Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity.

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42-3.59 µM. The most active compound 1 showed an IC50 value of 0.42 µM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 µM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

Inhibition of Mycobacterium tuberculosis InhA by 3-nitropropanoic acid.

3-Nitropropanoic acid (3NP), a bioactive fungal natural product, was previously demonstrated to inhibit growth of Mycobacterium tuberculosis. Here we demonstrate that 3NP inhibits the 2-trans-enoyl-acyl carrier protein reductase (InhA) from Mycobacterium tuberculosis with an IC50 value of 71 µM, and present the crystal structure of the ternary InhA-NAD+ -3NP complex. The complex contains the InhA substrate-binding loop in an ordered, open conformation with Tyr158, a catalytically important residue whose orientation defines different InhA substrate/inhibitor complex conformations, in the "out" position. 3NP occupies a hydrophobic binding site adjacent to the NAD+ cofactor and close to that utilized by the diphenyl ether triclosan, but binds predominantly via electrostatic and water-mediated hydrogen-bonding interactions with the protein backbone and NAD+ cofactor. The identified mode of 3NP binding provides opportunities to improve inhibitory activity toward InhA.

Identification of Potent DNA Gyrase Inhibitors Active against Mycobacterium tuberculosis.

Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 µg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 µM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity.

Mycobacterium tuberculosis protein kinase B (PknB) is essential to mycobacterial growth and has received considerable attention as an attractive target for novel anti-tuberculosis drug development. Here, virtual screening, validated by biological assays, was applied to select candidate inhibitors of M. tuberculosis PknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitro biological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosis H37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 µg/mL, respectively. Two compounds, 2 and 10, inhibited M. tuberculosis PknB activity in vitro, with IC50 values of 14.4 and 12.1 µM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4 displayed anti-tuberculosis activity against M. tuberculosis H37Rv but showed no inhibition of PknB activity (IC50 > 128 µM). We hypothesize that hydrolysis of its ethyl ester to a carboxylate moiety generates an active species that inhibits other M. tuberculosis enzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10 bound to M. tuberculosis PknB indicated that compound 4 has a lower affinity for M. tuberculosis PknB than compounds 2 and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2 and 10 therefore represent candidate inhibitors of M. tuberculosis PknB that provide attractive starting templates for optimization as anti-tubercular agents.

α-Glucosidase Inhibitors from the Stems of Knema globularia.

Six new compounds, globunones A-F (1-6), and two new flavonoids (7 and 8) together with nine known compounds (9-17) were isolated from the stems of Knema globularia. The chemical structures of 1-8 were elucidated by an analysis of their NMR and high-resolution electrospray ionization mass spectrometry data as well as by comparison with literature values. The absolute configurations were determined using time-dependent density functional theory electronic circular dichroism (TD-DFT-ECD). Globunones A-E (1-5) represent the initial combined structures of a flavan-3-ol core and a 1,4-benzoquinone core. Globunone F (6) is the first flavanone-type compound bearing a 2-(2,4-dihydroxyphenyl)-2-oxoethyl group found to date in Nature. Compounds 1-3 and 6-17 were tested for their yeast α-glucosidase inhibitory activity. All compounds tested (except for 13 and 14) showed potent inhibition toward α-glucosidase with IC50 values in the range 0.4-26.6 µM. Calodenin A (15) was the most active compound with an IC50 value of 0.4 µM (the positive control, acarbose, IC50 93.6 µM). A kinetic analysis of 15 revealed that it is a noncompetitive inhibitor with a Ki value of 3.4 µM.

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 µg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 µM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

Simulations of Shikimate Dehydrogenase from Mycobacterium tuberculosis in Complex with 3-Dehydroshikimate and NADPH Suggest Strategies for MtbSDH Inhibition.

