Deciphering the AChE-binding mechanism with multifunctional tricyclic coumarin anti-Alzheimer's agents using biophysical and bioinformatics approaches and evaluation of their modulating effect on Amyloidogenic peptide assembly.

Investigating the drug-AChE binding mechanism is vital in understanding its cogent use in medical practice against Alzheimer's disease (AD). The production and accumulation of oligomers of ß-amyloid is a central event in the neuropathology of AD. Beside the inhibition of assembly process, modulation of the aggregation process of these proteins towards minimally toxic pathways may be a possible therapeutic strategy for AD. Hence, the present study aims to examine the effect of multifunctional fused tricyclic 7-hydroxy 4-methyl coumarin analogs (HMC1-5) on the self-induced aggregation of ß-amyloid using Thioflavin T (ThT) assay, scanning electron microscopic study, AlamarBlue and immune blotting assays and also the binding mechanism with AChE by fluorescence emission, conformational, molecular docking and molecular dynamic simulation studies under physiological pH 7.4. The ThT assay, FE-SEM study, cell line and western blots establish that the HMC1-5 molecules could irreversibly disrupt preformed Aß42 fibrils, accelerate the aggregates into micro size co-assembled structures, and effectively eliminate the cytotoxicity of Aß1-42. Fluorescence emission studies indicating a strong binding affinity between HMC1-5 and AChE with the binding constants of 1.04 × 105, 3.57 × 104, 1.97 × 104, 3.07 × 104 and 2.95 × 104 M-1, respectively and binding sites number found to be 1. CD studies disclosed a partial unfolding in the secondary structure of AChE upon binding with HMC1-5. Docking analysis inferred that the HMC1-5 were bound through hydrophobic and hydrophilic interactions to the AChE active site. Molecular dynamics simulations emphasized the stability of AChE-HMC1-5 complexes throughout the 100 ns simulations, and the local conformational changes of the residues of AChE validate the stability of complexes. These results provide new and unique complementary approach for modulating the biological effects of the Aß aggregates by coumarin analogs and new insights for further in vivo investigations as novel anti AD agents.

Improving the inhibition of ß-amyloid aggregation by withanolide and withanoside derivatives.

Here, we have studied the ameliorative effects of Withania somnifera derivatives (Withanolide A, Withanolide B, Withanoside IV, and Withanoside V) on the fibril formation of amyloid-ß 42 for Alzheimer's disease. We analyzed reduction in the aggregation of ß amyloid protein with these Ashwagandha derivatives by Thioflavin T assay in the oligomeric and fibrillar state. We have tested the cytotoxic activity of these compounds against human SK-N-SH cell line for 48 h, and the IC 50 value found to be 28.61 ± 2.91, 14.84 ± 1.45, 18.76 ± 0.76 and 30.14 ± 2.59 µM, respectively. After the treatment of the cells with half the concentration of IC 50 value, there was a remarkable decrease in the number of apoptotic cells stained by TUNEL assay indicating the DNA damage and also observed significant decrease of reactive oxygen species. Also, the binding and molecular stability of these derivatives with amyloid ß was also studied using bioinformatics tools where these molecules were interacted at LVFFA region which is inhibition site of amyloid-ß1 42. These studies revealed that the Withanolides and Withanosides interact with the hydrophobic core of amyloid-ß 1-42 in the oligomeric stage, preventing further interaction with the monomers and diminishing aggregation.

Unraveling the stability of plasma proteins upon interaction of synthesized uridine products: biophysical and molecular dynamics approach.

Most of the drugs binding to human serum albumin (HSA) are transported to various parts of the body. Here, we have studied the molecular interaction between HSA and synthesized uridine derivatives, 1-[(3R, 4S, 5 R)-2-methyl-3, 4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dion.)(C-MU); [(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyl-tetrahydrofuran-2-yl] methyl methyl phosphochloridate (CM-MU) and [(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-methyl-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate (P-MU). Cytotoxic studies of these synthesized compounds with mouse macrophages (RAW 246.7) and HeLa cells (human cervical cancer cells) and binding mechanism of these uridine derivatives with HSA were performed. Subsequently, fluorescence quenching was observed upon titration of uridine derivatives with HSA via static mode of quenching, and the binding constants (K2-C-MU = 4 ± 0.03 × 104M-1, K5-CM-MU = 1.95 ± 0.03 × 104 M-1 and K5-P-MU =1.56 ± 0.03 × 104 M-1) were found to be in sync with the computational results. Further, molecular displacement and molecular docking data revealed that all the derivatives are binding in the subdomain IIA and IIB regions of HSA. The protein secondary structure of complexes was determined by circular dichroism, indicating partial unfolding of the protein upon addition of the uridine derivatives. Furthermore, atomic force microscopy data reveal the change in topology upon binding of 2-C-MU, 5-CM-MU and 5-P-MU with HSA, indicating change in the microenvironment around tryptophan region. Additionally, cytotoxicity studies on HeLa and Raw Cell lines suggested that these molecules have significant anti-proliferative and anti-inflammatory properties. Hence, the study may be of help for development of new drugs based on uridine derivatives which may be helpful for combating various potential diseases.Communicated by Ramaswamy H. Sarma.

