Arun Bahadur Gurung

Identification of molecular descriptors for design of novel Isoalloxazine derivatives as potential Acetylcholinesterase inhibitors against Alzheimer's disease.

In Alzheimer’s disease (AD), the level of Acetylcholine (ACh) neurotransmitter is reduced. Since Acetylcholinesterase (AChE) cleaves ACh, inhibitors of AChE are very much sought after for AD treatment. The side effects of current inhibitors necessitate development of newer AChE inhibitors. Isoalloxazine derivatives have proved to be promising (AChE) inhibitors. However, their structure–activity relationship studies have not been reported till date. In the present work, various quantitative structure–activity relationship (QSAR) building methods such as multiple linear regression (MLR), partial least squares ,and principal component regression were employed to derive 3D-QSAR models using steric and electrostatic field descriptors. Statistically significant model was obtained using MLR coupled with stepwise selection method having r2 = .9405, cross validated r2 (q2) = .6683, and a high predictability (pred_r2 = .6206 and standard error, pred_r2se = .2491). Steric and electrostatic contribution plot revealed three electrostatic fields E_496, E_386 and E_577 and one steric field S_60 contributing towards biological activity. A ligand-based 3D-pharmacophore model was generated consisting of eight pharmacophore features. Isoalloxazine derivatives were docked against human AChE, which revealed critical residues implicated in hydrogen bonds as well as hydrophobic interactions. The binding modes of docked complexes (AChE_IA1 and AChE_IA14) were validated by molecular dynamics simulation which showed their stable trajectories in terms of root mean square deviation and molecular mechanics/Poisson–Boltzmann surface area binding free energy analysis revealed key residues contributing significantly to overall binding energy. The present study may be useful in the design of more potent Isoalloxazine derivatives as AChE inhibitors.

Binding of small molecules at interface of protein–protein complex – A newer approach to rational drug design

Protein–protein interaction is a vital process which drives many important physiological processes in the cell and has also been implicated in several diseases. Though the protein–protein interaction network is quite complex but understanding its interacting partners using both in silico as well as molecular biology techniques can provide better insights for targeting such interactions. Targeting protein–protein interaction with small molecules is a challenging task because of druggability issues. Nevertheless, several studies on the kinetics as well as thermodynamic properties of protein–protein interactions have immensely contributed toward better understanding of the affinity of these complexes. But, more recent studies on hot spots and interface residues have opened up new avenues in the drug discovery process. This approach has been used in the design of hot spot based modulators targeting protein–protein interaction with the objective of normalizing such interactions.

Human serum albumin reduces the potency of acetylcholinesterase inhibitor based drugs for Alzheimer's disease

Human serum albumin (HSA) induced modulation of acetylcholinesterase (AChE) inhibition activity of four well-known cholinergic inhibitors like tacrine hydrochloride (TAC), donepezil hydrochloride monohydrate (DON), (-) Huperzine A (HuPA), eserine (ESE) was monitored quantitatively by Ellmans method. Kinetic analysis of enzyme hydrolysis reaction revealed that while the mechanism of inhibition does not change significantly, the inhibition efficiency changes drastically in presence of HSA, particularly for DON and TAC. However, interestingly, no notable difference was observed in the cases of HuPA and/or ESE. For example, the IC50 value of AChE inhibition increases by almost 135% in presence of ∼250 μM HSA (IC50 = 159 ± 8 nM) while comparing with aqueous buffer solution of pH 8.0 (IC50 = 68 ± 4 nM) in DON. On the other hand, the change is almost insignificant (<10%) in case of HuPA under the similar condition. The experimentally observed difference in the extent of modulatory effect was correlated with the sequestration ability of HSA towards different drugs predicted from molecular docking calculations. The result in this study demonstrates the importance to consider the plasma protein binding tendency of a newly synthesized AD drug before claiming its potency over the existing one. Further, development of new and intelligent delivery medium that shields the administered drugs from serum adsorption may reduce the optimal drug dose requirement.

Interactome analysis and design of inhibitors against selected protein targets of Ser/Thr protein kinase (STPK) signaling pathways in Mycobacterium tuberculosis H37Rv.

