Probable Binding Site Research Articles

Modulation of the mitochondrial voltage-dependent anion channel (VDAC) by hydrogen peroxide and its recovery by curcumin.

The Voltage-Dependent Anion Channel (VDAC) plays a vital role in mitochondria-mediated transport of ions and metabolites. It is well established that mitochondria are a site for production of hydrogen peroxide (H2O2). Excess production of H2O2 is toxic to the cell and causes oxidative stress. Therefore, the effect of H2O2 on the single-channel conductance of VDAC was investigated. In vitro bilayer electrophysiology experiments were performed on VDAC isolated from rat brain mitochondria, which consists predominately of the isoform VDAC1. VDAC was treated with H2O2 on a planar bilayer membrane (BLM). The conductance of VDAC increased upon H2O2 treatment, whereas the same concentration of H2O2 was unable to affect the BLM (without protein) over a long period of time. Subsequently, the sequential addition of curcumin to H2O2-treated VDAC reduced the conductance. Experimental results (bilayer electrophysiology) demonstrate the role of curcumin in the restoration of the activity of VDAC affected by H2O2. In silico docking studies enables identification of the probable binding site of H2O2 on VDAC. We further find that the oligomerization of VDAC that results in its increased conductance is an effect of lipid oxidation by H2O2.

Characterization of dye-decolorizing peroxidase from Bacillus subtilis

The dye-decolorizing peroxidases (DyPs) belong to a unique heme peroxidase family for their biotechnological potential to detoxify synthetic dyes. In this work, we have biochemically and structurally characterized the dye-decolorizing peroxidase from Bacillus subtilis (BsDyP). The biochemical studies of BsDyP demonstrate that pH 4.0 is optimum for the oxidation of malachite green (MG) and methyl violet (MV). However, it oxidizes the MG with higher catalytic efficiency (kcat/Km = 6.3 × 102 M−1s−1), than MV (kcat/Km = 5.0 × 102 M−1s−1). While reactive black 5 (RB5) is oxidized at pH 3.0 with the catalytic efficiency of kcat/Km = 3.6 × 102 M−1s−1. The calculated thermodynamic parameters by isothermal titration calorimetry (ITC) reveal the feasibility and spontaneity of dyes binding with BsDyP. Further, the crystal structures of a HEPES bound and unbound of BsDyP provide insight into the probable binding sites of the substrates. In BsDyP-HEPES bound structure, the HEPES-1 molecule is found in the heme cavity at the γ-edge, and another HEPES-2 molecule is bound ~16 Å away from the heme that is fenced by Ile231, Arg234, Ser235, Asp239, Glu334, and surface-exposed Tyr335 residues. Furthermore, the molecular docking, simulation, and MMPBSA studies support the binding of dyes at both the sites of BsDyP and produce lower-energy stable BsDyP-dyes complexes. Here, the BsDyP study allows the identification of its two potential binding sites and shows the oxidation of a variety of dyes. Structural and functional insight of BsDyP will facilitate its engineering for the improved decolorization of dyes.

Activating Effect of 3-Benzylidene Oxindoles on AMPK: From Computer Simulation to High-Content Screening.

AMP-activated protein kinase (AMPK) is currently the subject of intensive study and active discussions. AMPK performs its functions both at the cellular level, providing the switch between energy-consuming and energy-producing processes, and at the whole body level, particularly, regulating certain aspects of higher nervous activity and behavior. Control of such a 'main switch' compensates dysfunctions and associated diseases. In the present paper, we studied the binding of 3-benzylidene oxindoles to the kinase domain of the AMPK α-subunit, which is thought to prevent its interaction with the autoinhibitory domain and thus result in the AMPK activation. For this purpose, we developed the cellular test system based on the AMPKAR plasmid, which implements the FRET effect, synthesized a number of 3-benzylidene oxindole compounds and simulated their binding to various sites of the kinase domain. The most probable binding site for the studied compounds was established by the correlation of calculated and experimental data. The obtained results allow to analyze various classes of AMPK activators using virtual and high-content screening.

