Synthesis,Spectroscopic Analysis,and in Vitro/in Silico Biological Studies of Novel Piperidine Derivatives Heterocyclic Schiff-Mannich Base Compounds

In the present study, 3-substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( S1-8 ) were synthesized by treating 4-hydroxybenzaldehyde ( B ) with eight different 3-substitued-4-amino-4,5-dihydro-1H-1,2,4-triazole-5-ones ( T1-8 ) in acetic acid medium, separately. The synthesized Schiff bases ( S ) were reacted with formaldehyde and secondary amine such as 4-piperidinecarboxyamide to afford novel heterocyclic bases. 3-Substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( T ) were treated with 4-piperidinecarboxyamide in the presence of formaldehyde to synthesize eight new 1-(4-piperidinecarboxyamide-1-yl - methyl)-3-substitued-4-(4-hydroxybenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones ( M1-8 ). The structure characterization of compounds was carried out using ¹H-NMR, IR, HR-MS, and ¹³C-NMR spectroscopic methods. The inhibitory properties of the newly synthesized compounds were calculated against the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and glutathione S-transferase (GST) enzymes. Ki values were calculated in the range of 20.06±3.11–36.86±6.17 μM for GST, 17.87±2.91–30.53±4.25 μM for AChE, 9.08±0.69–20.02±2.88 μM for BChE, respectively, Besides, IC₅₀ values were also calculated. Best binding scores of -inhibitors against used enzymes were calculated as −12.095 kcal/mol, −12.775 kcal/mol, and −9.336 kcal/mol, respectively. While 5-oxo-triazole piperidine-4-carboxamide moieties have a critical role in the inhibition of AChE and GST enzymes, hydroxy benzyl moiety is important for BChE enzyme inhibition.     

Participation of cAMP in tacrine-induced gastric smooth muscle relaxation

Tacrine, a well-known acetylcholinesterase inhibitor, applied in concentrations higher than 2×10⁻⁵ mol/l promoted Ca²⁺-independent relaxation of rat gastric smooth muscles in experiments in vitro. The relaxation was not cholinergic and was a result of influence of tacrine over intracellular signaling pathways regulating smooth muscle contraction/relaxation. The nature of this untypical muscle relaxation was studied by using smooth muscle strips isolated from rat stomach. Their bioelectrical and mechanical responses were recorded after treatment with tacrine and different activators or blockers of intracellular pathways involved in muscle contractility. Following the activation of adenylate cyclase with 1×10⁻⁶ mol/l forskolin and increase in the concentration of cyclic adenosine monophosphate (cAMP) after application of 4×10⁻⁵ mol/l SQ22536, a significant decrease in the muscle relaxation was observed. Theophylline (2×10⁻⁴ mol/l), a phosphodiesterase inhibitor, had no effect on the amplitude of tacrine-induced relaxation. The latter was also reduced by inhibition of protein kinase A (PKA) with 5×10⁻⁶ mol/l KT5720. These findings support the assumption that tacrine promoted smooth muscle relaxation through PKA-induced phosphorylation and inhibition of myosin light chain kinase activity. The reduction of spike-linked Ca²⁺ influx provoked by tacrine was probably a secondary contributing process, associated with an influence of increased cAMP level on Ca²⁺ channels.     

Antiproliferative Noscapinoids Bearing an Amidothiadiazole Scaffold as Apoptosis Inducers: Design,Synthesis and Molecular Docking

Noscapine an FDA-approved antitussive agent. With low cytotoxicity with higher concentrations, noscapine and its derivatives have been shown to have exceptional anticancer properties against a variety of cancer cell lines. In order to increase its potency, in this study, we synthesized a series of new amido-thiadiazol coupled noscapinoids and tested their cytotoxicity in vitro. All of the newly synthesised compounds demonstrated potent cytotoxic potential, with IC₅₀ values ranging from 2.1 to 61.2 μM than the lead molecule, noscapine (IC₅₀ value ranges from 31 to 65.5 μM) across all cell lines, without affecting normal cells (IC₅₀ value is>300 μM). Molecular docking of all these molecules with tubulin (PDB ID: 6Y6D, resolution 2.20 Å) also revealed better binding affinity (docking score range from −5.418 to −9.679 kcal/mol) compared to noscapine (docking score is −5.304 kcal/mol). One of the most promising synthetic derivatives 6aa (IC₅₀ value ranges from 2.5 to 7.3 μM) was found to bind tubulin with the highest binding affinity (ΔG_binding is −28.97 kcal/mol) and induced apoptosis in cancer cells more effectively.     