Shikimate dehydrogenase (SDH) from Mycobacterium tuberculosis ( MtbSDH), encoded by the aroE gene, is essential for viability of M. tuberculosis but absent from humans. Therefore, it is a potentially promising target for antituberculosis agent development. Molecular-level understanding of the interactions of MtbSDH with its 3-dehydroshikimate (DHS) substrate and NADPH cofactor will help in the design of novel and effective MtbSDH inhibitors. However, this is limited by the lack of relevant crystal structures for MtbSDH complexes. Here, molecular dynamics (MD) simulations were performed to generate these MtbSDH complexes and investigate interactions of MtbSDH with substrate and cofactor and the role of MtbSDH dynamics within these. The results indicate that, while structural rearrangements are not necessary for DHS binding, reorientation of individual side chains in the NADPH binding pocket is involved in ternary complex formation. The mechanistic roles for Lys69, Asp105, and Ala213 were investigated by generating Lys69Ala, Asp105Asn, and Ala213Leu mutants in silico and investigating their complexes with DHS and NADPH. Our results show that Lys69 plays a dual role, in positioning NADPH and in catalysis. Asp105 plays a crucial role in positioning both the ε-amino group of Lys69 and nicotinamide ring of NADPH for MtbSDH catalysis but makes no direct contribution to DHS binding. Ala213 is the selection key for NADPH binding with the nicotinamide ring in the proS, rather than proR, conformation in the MtbSDH complex. Our results identify three strategies for MtbSDH inhibition: prevention of MtbSDH binary and ternary complex formation by blocking DHS and NADPH binding (first and second strategies, respectively) and the prevention of MtbSDH complex formation with either DHS or NADPH by blocking both DHS and NADPH binding (third strategy). Further, based on this third strategy, we propose guidelines for the rational design of "hybrid" MtbSDH inhibitors able to bind in both the substrate (DHS) and cofactor (NADPH) pockets, providing a new avenue of exploration in the search for anti-TB therapeutics.

Structure-based drug design of novel M. tuberculosis InhA inhibitors based on fragment molecular orbital calculations.

2-trans enoyl-acyl carrier protein reductase (InhA) is a promising target for developing novel chemotherapy agents for tuberculosis, and their inhibitory effects on InhA activity were widely investigated by the physicochemical experiments. However, the reason for the wide range of their inhibitory effects induced by similar agents was not explained by only the difference in their chemical structures. In our previous molecular simulations, a series of heteroaryl benzamide derivatives were selected as candidate inhibitors against InhA, and their binding properties with InhA were investigated to propose novel derivatives with higher binding affinity to InhA. In the present study, we extended the simulations for a series of 4-hydroxy-2-pyridone derivatives to search widely for more potent inhibitors against InhA. Using ab initio fragment molecular orbital (FMO) calculations, we elucidated the specific interactions between InhA residues and the derivatives at an electronic level and highlighted key interactions between InhA and the derivatives. The FMO results clearly indicated that the most potent inhibitor has strong hydrogen bonds with the backbones of Tyr158, Thr196, and NADH of InhA. This finding may provide informative structural concepts for designing novel 4-hydroxy-2-pyridone derivatives with higher binding affinity to InhA. Our previous and present molecular simulations could provide important guidelines for the rational design of more potent InhA inhibitors.

Elucidating specific interactions for designing novel pyrrolamide derivatives as potential GyrB inhibitors based on ab initio fragment molecular orbital calculations.

Tuberculosis (TB), the second leading infectious killer, causes serious public health problems worldwide. To develop novel anti-TB agents, many biochemical studies have targeted the subunit B of DNA gyrase (GyrB), which captures a second DNA segment and responses for ATP hydrolysis. Here, we investigated specific interactions between GyrB residues and existing pyrrolamide derivatives at an electronic level using ab initio fragment molecular orbital (FMO) calculations and designed potent inhibitors against GyrB. The evaluated binding affinities between GyrB and pyrrolamides were confirmed to be consistent with the IC50 values obtained from previous experiments. Thus, we employed the most potent pyrrolamide (compound 1) as a lead compound and proposed novel pyrrolamide derivatives. The specific interactions between GyrB and these derivatives were investigated using molecular mechanic optimizations and FMO calculations. The results revealed that our proposed derivatives had strong hydrogen bonds with Asp79 and Arg141 and exhibited electrostatic interactions with Glu56 and Ile84 of GyrB. In addition, the binding affinity between GyrB and compound 1 was enhanced significantly by the replacement at the R3 site of compound 1. The present results may provide structural concepts for the rational design of potent GyrB inhibitors as anti-TB agents.Communicated by Ramaswamy H. Sarma.

In silico multiscale drug design to discover key structural features of potential JAK2 inhibitors.