New Protocol for Predicting the Ligand-Binding Site and Mode Based on the 3D-RISM/KH Theory.

An efficient algorithm to find the binding position and mode of small ligands bound at an active site of protein is proposed based on the spatial distribution function (SDF) obtained from the three-dimensional reference interaction site model (3D-RISM) theory with the Kovalenko-Hirata (KH) closure relation. The ligand examined includes hydrophobic, acidic, and basic molecules and zwitterions. Eighteen different types of proteins, which serve as targets for those ligands, are selected to examine the robustness of the algorithm. An imaginary atom, referred to as an "anchor site", is defined at the center of geometry of a ligand molecule that serves as a center for searching the binding position and mode of the ligand molecule in the translational and rotational spaces. The probable binding sites (PBSs) are identified based on the SDFs of the ligand molecules around the protein, and the PBS is ranked according to the peak height of SDF. The deviations from the mean height of the peak values of SDFs for 50 PBSs are analyzed based on the z-score, which is a measure of prominence of the site. The PBS found at the closest distance from the anchor site of the crystal structure is referred to as the "nearest site". The orientation of the ligand molecule at each PBS is explored by changing the Euler angles, and the most probable binding mode is determined based on the superposition approximation. The binding position of ligand molecules is successfully predicted as one of the distinct peaks in SDF of the anchor site, with a few exceptions. The binding mode of the ligand molecule predicted based on the superposition approximation is consistent with the X-ray crystal structure in nine systems, a half of the systems investigated. The significance of the results is discussed in detail. An application of the new protocol to fragment-based drug discovery is suggested.

A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

Peripheral Binding of Hexokinase-2 to the Rim of VDAC1 Mediated by the Mitochondrial Outer Membrane

Complex formation between hexokinase-2 (HK2) and the mitochondrial channel VDAC1 plays a crucial role in regulating cell death, however, structural details of this complex remain elusive. While previous protein-protein docking studies proposed direct insertion of HK2 into the VDAC1 pore, several experimental studies suggest that this process might be facilitated by an initial insertion of HK2 into the mitochondrial outer membrane. Therefore, in order to systematically model the complex of membrane-embedded VDAC1 with membrane-bound HK2, we adopted a hybrid approach combining molecular dynamics (MD) and Brownian dynamics (BD) simulations, first describing membrane binding of HK2 with MD, and then formation of its complex with VDAC1 with BD. Spontaneous insertion of HK2 N-terminal helix was consistently captured in multiple independent MD simulations. The positioning (insertion and orientation) of HK2 in the membrane were then used as restraints in 100 independent BD simulations (totaling 2 milliseconds), where binding of HK2 to a membrane-embedded VDAC1 was studied. Clustering analysis identified VDAC1 outer rim as the most probable HK2 binding site. The complex was further refined with MD simulations, during which additional salt-bridges formed to strengthen the complex. A major feature in the complex is the blockade of VDAC1's permeation pathway by HK2, a result supported by electrophysiological measurements. The HK2-VDAC1 interface is highlighted by the presence of a putative GSK3β phosphorylation site, S215, whose phosphorylation disrupts the most probable binding site of HK2 in VDAC1. Consistent with the simulation results, electrophysiological experiments show that HK2 blocks ion conduction in wildtype VDAC1 but not in the phosphomimetic S215E mutant. Furthermore, the S215E mutation reduces cell survival rate under hypoxia-reoxygenation stress, possibly due to reduced HK2 binding. Together, our results provide a novel, experimentally validated model for membrane-bound HK2-VDAC1 complex.

Novel Quinoline Compound Derivatives of NSC23925 as Potent Reversal Agents Against P-Glycoprotein-Mediated Multidrug Resistance.