Protoberberine Alkaloids Berberine,Palmatine, and Coralyne Binding to Poly(dT)⋅(Poly(dA)⋅Poly(dT)) Triplex: Comparative Structural Aspects and Energetics Profiles of the Interaction

The interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT)⋅(poly(dA)⋅poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29×10⁷ M ⁻¹) than that of berberine (3.43×10⁵ M ⁻¹) and palmatine (2.34×10⁵ M ⁻¹). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (ΔH=−3.34 kcal/mol) with favorable entropy contribution (TΔS = 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (ΔH =−3.88 kcal/mol) and entropy changes (TΔS = 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (ΔH =−25.62 kcal/mol and TΔS =−15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.     

Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4’ phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (?.164 kcal/mol) and B (?.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: ?5.872 kcal/mol and B form: ?5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.     

Development of tacrine clusters as positively cooperative systems for the inhibition of acetylcholinesterase

The synthesis of four tetra-tacrine clusters where the tacrine binding units are attached to a central scaffold via linkers of variable lengths is described. The multivalent inhibition potencies for the tacrine clusters were investigated for the inhibition of acetylcholinesterase. Two of the tacrine clusters displayed a small but significant multivalent inhibition potency in which the binding affinity of each of the tacrine binding units increased up to 3.2 times when they are connected to the central scaffold.     

New Anthranilic Acid Hydrazones as Fenamate Isosteres: Synthesis,Characterization, Molecular Docking,Dynamics & in Silico ADME,in Vitro Anti-Inflammatory and Anticancer Activity Studies

In this study, twenty new anthranilic acid hydrazones 6 – 9 ( a – e ) were synthesized and their structures were characterized by Fourier-transform Infrared (FT-IR), Nuclear Magnetic Resonance (¹H-NMR – ¹³C-NMR), and High-resolution Mass Spectroscopy (HR-MS). The inhibitory effects of the compounds against COX-II were evaluated. IC₅₀ values of the compounds were found in the range of >200–0.32 μM and compounds 6e , 8d , 8e , 9b , 9c , and 9e were determined to be the most effective inhibitors. Cytotoxic effects of the most potent compounds were investigated against human hepatoblastoma (Hep-G2) and human healthy embryonic kidney (Hek-293) cell lines. Doxorubicin (IC₅₀: 8.68±0.16 μM for Hep-G2, 55.29±0.56 μM for Hek-293) was used as standard. 8e is the most active compound, with low IC₅₀ against Hep-G2 (4.80±0.04 μM), high against Hek-293 (159.30±3.12), and high selectivity (33.15). Finally, molecular docking and dynamics studies were performed to understand ligand-protein interactions between the most potent compounds and COX II, Epidermal Growth Factor Receptor (EGFR), and Transforming Growth Factor beta II (TGF-βII). The docking scores were calculated in the range of −10.609–−6.705 kcal/mol for COX-II, −8.652–−7.743 kcal/mol for EGFR, and −10.708–−8.596 kcal/mol for TGF-βII.     

Dual-Knockout of Mutant Isocitrate Dehydrogenase 1 and 2 Subtypes Towards Glioma Therapy: Structural Mechanistic Insights on the Role of Vorasidenib

Recently, Vorasidenib (AG-881) has been reported as a therapeutic alternative that exerts potent dual inhibitory activity against mIDH1/2 towards the treatment of low-grade glioma. However, structural and dynamic events associated with its dual inhibition mechanism remain unclear. As such, we employ integrative computer-assisted atomistic techniques to provide thorough structural and dynamic insights. Our analysis proved that the dual-targeting ability of AG-881 is mediated by Val255/Val294 within the binding pockets of both mIDH1 and mIDH2 which are shown to elicit a strong intermolecular interaction, thus favoring binding affinity. The structural orientations of AG-881 within the respective hydrophobic pockets allowed favorable interactions with binding site residues which accounted for its high binding free energy of −28.69 kcal/mol and −19.89 kcal/mol towards mIDH1 and mIDH2, respectively. Interestingly, upon binding, AG-881 was found to trigger systemic alterations of mIDH1 and mIDH2 characterized by restricted residue flexibility and a reduction in exposure of residues to the solvent surface area. As a result of these structural alterations, crucial interactions of the mutant enzymes were inhibited, a phenomenon that results in a suppression of the production of oncogenic stimulator 2-HG. Findings therefore provide thorough structural and dynamic insights associated with the dual inhibitory activity of AG-881 towards glioma therapy.     