Background: JAK2 inhibitors have been proposed as a new therapeutic option for thalassemia therapy. The objective of this study was to discover the key structural features for improving 2-aminopyrimidine derivatives as potential JAK2 inhibitors. Materials & methods: Quantitative structure-activity relationship (QSAR) approaches (hologram QSAR and comparative molecular similarity indices analysis), molecular dynamics simulations, binding energy calculations and pharmacokinetic predictions were employed. Results: Reliable QSAR models, binding mode and binding interactions of JAK2 inhibitors were obtained and these obtained results were used as the key information for rational design of highly potent JAK2 inhibitors. Conclusion: The concept of new potential JAK2 inhibitors integrated from the obtained results was proved, producing two newly designed compounds, D01 and D02, with potential for use as JAK2 inhibitors.

In silico design of novel quinazoline-based compounds as potential Mycobacterium tuberculosis PknB inhibitors through 2D and 3D-QSAR, molecular dynamics simulations combined with pharmacokinetic predictions.

Serine/threonine protein kinase B (PknB) is essential to Mycobacterium tuberculosis (M. tuberculosis) cell division and metabolism and a potential anti-tuberculosis drug target. Here we apply Hologram Quantitative Structure Activity Relationship (HQSAR) and three-dimensional QSAR (Comparative Molecular Similarity Indices Analysis (CoMSIA)) methods to investigate structural requirements for PknB inhibition by a series of previously described quinazoline derivatives. PknB binding of quinazolines was evaluated by molecular dynamics (MD) simulations of the catalytic domain and binding energies calculated by Molecular Mechanics/Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) methods. Evaluation of a training set against experimental data showed both HQSAR and CoMSIA models to reliably predict quinazoline binding to PknB, and identified the quinazoline core and overall hydrophobicity as the major contributors to affinity. Calculated binding energies also agreed with experiment, and MD simulations identified hydrogen bonds to Glu93 and Val95, and hydrophobic interactions with Gly18, Phe19, Gly20, Val25, Thr99 and Met155, as crucial to PknB binding. Based on these results, additional quinazolines were designed and evaluated in silico, with HQSAR and CoMSIA models identifying sixteen compounds, with predicted PknB binding superior to the template, whose activity spectra and physicochemical, pharmacokinetic, and anti-M. tuberculosis properties were assessed. Compound, D060, bearing additional ortho- and meta-methyl groups on its R2 substituent, was superior to template regarding PknB inhibition and % caseum fraction unbound, and equivalent in other aspects, although predictions identified hepatotoxicity as a likely issue with the quinazoline series. These data provide a structural basis for rational design of quinazoline derivatives with more potent PknB inhibitory activity as candidate anti-tuberculosis agents.

Discovery of novel and potent InhA inhibitors by an in silico screening and pharmacokinetic prediction.

Aim: In silico screening approaches were performed to discover novel InhA inhibitors. Methods: Candidate InhA inhibitors were obtained from the combination of virtual screening and pharmacokinetic prediction. In addition, molecular mechanics Poisson-Boltzmann surface area, molecular mechanics Generalized Born surface area and WaterSwap methods were performed to investigate the binding interactions and binding energy of candidate compounds. Results: Four candidate compounds with suitable physicochemical, pharmacokinetic and antibacterial properties are proposed. The crucial interactions of the candidate compounds were H-bond, pi-pi and sigma-pi interactions observed in the InhA binding site. The binding affinity of these compounds was improved by hydrophobic interactions with hydrophobic side chains in the InhA pocket. Conclusion: The four newly identified InhA inhibitors reported in this study could serve as promising hit compounds against Mycobacterium tuberculosis and may be considered for further experimental studies.

Specific interactions between 2-trans enoyl-acyl carrier protein reductase and its ligand: Protein-ligand docking and ab initio fragment molecular orbital calculations.

2-trans enoyl-acyl carrier protein reductase (InhA) has been identified as a promising target for the development of novel chemotherapy for tuberculosis. In the present study, a series of heteroaryl benzamide derivatives were selected as potent inhibitors against InhA, and their binding properties with InhA were investigated at atomic and electronic levels by ab initio molecular simulations based on protein-ligand docking, classical molecular mechanics optimizations and ab initio fragment molecular orbital (FMO) calculations. The results evaluated by FMO highlight some key interactions between InhA and the derivatives, indicating that the most potent derivative has strong hydrogen bonds with the Met98 side chain of InhA and strong electrostatic interactions with the nicotinamide adenine dinucleotide cofactor. These findings provide informative structural concepts for designing novel heteroaryl benzamide derivatives with higher binding affinity to InhA.