Multidrug resistance is a serious problem and a common cause of cancer treatment failure, leading to patient death. Although numerous reversal resistance inhibitors have been evaluated in preclinical or clinical trials, efficient and low-toxicity reversal agents have not been identified. In this study, a series of novel quinoline compound derivatives from NSC23925 were designed to inhibit P-glycoprotein (P-gp). Among them, YS-7a showed a stronger inhibitory effect against P-gp than verapamil, as a positive control, when co-incubated with chemotherapy drugs at minimally cytotoxic concentrations. YS-7a suppressed the P-gp transport function without affecting the expression of P-gp but stimulated the ATPase activity of P-gp in a dose-dependent manner. Next, molecular docking was used to predict the six most probable binding sites, namely, SER270, VAL273, VAL274, ILE354, VAL357, and PHE390. Moreover, YS-7a had no effect on cytochrome P450 3A4 activity and showed little toxicity to normal cells. In addition, combined treatment of YS-7a with vincristine showed a better inhibitory effect than the positive control verapamil in vivo without a negative effect on mouse weight. Overall, our results showed that YS-7a could reverse cancer multidrug resistance through the inhibition of P-gp transport function in vitro and in vivo, suggesting that YS-7a may be a novel therapeutic agent.

Biological evaluation, molecular docking and DFT studies of charge transfer complexes of quinaldic acid with heterocyclic carboxylic acid

The quinaldic acid based charge transfer (CT) complexes with the ionic counterpart of furan (QCFC), thiophene (QCTC) and pyridine-2-carboxylic acid (QCPC) were prepared and successfully characterized by 1H NMR, 13C NMR, FT-IR spectroscopic techniques. In addition, the QCFC was characterized by Dept-135 and 2D NMR (Cosy, HQSC and HMBC) techniques. The electrical conductivity of synthesized CT complexes has also been investigated. The in vitro anti-inflammatory and antidiabetic activities were studied and the results were compared with reference drugs. The QCFC exhibited better anti-inflammatory and antidiabetic activity than QCTC and QCPC. Good stability under physiological conditions was observed for the CT complexes synthesized. The molecular docking studies were also performed with an α-amylase enzyme (1HNY.pdb) to identify the probable binding site of the complexes. All the CT complexes have similar binding score and the QCFC showed 4 hydrogen bonding interactions with α-amylase enzyme. Also, the 3D structure of the complexes was optimized using the Gaussian09 W program and DFT calculations were performed to find the significant regions that are necessary for enzyme inhibition.

M61 VALIDATION, CHARACTERIZATION OF OBSESSIVE-COMPULSIVE DISORDER CASE SAMPLES IDENTIFIED IN LARGE US BIOBANK

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.

Spectral and computational attributes: Binding of a potent anticancer agent, dasatinib to a transport protein

The present study describes the insights in to the mechanism of interaction between a model protein and an anticancer drug, dasatinib (DAS). DAS is a multi-target anticancer drug which controls the proliferation of mutant expressing cells. Human serum albumin (HSA) is the ubiquitous protein of blood plasma (40–50 g/L) and able to bind a wide range of exogenous and endogenous ligands. Binding mechanism of DAS to HSA was studied under physiological conditions by multi-spectroscopic techniques viz., steady state fluorescence, time resolved fluorescence, fluorescence (Fӧrster) resonance energy transfer (FRET), UV–visible and FTIR besides molecular modeling studies. Fluorescence measurements showed that the fluorescence intensity of HSA was quenched significantly by DAS with a red shift (from 342 to 356 nm) in its emission wavelength. The values of binding constant, number of binding sites and Stern-Volmer quenching constant (Ksv) were calculated for DAS-HSA interactions at different temperatures. Decreased Ksv values with increase in temperature suggested the presence of static quenching mechanism. This was also evident from time resolved fluorescence studies. The probable binding site for DAS in the hydrophobic pocket (sub-domain IIA) of HSA was revealed by both site probe experiments and molecular docking studies. Negative values of ΔG° revealed the spontaneity of DAS-HSA interaction while negative ΔH° and ΔS° values indicated the participation of hydrogen bonding and van der Waals forces in the stabilization of DAS-HSA complex. Further, conformational change in the protein backbone upon the addition of DAS was evident from absorption, FT-IR and synchronous fluorescence spectral results. The effect of essential metal ions on the binding of DAS to protein was also examined.