Nostotrebin 6, a bis(cyclopentenedione) with cholinesterase inhibitory activity isolated from Nostoc sp. str. Lukešová 27/97

Nostotrebin 6, a new polyphenolic compound with a fully substituted 2,2′-bis(cyclopent-4-en-1,3-dione) skeleton, was isolated from a methanolic extract of the cyanobacterial strain Nostoc sp. str. Luke?ová 27/97. The structure of this compound was determined using X-ray crystallography and further supported by NMR, IR spectroscopy, and MS. Nostotrebin 6 is an S-parabolic I-parabolic noncompetitive inhibitor of acetylcholinesterase (IC₅₀ = 5.5?μM) and an S-parabolic I-parabolic mixed inhibitor of butyrylcholinesterase (IC₅₀ = 6.1–7.5?μM). The inhibitory potency of nostotrebin 6 was compared with that of tacrine and galanthamine.     

Novel mechanism-based substrates of dihydrofolate reductase and the thermodynamics of ligand binding: A comparison of theory and experiment for 8-methylpterin and 6,8-dimethylpterin

Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of the designed mechanism-based pterins, 8-methylpterin and 6,8-dimethylpterin, to dihydrofolate reductase (DHFR), with co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The calculated free energy differences suggest that DHFR.NADPH.6,8-dimethylpterin is thermodynamically more stable than DHFR.NADPH.8-methylpterin by 2.4 kcal/mol when the substrates are protonated and by 1.3 kcal/mol when neutral. The greater binding strength of 6,8-dimethylpterin may be attributed largely to hydration effects. In terms of an appropriate model for the pH-dependent kinetic mechanism, these differences can be interpreted consistently with experimental data obtained from previous kinetic studies, i.e., 6,8-dimethylpterin is a more efficient substrate of vertebrate DHFRs than 8-methylpterin. The kinetic data suggest a value of 6.6 ± 0.2 for the pK_a of the active site Glu-30 in DHFR.NADPH. We have also used experimental data to estimate absolute values for thermodynamic dissociation constants of the active (i.e., protonated) forms of the substrates: these are of the same order as for the binding of folate (0.1–10 μM). The relative binding free energy calculated from the empirically derived dissociation constants for the protonated forms of 8-methylpterin and 6,8-dimethylpterin is 1.4 kcal/mol, a value which compares reasonably well with the theoretical value of 2.4 kcal/mol. © 1993 Wiley-Liss, Inc.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

Phytochemical Profiling and Pharmacological Evaluation of Leaf Extracts of Ruellia tuberosa L.: An In Vitro and In Silico Approach

The present study was designed to appraise the photoprotective, antioxidant, and antibacterial bioactivities of Ruellia tuberosa leaves extracts (RtPE, RtChl, RtEA, RtAc, RtMe, and RtHMe). The results showed that, RtHMe extracts of R. tuberosa was rich in total phenolic content, i. e., 1.60 mgGAE/g dry extract, while highest total flavonoid content was found in RtAc extract, i. e., 0.40 mgQE/g. RtMe showed effective antioxidant activity (%RSA: 58.16) at the concentration of 120 μL. RtMe, RtEA and RtHMe exhibited effective in vitro antibacterial activity against Gram-negative bacteria (E. coli). In silico docking studies revealed that paucifloside (−11.743 kcal/mol), indole-3-carboxaldehyde (−7.519 kcal/mol), nuomioside (−7.275 kcal/mol), isocassifolioside (−6.992 kcal/mol) showed best docking score against PDB ID 2EX8 [penicillin binding protein 4 (dacB) from Escherichia coli, complexed with penicillin-G], PDB ID 6CQA (E. coli dihydrofolate reductase protein complexed with inhibitor AMPQD), PDB ID 2Y2I [Penicillin-binding protein 1B in complex with an alkyl boronate (ZA3)] and PDB ID 2OLV (from S. aureus), respectively. Docked phytochemicals also showed good drug likeness properties.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

The green synthesis and molecular docking of novel N-substituted rhodanines as effective inhibitors for carbonic anhydrase and acetylcholinesterase enzymes