Comparative binding of Swertiamarin with human serum albumin and α-1 glycoprotein and its cytotoxicity against neuroblastoma cells.

Communicated by Ramaswamy H. Sarma.

Enzymatic Textile Dyes Decolorization by In vitro and In silico Studies.

BACKGROUND: Laccase, a multicopper oxidoreductase (EC: 1.10.3.2), is a widely used enzyme in bioremediation of textile dye effluents. Fungal Laccase is preferably used as a remediating agent in the treatment and transformation of toxic organic pollutants. In this study, crude laccase from a basidiomycetes fungus, Phanerochaete sordida, was able to decolorize azo, antroquinone and indigoid dyes. In addition, interactions between dyes and enzyme were analysed using molecular docking studies. METHODS: In this work, a white rot basidiomycete's fungus, Phanerochaete sordida, was selected from forest soil isolates of Eastern Ghats, and Tirumala and lignolytic enzymes production was assayed after 7 days of incubation. The crude enzyme was checked for decolourisation of various synthetic textile dyes (Vat Brown, Acid Blue, Indigo, Reactive Blue and Reactive Black). Molecular docking studies were done using Autodock-4.2 to understand the interactions between dyes and enzymes. RESULTS: Highest decolourisation efficiency was achieved with the crude enzyme in case of vat brown whereas the lowest decolourisation efficiency was achieved in Reactive blue decolourisation. Similar results were observed in their binding affinity with lignin peroxidase of Phanerochaete chrysosporium through molecular docking approach. CONCLUSION: Thus, experimental results and subsequent in silico validation involving an advanced remediation approach would be useful to reduce time and cost in other similar experiments.

Spectroscopic evaluation of synthesized 5ß-dihydrocortisol and 5ß-dihydrocortisol acetate binding mechanism with human serum albumin and their role in anticancer activity.

Our study focus on the biological importance of synthesized 5ß-dihydrocortisol (Dhc) and 5ß-dihydrocortisol acetate (DhcA) molecules, the cytotoxic study was performed on breast cancer cell line (MCF-7) normal human embryonic kidney cell line (HEK293), the IC50 values for MCF-7 cells were 28 and 25 µM, respectively, whereas no toxicity in terms of cell viability was observed with HEK293 cell line. Further experiment proved that Dhc and DhcA induced 35.6 and 37.7% early apoptotic cells and 2.5, 2.9% late apoptotic cells, respectively, morphological observation of cell death through TUNEL assay revealed that Dhc and DhcA induced apoptosis in MCF-7 cells. The complexes of HSA-Dhc and HSA-DhcA were observed as static quenching, and the binding constants (K) was 4.7 ± .03 × 104 M-1 and 3.9 ± .05 × 104 M-1, and their binding free energies were found to be -6.4 and -6.16 kcal/mol, respectively. The displacement studies confirmed that lidocaine 1.4 ± .05 × 104 M-1 replaced Dhc, and phenylbutazone 1.5 ± .05 × 104 M-1 replaced by DhcA, which explains domain I and domain II are the binding sites for Dhc and DhcA. Further, FT-IR, synchronous spectroscopy, and CD results revealed that the secondary structure of HSA was altered in the presence of Dhc and DhcA. Furthermore, the atomic force microscopy and transmission electron microscopy showed that the dimensions like height and molecular size of the HSA-Dhc and HSA-DhcA complex were larger compared to HSA alone. Detailed analysis through molecular dynamics simulations also supported greater stability of HSA-Dhc and HSA-DhcA complexes, and root-mean-square-fluctuation interpreted the binding site of Dhc as domain IB and domain IIA for DhcA. This information is valuable for further development of steroid derivative with improved pharmacological significance as novel anti-cancer drugs.