Anti-HIV-1 activity of quinic acid isolated from Helichrysum mimetes using NMR-based metabolomics and computational analysis

Helichrysum species are widely used in traditional medicine with potential against infectious diseases. Extraction of the aerial parts of 32 Helichrysum species was done using polar (methanol:water (1:1)) and non-polar (hexane, dichloromethane and acetone) solvent systems. Anti-human immunodeficiency virus (HIV) bioassays revealed that polar extracts of H. mimetes and H. chrysargyrum (2.5 μg/mL), polar and non-polar extracts of H. cephaloideum (25 μg/mL) and polar and non-polar extracts of H. zeyheri, H. setosum, H. platypterum and H. kraussii at (2.5 μg/mL), had greater than 90% inhibitory activity. The polar extract of H. mimetes (100 μg/mL) exhibited 55.93% reverse transcriptase (RT) inhibition as a possible indication of the mechanism of action. Proton NMR spectra of the polar extracts exhibited the presence of aromatic compounds and carbohydrate moieties. Principal component analysis (PCA) of the polar extracts showed clustering related to the activity of the extracts with good predictability scores (Q2 > 0.5). However, orthogonal projections to latent structures discriminant analysis (OPLS-DA) predictability of the model was low based on the Q2 at approximately 0.25. Quinic acid (QA) isolated from H. mimetes showed promising anti-RT activity (IC50 = 53.82 μg/mL) comparable to the positive drug control, doxorubicin (IC50 = 40.31 μg/mL). A molecular docking study revealed the probable binding site and conformation of QA within cavity 4, with a docking score of −8.03. The docking score of doxorubicin within cavity 4 was −7.87. These are the first reports of QA's HIV-1-RT activity, as well as doxorubicin's docking characteristics on this enzyme.

Identification of potential Aurora kinase-C protein inhibitors: an amalgamation of energy minimization, virtual screening, prime MMGBSA and AutoDock

Genomic instability is a trademark of cancer evolution, there is still a wide necessity to discover safe and effective anti-cancer drugs. Aurora kinase-C protein is a member of the Aurora kinase family involved in regulating mitosis phases of the cell cycle. Aurora kinase-C protein has an aberrant expression at spindle assembly checkpoint leading to human cervical cancer and colorectal cancer. Knowledge of 3D structure of the protein is vital for identify possible inhibitors. In the present study, 3D structure prediction of Aurora kinase-C protein is taken up by using comparative homology modeling techniques and minimized by NAMD-VMD Software. The quality of modeled protein was validated using ProSA and Ramachandran plot. Probable binding site in protein was identified from SiteMap and molecular docking based virtual screening was carried out using Asinex database to identify lead molecules. The obtained lead molecules were further optimized using PrimeMM-GBSA and AutoDock. ADME properties of the leads were calculated to ascertain the drug-like properties at lead molecules.Communicated by Ramaswamy H. Sarma

Evaluation of DNA, BSA binding, DNA cleavage and antimicrobial activity of ytterbium(III) complex containing 2,2'-bipyridine ligand

In order to estimate the biological potential of a synthesized complex [Yb(bpy)2Cl3.OH2] where bpy is 2,2'-bipyridine, its binding behavior with fish salmon-DNA (FS-DNA) and bovine serum albumin (BSA) were studied by different kinds of spectroscopy and molecular modeling methods. This complex was selected for its antibacterial and antifungal activities as well as the DNA cleavage activities were examined by agarose gel electrophoresis. The analyses of fluorescence data at four temperatures were done in order to evaluate the binding and thermodynamic parameters of the interaction of Yb(III) complex with DNA and BSA. The experimental results indicated that the major binding modes were based on groove binding with DNA and BSA. In addition, iodide quenching studies, ethidium bromide (EtBr) exclusion assay, ionic strength effect, circular dichroism, and viscosity studies reflected the binding of Yb(III) complex explicitly with the FS-DNA mainly in a groove binding mode. Moreover, molecular docking studies indicated that this complex was bound to the minor groove of DNA and to polar and apolar residues located in the subdomain IB of BSA (site 3). Also, the results of competitive experiments assessed site 3 of BSA as the most probable binding site for this complex. The molecular docking results were in good agreement with our experimental results. From both experimental and docking results, the binding constant values displayed the remarkably high affinity of Yb(III) complex to DNA as well as BSA.Communicated by Ramaswamy H. Sarma

Rational in silico design of aptamers for organophosphates based on the example of paraoxon

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.

Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos

BackgroundThe CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined.ResultsFirst, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events.ConclusionsThe prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.

Streptozocin; a GLUT2 binding drug, interacts with human serum albumin at loci h6DOM3-h7DOM3.

Streptozocin (STZ) is a broad range antibiotic, highly genotoxic, antineoplastic and hyperglycemic. HSA is the most abundant protein in physiology and it binds to almost all exogenic and endogenic ligands, including drugs. STZ-induced fluorescence quenching of HSA has been done at pH 7.4, pH 3.5 and at pH 7.4 with 4.5 M urea at temperatures 286 K, 291 K, and 306 K. Ksv found to be 103 M-1, binding constant 1.5X103M-1 and binding sites ~1. But, Ksv for HSA and glucopyranose interaction was found lesser than that of HSA-STZ binding. Binding of STZ/glucopyranose on HSA seems to result in complex formation as calculated Kq > 1010 M-1 s-1. The number of binding sites, binding constants, and binding energies were increased with temperature. The ΔG0, ΔH0, and ΔS0 for HSA-STZ interaction were found to be -17.7 × 103 J·mol-1; 2.34 × 105 J·mol-1 and 841 JK-1 mol-1 respectively at pH 7.4 and 291 K. The comparative bindings of N, F and I states of HSA with STZ and their molecular docking analyses indicate that IIIA-B junction (i.e., inter-helix h6DOM3-h7DOM3) is the probable binding site, a locus close to fatty acid binding site-5. These results could be useful for therapeutic and analytical exploitation of STZ, as albumin used as the vehicle for drug delivery.

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface.

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

Coumarinyl Aryl/Alkyl Sulfonates with Dual Potential: Alkaline Phosphatase and ROS Inhibitory Activities: In-Silico Molecular Modeling and ADME Evaluation

Background: Alkaline Phosphatase (AP) is a physiologically important metalloenzyme that belongs to a large family of ectonucleotidase enzymes. Over-expression of tissue non-specific alkaline phosphatase has been linked with ectopic calcification including vascular and aortic calcification. In Vascular Smooth Muscles Cells (VSMCs), the high level of Reactive Oxygen Species (ROS) resulted in the up-regulation of TNAP. Accordingly, there is a need to identify highly potent and selective inhibitors of APs for treatment of disorders related to hyper activity of APs. </P><P> Methods: Herein, a series of coumarinyl alkyl/aryl sulfonates (1-40) with known Reactive Oxygen Species (ROS) inhibition activity, was evaluated for alkaline phosphatase inhibition against human Tissue Non-specific Alkaline Phosphatase (hTNAP) and Intestinal Alkaline Phosphatase (hIAP). </P><P> Results: With the exception of only two compounds, all other compounds in the series exhibited excellent AP inhibition. For hIAP and hTNAP inhibition, IC50 values were observed in the range 0.62-23.5 µM, and 0.51-21.5 µM, respectively. Levamisole (IC50 = 20.21 ± 1.9 µM) and Lphenylalanine (IC50 = 100.1 ± 3.15 µM) were used as standards for hIAP and hTNAP inhibitory activities, respectively. 4-Substituted coumarinyl sulfonate derivative 23 (IC50 = 0.62 ± 0.02 µM) was found to be the most potent hIAP inhibitor. Another 4-substituted coumarinyl sulfonate derivative 16 (IC50 = 0.51 ± 0.03 µM) was found to be the most active hTNAP inhibitor. Some of the compounds were also found to be highly selective inhibitors of APs. Detailed Structure-Activity Relationship (SAR) and Structure-Selectivity Relationship (SSR) analysis were carried out to identify structural elements necessary for efficient and selective AP inhibition. Molecular modeling and docking studies were carried out to rationalize the most probable binding site interactions of the inhibitors with the AP enzymes. In order to evaluate drug-likeness of compounds, in silico ADMETox evaluation was carried out, most of the compounds were found to have favorable ADME profiles with good predicted oral bioavailability. X-ray crystal structures of compounds 38 and 39 were also determined. </P><P> Conclusion: Compounds from this series may serve as lead candidates for future research in order to design even more potent, and selective inhibitors of APs.

