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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. tuberculosisGyrB 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. tuberculosisDNA gyrase relative to those from other bacterial species.

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosisinfections. Identification of new agents that inhibit M. tuberculosisDNA 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. tuberculosisDNA gyrase ATPase activity. This yielded the modified compound R3-13with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosisDNA gyrase. Utilization of compound R3-13as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosisDNA gyrase ATPase inhibitors with IC50values in the range of 0.42–3.59 μM. The most active compound 1showed an IC50value of 0.42 μM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 μM). Compound 1showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosisDNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1to M. tuberculosisGyrB 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 1represents a potential new scaffold for further exploration and optimization as a M. tuberculosisDNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

Mycobacterium tuberculosisDNA 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. tuberculosisDNA 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, G24and G26, inhibited the growth of M. tuberculosisH37Rv with a minimal inhibitory concentration of 12.5 μg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50values of 2.69 and 2.46 μM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24and G26bound to the M. tuberculosisDNA 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 G24and G26provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Mycobacterium tuberculosisis the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosisDNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosisGyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosisDNA gyrase ATPase inhibitors. IC50values of compounds 8, 11, and 14(0.26, 0.56, and 0.08 μM, respectively) for inhibition of M. tuberculosisDNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosisH37Ra are 25.0, 3.1, and 6.2 μg/mL, respectively, superior to novobiocin (MIC > 100.0 μg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14to M. tuberculosisGyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosisDNA gyrase ATPase activity in vitro and growth of M. tuberculosisbacteria.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosisis 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 1and 7) showed MIC of 12.5 and 25 μg/mL against M. tuberculosisH37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50values 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. tuberculosisthat act by targeting InhA.

Shikimate dehydrogenase (SDH) from Mycobacterium tuberculosis(MtbSDH), encoded by the aroEgene, is essential for viability of M. tuberculosisbut 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 silicoand 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.

In this work, water treatment sludge from the water treatment plant was used to synthesize zeolite material. In the synthesis, the washed sludge was mixed with sodium hydroxide and then heated at 600ºC for 6 h. After agitation, the aged material was heated in a water bath at 80ºC for different period of aging times. Based on XRD and SEM analyses, Faujasite zeolite was obtained. Then, the feasibility of employing the obtained zeolite as adsorbent for Brilliant Green (BG) removal was investigated. The effect of adsorbent dosage and contact time were examined. Adsorption kinetics and isotherm were also evaluated. The results showed that the obtained zeolite has potential for applying as low-cost adsorbent for the removal of BG from wastewater with higher than 97% adsorption efficiency.

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 r2of 0.99 and q2of 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.

The on-chip platinum micro-heater prototypes for thermal cycling equipped with platinum temperature sensor are fabricated. The device has been designed, fabricated and characterized to explore the feasibility of the micro-heater for a fully integrated disposable lab-on-a-chip with the PCR module. The on-chip micro-heater demonstrates that the temperature transitions are shorter by comparison with the conventional PCR temperature routines.

Six new compounds, globunones A–F (1–6), and two new flavonoids (7and 8) together with nine known compounds (9–17) were isolated from the stems of Knema globularia.The chemical structures of 1–8were 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 1–3and 6–17were tested for their yeast α-glucosidase inhibitory activity. All compounds tested (except for 13and 14) showed potent inhibition toward α-glucosidase with IC50values in the range 0.4–26.6 μM. Calodenin A (15) was the most active compound with an IC50value of 0.4 μM (the positive control, acarbose, IC5093.6 μM). A kinetic analysis of 15revealed that it is a noncompetitive inhibitor with a Kivalue of 3.4 μM.

Mycobacterium tuberculosisprotein 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. tuberculosisPknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitrobiological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosisH37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 μg/mL, respectively. Two compounds, 2and 10, inhibited M. tuberculosisPknB activity in vitro, with IC50values of 14.4 and 12.1 μM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4displayed anti-tuberculosis activity against M. tuberculosisH37Rv 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. tuberculosisenzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10bound to M. tuberculosisPknB indicated that compound 4has a lower affinity for M. tuberculosisPknB than compounds 2and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2and 10therefore represent candidate inhibitors of M. tuberculosisPknB that provide attractive starting templates for optimization as anti-tubercular agents.