Exploration of binding studies of ß-oxalyldiamino propionic acid (ß-ODAP), a non-protein amino acid with human serum albumin-biophysical and computational approach.

Communicated by Ramaswamy H. Sarma.

Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin.

Withania somnifera (Ashwagandha) is an efficient medicinal plant known in Ayurveda and Chinese medicine since ancient times, whose extracts are consumed orally as food supplement or as a health tonic owing to its several restorative properties for various CNS disorders, inflammation, tumour, stress, rheumatism etc. In this study, we have analyzed the binding interaction of four derivatives of Withania somnifera (Withanolide A, Withanolide B, Withanoside IV and Withanoside V) with HSA because of their important pharmacological properties. To unravel the binding between derivatives of Withania somnifera and HSA, fluorescence spectroscopy was used. Binding studies were further studied by molecular docking and dynamics and results confirmed greater stability upon binding of derivatives with HSA. Circular dichroism data illustrated change in the secondary structure of protein upon interaction with these derivatives, particularly the helical structure was increased and ß-sheets and random coils were decreased. Furthermore, morphological and topological changes were observed using AFM and TEM upon binding of ligands with HSA indicating that HSA-withnoside/withanolide complexes were formed. All the results cumulatively demonstrate strong binding of withanosides and withanolides derivatives with serum albumin, which should further be explored to study the pharmacokinetics and pharmacodynamics of these derivatives.

Characterization and molecular modeling of polygalacturonase isoforms from Saccharomyces cerevisiae.

Earlier, low-temperature-active polygalacturonase isoforms from Saccharomyces cerevisiae PVK4 were isolated and purified. Substrate specificity of polygalacturonase isoforms indicated high affinity for pectins and very low enzyme activity towards non-pectic polysaccharides. To characterize the polygalacturonase isoforms, biochemical, spectral, and in silico approaches were used. The apparent Km and Vmax values for hydrolysis of pectin and galacturonic acid were 0.31 mg/ml and 3.15 mmol min/mg, respectively. Interestingly, the polygalacturonase isoforms were found to be more stable at optimal pH and temperature of 4.5 and 40 °C, respectively. These isoforms were reacted with different metal ions; Cd2+ and Ni2+ severely inhibited the enzyme activity, while Mg2+, Zn2+, Cd2+, Fe2+ Cu2+, and Ni2+ inhibited to a lesser extent, which clearly demonstrated that variations in enzyme activity were due to their differential binding affinity of metal ions. Furthermore, decrease in the viscosity of polygalacturonic acid and citrus pectin by these isoforms was approximately four and six times higher than the rate of release of reducing sugars. This indicates that polygalacturonase isoforms have an endo-mode of action. In addition to the above, thermostability of purified polygalacturonase isoforms was studied by circular dichroism and fluorescence spectroscopy. Circular dichroism showed 18% alpha helix and 57% beta sheets at pH 5, while at pH 7, 8, and 9 there was an increase of random coil. Fluorescence studies revealed small conformational changes, which were observed at 30-50 °C, while unfolding transition region was noticed between 60 and 70 °C. The purified enzyme fractions were analyzed by MALDI-TOF MS. Finally, 3D model structures for isoenzymes of polygalacturonase of S. cerevisiae were generated and validated as good quality models, which are also suitable for molecular interaction studies.

In Silico Approach to Support that p-Nitrophenol Monooxygenase from Arthrobacter sp. Strain JS443 Catalyzes the Initial Two Sequential Monooxygenations.

p-Nitrophenol (PNP), used primarily for manufacturing pesticides and dyes, has been recognized as a priority environmental pollutant. It is therefore important to reduce the input of this toxicant into the environment and to establish approaches for its removal from the contaminated sites. PNP monooxygenase, a novel enzyme from Gram-positive bacteria like Arthrobacter sp. and Bacillus sp., that comprises two components, a flavoprotein reductase and an oxygenase, catalyzes the initial two sequential monooxygenations to convert PNP to trihydroxybenzene. Accurate and reliable prediction of this enzyme-substrate interactions and binding affinity are of vital importance in understanding these catalytic mechanisms of the two sequential reactions. As crystal structure of the enzyme has not yet been published, we built a homology model for PNP monooxygenase using crystallized chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100 (3HWC) as the template. The model was assessed for its reliability using PROCHECK, ERRAT and ProSA. Molecular docking of the physiological substrates, PNP and 4-nitrocatechol (4-NC), was carried out using Glide v5.7 implemented in Maestro v9.2, and the binding energies were calculated to substantiate the prediction. Docking complexes formed by molecular level interactions of PNP monooxygenase-PNP/4-NC without or with the cofactors, FAD and NADH, showed good correlation with the established experimental evidence that the two-component PNP monooxygenase catalyzes both the hydroxylation of PNP and the oxidative release of nitrite from 4-NC in B. sphaericus JS905. Furthermore, molecular dynamics simulations performed for docking complexes using Desmond v3.0 showed stable nature of the interactions as well.