Probing interaction of bovine serum albumin (BSA) with the biodegradable version of cationic gemini surfactants

A multi-technique approach has been used to probe the interaction of model protein bovine serum albumin (BSA) with a series of substantially biodegradable and less cytotoxic ethylene-oxide linked ester-functionalized gemini surfactants (designated as Cm-E2O-Cm, m = 12, 14 and 16). Binding of BSA with the gemini surfactants has been deduced on the basis of steady state fluorescence measurements. Synchronous fluorescence spectra indicate an exposure of the tyrosine residues and entrapment of the tryptophan residues into the hydrophobic cloud of the gemini surfactants. Post surfactant addition, pyrene fluorescence studies depict mild fluctuations in the micropolarity experienced by the probe molecules encapsulated in the protein. Temperature variation fluorescence and UV absorption studies confirm ground state complexation between BSA and the concerned gemini surfactants. Far-UV CD spectra are indicative of slight uncoiling of the alpha helices. These experimental findings are corroborated by the molecular docking results which predict that the probable binding sites for Cm-E2O-Cm gemini surfactants are in the vicinity of aromatic residues. Thus, the investigations presented herein elucidate the binding of serum albumin with surfactants of optimized architecture. Therefore, with further exhaustive screening tests, the Cm-E2O-Cm gemini surfactants may be potentially utilized in pharmaceuticals, cosmetics and other broad application fields.

Molecular modelling and simulation studies of the Mycobacterium tuberculosis multidrug efflux pump protein Rv1258c.

Mycobacterial efflux pumps play a major role in the emergence of antimycobacterial drug resistance. Of particular interest is the proteinaceous multi-drug efflux pump protein Rv1258c that encodes a tetracycline/aminoglycoside resistance (TAP-2)-like efflux pump which is active in susceptible and drug resistant Mycobacterium tuberculosis. Rv1258c is implicated in drug resistance to numerous antimycobacterials including first line drugs rifampicin and isoniazid as well as fluoroquinolone and aminoglycoside antibiotic classes. To date, compounds like verapamil and piperine have been shown to inhibit Rv1258c but no direct evidence for binding or mode of action exist. Therefore in the present study we generated an accurate 3D model of Rv1258c using MODELLER and validated its structure using molecular dynamic simulation studies with GROMACS software. The 3D-structures of Rv1258c and the homologous template 1pw4 were simulated within a POPE/POPG lipid bilayer and found to behave similar. Another important finding was the identification of one local energy minima state of the apo protein, which speaks to the flexibility of the protein and will be investigated further. Extraction of one of the open channel conformations of Rv1258c and blind docking of various structurally diverse putative inhibitors and substrates, allowed for the identification of a probable binding site. Spectinamide was found to bind to a different location on the outside surface of the protein suggesting its ability to avoid the efflux channel. We further identified 246 putative compounds that showed higher binding affinity values to Rv1258c compared to piperine and verapamil. Interaction analysis of the top 20 purchasable compounds identified crucial hydrogen bond interactions with Ser26, Ser45 and Glu243 as well as a pi-pi stacking interaction with Trp32 that accounted for the strong affinity of these compounds for Rv1258c. Future studies will entail purchasing a number of compounds for in vitro activity testing against Mycobacterium tuberculosis.