Tuberculosis continues to be a major cause of mortality worldwide despite significant advances in chemotherapy and development of the BCG vaccine. Although curable, the tuberculosis treatment period (6-9 months) presents many concerns, including patient noncompliance and the development of drug toxicity and drug resistance. This study aimed to understand the protein-protein interactions of key proteins involved in the Mycobacterium tuberculosis STPK signal transduction pathway (such as PknB, PknE, and PstP); in addition, we attempted to identify promising leads for the inhibition of protein-protein interactions. Interactome analyses revealed the interactions of these protein targets with several other proteins, including PknG and PbpA. Drug-like candidates were screened based on Lipinskis rule of five and the absorption digestion metabolism excretion toxicity. Molecular docking of the target proteins with the selected ligands identified cryptolepine HCl to be a common molecule interacting with all protein targets (with a good docking score). The generation of a pharmacophore model for cryptolepine HCl revealed three pharmacophoric regions: aromatic hydrocarbon, hydrogen bond acceptor, and hydrogen bond donor, which play important roles in its interaction with the protein targets. Therefore, cryptolepine HCl appears to be a promising drug candidate for further optimization and validation against M. tuberculosis.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Target fishing of glycopentalone using integrated inverse docking and reverse pharmacophore mapping approach

Glycopentalone isolated from Glycosmis pentaphylla (family Rutaceae) has cytotoxic and apoptosis inducing effects in various human cancer cell lines; however, its mode of action is not known. Therefore, target fishing of glycopentalone using a combined approach of inverse docking and reverse pharmacophore mapping approach was used to identify potential targets of glycopentalone, and gain insight into its binding modes against the selected molecular targets, viz., CDK-2, CDK-6, Topoisomerase I, Bcl-2, VEGFR-2, Telomere:G-quadruplex and Topoisomerase II. These targets were chosen based on their key roles in the progression of cancer via regulation of cell cycle and DNA replication. Molecular docking analysis revealed that glycopentalone displayed binding energies ranging from -6.38 to -8.35 kcal/mol and inhibition constants ranging from 0.758 to 20.90 μM. Further, the binding affinities of glycopentalone to the targets were in the order: Telomere:G-quadruplex > VEGFR-2 > CDK-6 > CDK-2 > Topoisomerase II > Topoisomerase I > Bcl-2. Binding mode analysis revealed critical hydrogen bonds as well as hydrophobic interactions with the targets. The targets were validated by reverse pharmacophore mapping of glycopentalone against a set of 2241 known human target proteins which revealed CDK-2 and VEGFR-2 as the most favorable targets. The glycopentalone was well mapped to CDK-2 and VEGFR-2 which involve six pharmacophore features (two hydrophobic centers and four hydrogen bond acceptors) and nine pharmacophore features (five hydrophobic, two hydrogen bond acceptors and two hydrogen bond donors), respectively. The present computational approach may aid in rational identification of targets for small molecules against large set of candidate macromolecules before bioassays validation.

Impact of tyrosine nitration at positions Tyr307 and Tyr335 on structural dynamics of Lipoprotein-associated phospholipase A2–A therapeutically important cardiovascular biomarker for atherosclerosis

Protein tyrosine nitration (PTN) is a post translational event which results in the generation of 3-Nitrotyrosine (3-NT). High levels of 3-NT were reported in several human diseases such as Parkinsons disease, Alzheimers disease, amylotrophic lateral sclerosis and coronary artery disease. It was reported that PTN at positions 307 and 335 of Lipoprotein-associated phospholipase A2 (Lp-PLA2) curtails its enzymatic activity but the mechanism of inhibition at the structure level is still incomprehensible. The present study is an in silico endeavor to understand nitrative stress induced structural changes in Lp-PLA2. Molecular docking studies revealed a decreased binding affinity of substrate, Platelet Activating Factor (PAF) with the nitrated forms of Lp-PLA2 (NT-Tyr307 and NT-Tyr335) compared to the wild type, due to differences in the hydrogen bond interaction patterns. Molecular dynamics (MD) simulation studies suggests higher flexibility of nitrated forms compared to wild type, disorientation of the catalytic triad and decreased molecular interactions of NT-Tyr307 and NT-Tyr335 with other residues of the protein. Essential dynamics (ED) further confirmed the enhanced structural flexibility of nitrated forms of Lp-PLA2. Our findings would help understand the molecular mechanism of nitrative stress induced inhibition of Lp-PLA2 which may further assist in designing of therapeutics having protective functions against PTN.