Key Structures and Interactions for Binding of Mycobacterium tuberculosis Protein Kinase B Inhibitors from Molecular Dynamics Simulation.

Substituted aminopyrimidine inhibitors have recently been introduced as antituberculosis agents. These inhibitors show impressive activity against protein kinase B, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the protein kinase B enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent protein kinase B inhibitors in the future. Here, molecular dynamics simulations, in conjunction with molecular mechanics/Poisson-Boltzmann surface area binding free-energy analysis, were employed to gain insight into the complex structures of the protein kinase B inhibitors and their binding energetics. The complex structures obtained by the molecular dynamics simulations show binding free energies in good agreement with experiment. The detailed analysis of molecular dynamics results shows that Glu93, Val95, and Leu17 are key residues responsible to the binding of the protein kinase B inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of protein kinase B inhibitors with highly increased antagonistic activity.

Elucidating the structural basis of diphenyl ether derivatives as highly potent enoyl-ACP reductase inhibitors through molecular dynamics simulations and 3D-QSAR study.

Diphenyl ether derivatives are good candidates for anti-tuberculosis agents that display a promising potency for inhibition of InhA, an essential enoyl-acyl carrier protein (ACP) reductase involved in fatty acid biosynthesis pathways in Mycobacterium tuberculosis. In this work, key structural features for the inhibition were identified by 3D-QSAR CoMSIA models, constructed based on available experimental binding properties of diphenyl ether inhibitors, and a set of four representative compounds was subjected to MD simulations of inhibitor-InhA complexes for the calculation of binding free energies. The results show that bulky groups are required for the R1 substituent on the phenyl A ring of the inhibitors to favor a hydrophobic pocket formed by residues Phe149, Met155, Pro156, Ala157, Tyr158, Pro193, Met199, Val203, Leu207, Ile215, and Leu218. Small substituents with a hydrophilic property are required at the R3 and R4 positions of the inhibitor phenyl B rings to form hydrogen bonds with the backbones of Gly96 and Met98, respectively. For the R2 substituent, small substituents with simultaneous hydrophilic or hydrophobic properties are required to favor the interaction with the pyrophosphate moiety of NAD(+) and the methyl side chain of Ala198, respectively. The reported data provide structural guidance for the design of new and potent diphenyl ether-based inhibitors with high inhibitory activities against M. tuberculosis InhA.

Key Structural Features of Azanaphthoquinone Annelated Pyrrole Derivative as Anticancer Agents Based on the Rational Drug Design Approaches.

Azanaphthoquinone annelated pyrrole derivatives have been developed and synthesized with a continuous attempt to develop novel DNA intercalating agents as anti-cancer compounds with lower organ toxicity. With the remarkable antiproliferative activity of synthesized azanaphthoquinone annelated pyrrole derivatives, a structurally novel scaffold of these compounds is appropriated for further development of novel anti-cancer agents. Therefore, in the present study, 3D QSAR study (CoMSIA) was applied on 28 azanaphthoquinone annelated pyrrole derivatives to evaluate the structural requirement of these compounds. The resulting CoMSIA model is satisfied with r(2) of 0.99 and q(2) of 0.65. The interpretation of CoMSIA contours reveals the significant importance of steric, electrostatic, hydrophobic and hydrogen acceptor descriptors on the activities of azanaphthoquinone annelated pyrrole derivatives. Remarkably, the structural requirement of six substituent positions on the azanaphthoquinone annelated pyrrole scaffold was elucidated here. This result is the useful concept for design of new and more active azanaphthoquinone annelated pyrrole derivatives. Moreover, MD simulations using AMBER program were performed to model the binding of azanaphthoquinone annelated pyrrole derivatives in the intercalation site of the DNA duplex. Based on MD simulations, the information in terms of ligand-DNA interaction, complex structure and binding free energy was provided in this work. Therefore, the integrated results are informative for further modification of azanaphthoquinone annelated pyrrole scaffold leading to gain novel azanaphthoquinone annelated pyrrole derivatives possessing better antiproliferative activity.