Comparative binding mechanism of lupeol compounds with plasma proteins and its pharmacological importance.

Lupeol, a triterpene, possesses beneficial effects like anti-inflammatory and anti-cancer properties. Binding of lupeol and its derivative (phytochemicals) to plasma proteins such as human serum albumin (HSA) and α-1-acid glycoprotein (AGP) is a major determinant in the disposition of drugs. Cytotoxic studies with mouse macrophages (RAW 246.7) and HeLa cell lines revealed anti-inflammatory and anti-cancer properties for both lupeol and lupeol derivative. Both molecules reduced the expression of pro-inflammatory cytokines in LPS induced macrophages. Further, apoptosis was observed in HeLa cell lines when they were incubated with these molecules for 24 h. The fluorescence quenching of HSA was observed upon titration with different concentrations of lupeol and lupeol derivative; their binding constants were found to be 3 ± 0.01 × 10(4) M(-1) and 6.2 ± 0.02 × 10(4) M(-1), with binding free energies of -6.59 kcal M(-1) and -7.2 kcal M(-1). With AGP, however, the lupeol and lupeol derivative showed binding constants of 0.9 ± 0.02 × 10(3) M(-1) and 2.7 ± 0.01 × 10(3) M(-1), with free energies of -4.6 kcal M(-1) and -5.1 kcal M(-1) respectively. Molecular displacement studies based on competition with site I-binding phenylbutazone (which binds site I of HSA) and ibuprofen (which binds site II) suggest that lupeol binds site II and the lupeol derivative site I. Molecular docking studies also confirmed that lupeol binds to the IIIA and the lupeol derivative to the IIA domain of HSA. Secondary structure changes were observed upon formation of HSA-lupeol/lupeol derivative complexes by circular dichroism spectroscopy. Molecular dynamics simulations support greater stability of HSA-lupeol and HSA-lupeol derivative complexes compared to that of HSA alone.

In silico approach to support that p-nitrophenol monooxygenase from Arthrobacter sp. strain JS443 catalyzes the initial two sequential monooxygenations.

p-Nitrophenol (PNP), used primarily for manufacturing pesticides and dyes, has been recognized as a priority environmental pollutant. It is therefore important to reduce the input of this toxicant into the environment and to establish approaches for its removal from the contaminated sites. PNP monooxygenase, a novel enzyme from Gram-positive bacteria like Arthrobacter sp. and Bacillus sp., that comprises two components, a flavoprotein reductase and an oxygenase, catalyzes the initial two sequential monooxygenations to convert PNP to trihydroxybenzene. Accurate and reliable prediction of this enzyme-substrate interactions and binding affinity are of vital importance in understanding these catalytic mechanisms of the two sequential reactions. As crystal structure of the enzyme has not yet been published, we built a homology model for PNP monooxygenase using crystallized chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100 (3HWC) as the template. The model was assessed for its reliability using PROCHECK, ERRAT, WHATCHECK and ProSA. Molecular docking of the physiological substrates, PNP and 4-nitrocatechol (4-NC), was carried out using Glide v5.7 implemented in Maestro v9.2, and the binding energies were calculated to substantiate the prediction. Docking complexes formed by molecular level interactions of PNP monooxygenase-PNP/4-NC without or with the cofactors, FAD and NADH, showed good correlation with the established experimental evidence that the two-component PNP monooxygenase catalyzes both the hydroxylation of PNP and the oxidative release of nitrite from 4-NC in B. sphaericus JS905. Furthermore, molecular dynamics simulations performed for docking complexes using Desmond v3.0 showed stable nature of the interactions as well.

Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein.

Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be K(piperine) = 5.7 ± .2 × 10(5) M(-1) and K(piperine) = 9.3± .25 × 10(4) M(-1) which correspond to the free energy of -7.8 and -6.71 kcal M(-1)at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA-piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA-piperine complex reaches equilibration state at around 3 ns, which prove that the HSA-piperine complex is stable in nature.