK 3.0.5. The more relevant docked conformations were then used to compute the interaction energy between the ligands and the InhA cavity. The AM1 Hamiltonian and the QM/MM ONIOM methodologies were used and the results compared. The various tautomers were found docked in almost the same place where INH-NAD was present as predicted by earlier X-ray crystallographic studies. However, some changes of ligand conformation and of the interactions ligand–protein were evidenced. The lower binding energy was observed for the 4 S chain adduct that probably represents the effective active form of the INH-NAD adducts, as compared to the 4 R epimer. The two 4 S,7 R and 4 R,7 S ring tautomers show intermediate and similar binding energies contrasting with their different experimental inhibitory potency on InhA. As a possible explanation based on calculated conformations, we formulated the hypothesis of an initial binding of the two ring tautomers to InhA followed by opening of only the ring hemiamidal 4 S,7 R tautomer (possibly catalyzed by Tyr158 phenolate basic group) to give the 4 S chain INH-NAD tight-binding inhibitor. The predictions of ligand–protein interactions at the molecular level can be of primary importance in elucidating the mechanisms of action of isoniazid and InhA-related resistances, in identifying the effective mycobactericidal entities and, in further step, in the design of a new generation of antitubercular drugs.

Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

Enoyl-ACP reductases participate in fatty acid biosynthesis by utilizing NADH to reduce the trans double bond between positions C2 and C3 of a fatty acyl chain linked to the acyl carrier protein. The enoyl-ACP reductase from Mycobacterium tuberculosis, known as InhA, is a member of an unusual FAS-II system that prefers longer chain fatty acyl substrates for the purpose of synthesizing mycolic acids, a major component of mycobacterial cell walls. The crystal structure of InhA in complex with NAD+ and a C16 fatty acyl substrate, trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester, reveals that the substrate binds in a general "U-shaped" conformation, with the trans double bond positioned directly adjacent to the nicotinamide ring of NAD+. The side chain of Tyr158 directly interacts with the thioester carbonyl oxygen of the C16 fatty acyl substrate and therefore could help stabilize the enolate intermediate, proposed to form during substrate catalysis. Hydrophobic residues, primarily from the substrate binding loop (residues 196-219), engulf the fatty acyl chain portion of the substrate. The substrate binding loop of InhA is longer than that of other enoyl-ACP reductases and creates a deeper substrate binding crevice, consistent with the ability of InhA to recognize longer chain fatty acyl substrates.