An in silico approach to understand the structure-function properties of a serine protease (Bacifrinase) from Bacillus cereus and experimental evidence to support the interaction of Bacifrinase with fibrinogen and thrombin

Microbial fibrinogenolytic serine proteases find therapeutic applications in the treatment of thrombosis- and hyperfibrinogenemia-associated disorders. However, analysis of structure–function properties of an enzyme is utmost important before its commercial application. In this study, an attempt has been made to understand the structure of a fibrinogenolytic protease enzyme, “Bacifrinase” from Bacillus cereus strain AB01. From the molecular dynamics trajectory analysis, the modelled three-dimensional structure of the protease was found to be stable and the presence of a catalytic triad made up of Asp102, His83 and Ser195 suggests that it is a serine protease. To understand the mechanism of enzyme–substrate and enzyme–inhibitor interactions, the equilibrated protein was docked with human fibrinogen (the physiological substrate of this enzyme), human thrombin and with ten selective protease inhibitors. The Bacifrinase–chymostatin interaction was the strongest among the selected protease inhibitors. The serine protease inhibitor phenyl methane sulphonyl fluoride was found to interact with the Ser134 residue of Bacifrinase. Furthermore, protein–protein docking study revealed the fibrinogenolytic property of Bacifrinase and its interaction with Aα-, Bβ- and Cγ-chains human fibrinogen to a different extent. However, biochemical analysis showed that Bacifrinase did not hydrolyse the γ-chain of fibrinogen. The in silico and spectrofluorometric studies also showed interaction of Bacifrinase with thrombin as well as fibrinogen with a Kd value of 16.5 and .81 nM, respectively. Our findings have shed light on the salient structural features of Bacifrinase and confirm that it is a fibrinogenolytic serine protease.

Disruption of redox catalytic functions of peroxiredoxin-thioredoxin complex in Mycobacterium tuberculosis H37Rv using small interface binding molecules

Mycobacterium tuberculosis has distinctive ability to detoxify various microbicidal superoxides and hydroperoxides via a redox catalytic cycle involving thiol reductants of peroxiredoxin (Prx) and thioredoxin (Trx) systems which has conferred on it resistance against oxidative killing and survivability within host. We have used computational approach to disrupt catalytic functions of Prx-Trx complex which can possibly render the pathogen vulnerable to oxidative killing in the host. Using protein-protein docking method, we have successfully constructed the Prx-Trx complex. Statistics of interface region revealed contact area of each monomer less than 1500Å2 and enriched in polar amino acids indicating transient interaction between Prx and Trx. We have identified ZINC40139449 as a potent interface binding molecule through virtual screening of drug-like compounds from ZINC database. Molecular dynamics (MD) simulation studies showed differences in structural properties of Prx-Trx complex both in apo and ligand bound states with regard to root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuations (RMSF), solvent accessible surface area (SASA) and number of hydrogen bonds (NHBs). Interestingly, we found stability of two conserved catalytic residues Cys61 and Cys174 of Prx and conserved catalytic motif, WCXXC of Trx upon binding of ZINC40139449. The time dependent displacement study reveals that the compound is quite stable in the interface binding region till 30ns of MD simulation. The structural properties were further validated by principal component analysis (PCA). We report ZINC40139449 as promising lead which can be further evaluated by in vitro or in vivo enzyme inhibition assays.

Impact of a non-synonymous Q281R polymorphism on structure of human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2)

Non‐synonymous single nucleotide polymorphisms (nsSNPs) are genetic variations at single base resulting in an amino acid change which have been associated with various complex human diseases. The human Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) gene harbours a rare Q281R polymorphism which was previously reported to cause loss of enzymatic function. Lp‐PLA2 is an important enzyme which catalyzes the hydrolysis of polar phospholipids releasing pro‐atherogenic and pro‐inflammatory mediators involved in the pathogenesis of atherosclerosis. Our current study is aimed at elucidating the structural and functional consequences of Q281R polymorphism on Lp‐PLA2. The Q281R mutation is classified as deleterious and causes protein instability as deduced from evolutionary, folding free energy changes and Support vector machine (SVM)‐based methods. A Q281R mutant structure was deciphered using homology modelling approach and was validated using phi and psi dihedral angles distribution, ERRAT, Verify_3D scores, Protein Structure Analysis (ProSA) energ,y and Z‐score. A decreased hydrophobic interactions and weaker substrate binding affinity was observed in the mutant compared to the wild‐ type (WT) using molecular docking. Further, the mutant displayed enhanced structural flexibility particularly in the low density lipoprotein (LDL) binding domain, decreased solvent accessibility of catalytic residues‐Phe274 and Ser273 and increased Cɑ distance between Phe274 and Leu153 and large conformational entropy change as inferred from all‐atom molecular dynamics (MD) simulation and essential dynamics (ED) studies. Our results corroborate well with previous experimental studies and thus these aberrations in the Q281R mutant structure may help explain the molecular basis of loss of enzyme activity.