Recently, inhibition effects of enzymes such as acetylcholinesterase (AChE) and carbonic anhydrase (CA) has appeared as a promising approach for pharmacological intervention in a variety of disorders such as epilepsy, Alzheimer’s disease and obesity. For this purpose, novel N-substituted rhodanine derivatives (RhAs) were synthesized by a green synthetic approach over one-pot reaction. Following synthesis the novel compounds, RhAs derivatives were tested against AChE and cytosolic carbonic anhydrase I, and II (hCAs I, and II) isoforms. As a result of this study, inhibition constant (Ki) were found in the range of 66.35 ± 8.35 to 141.92 ± 12.63 nM for AChE, 43.55 ± 14.20 to 89.44 ± 24.77 nM for hCA I, and 16.97 ± 1.42 to 64.57 ± 13.27 nM for hCA II, respectively. Binding energies were calculated with docking studies as −5.969, −5.981, and −9.121 kcal/mol for hCA I, hCA II, and AChE, respectively.     

Osthenol,a prenylated coumarin,as a monoamine oxidase A inhibitor with high selectivity

Osthenol (6), a prenylated coumarin isolated from the dried roots of Angelica pubescens, potently and selectively inhibited recombinant human monoamine oxidase-A (hMAO-A) with an IC₅₀ value of 0.74?µM and showed a high selectivity index (SI?>?81.1) for hMAO-A versus hMAO-B. Compound 6 was a reversible competitive hMAO-A inhibitor (K_i?=?0.26?µM) with a potency greater than toloxatone (IC₅₀?=?0.93?µM), a marketed drug. Isopsoralen (3) and bakuchicin (1), furanocoumarin derivatives isolated from Psoralea corylifolia L., showed slightly higher IC₅₀ values (0.88 and 1.78?µM, respectively) for hMAO-A than 6, but had low SI values (3.1 for both). Other coumarins tested did not effectively inhibit hMAO-A or hMAO-B. A structural comparison suggested that the 8-(3,3-dimethylallyl) group of 6 increased its inhibitory activity against hMAO-A compared with the 6-methoxy group of scopoletin (4). Molecular docking simulations revealed that the binding affinity of 6 for hMAO-A (?8.5?kcal/mol) was greater than that for hMAO-B (?5.6?kcal/mol) and that of 4 for hMAO-A (?7.3?kcal/mol). Docking simulations also implied that 6 interacted with hMAO-A at Phe208 and with hMAO-B at Ile199 by carbon hydrogen bondings. Our findings suggest that osthenol, derived from natural products, is a selective and potent reversible inhibitor of MAO-A, and can be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.     

Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values of methyl benzoates were determined. These compounds inhibited LPO with K_i values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (K_i=0.033±0.004 μM). The most potent inhibitor ( 1 a ) showed with a docking score of −3.36 kcal/mol and an MM-GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives ( 1 a – 16 a ) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).     

Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 10⁴M^−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK _ais 1.0 ± 0.1 × 10⁴M⁻¹ as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK _avalue is 1.38 ± 0.1 × 1 0 4M⁻¹ resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T _mof the protein decreases from 76°C to 72°C at 1 × 10⁻³M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK _aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.     

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ ?30 to ?35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ ?22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR’s (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.     

In-silico analysis of Sirt2 from Schistosoma mansoni: structures,conformations, and interactions with inhibitors

Sirtuins are NAD+-dependent lysine deacetylases member of the class III HDAC family. These are demonstrated to be therapeutic targets in parasitic diseases like schistosomiasis. Observations suggested that sirtuin enzyme is necessary for the functionality of fe/male reproductive system, due to which SmSirt2 is treated as a potential therapeutic target. There are no structural and molecular features of SmSirt2 have been reported yet. In this study, homology modeling has been used to determine the three-dimensional features of the SmSITRT2. Further, structure validation has been performed by energy minimization and Ramachandran plot. Validated structures are further subjected to molecular docking and virtual screening to find the best lead molecules for downstream analysis. Ten lead molecules were selected while comparing virtual screening of hSirt2 and SmSirt2 both. These leads are further compared with AKG2 which is known inhibitor of hSirt2 (?8.8 kcal/mol). Out of selected 10 leads, four of them (ZINC23995485 (?9.5 kcal/mol), ZINC53298162 (?9.4 kcal/mol), ZINC70927268 (?10.0 kcal/mol), ZINC89878705 (?11.2 kcal/mol)) have shown better interaction with SmSirt2, in which ZINC89878705 (?11.2 kcal/mol) shows a more compact packing as compared to AKG2 and rest of ligands. These molecules could be further subject to in vitro study and model of SmSirt2 has been proposed for further structure-based drug design projects concerning sirtuins from Schistosoma mansoni.