Identification of Novel Potential Anti-Diabetic Candidates targeting Human Pancreatic ɑ-Amylase and Human ɑ-Glycosidase: An Exhaustive Structure-based Screening

Diabetes is a major health issue that half a billion people affected worldwide. It is a serious, long-term medical condition majorly impacting the lives and well-being of individuals, families, and societies at large. It is amongst the top 10 diseases responsible for the death amongst adults with an expected rise to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045. The carbohydrates absorbed into the body are hydrolyzed by pancreatic α-amylase and other enzymes, human α-glucosidase. The α-amylase and α-glucosidase are validated therapeutic targets in the treatment of Type II diabetes (T2DM) as they play a vital role in modulating the blood glucose post meal. Herein, we report novel and diverse molecules as potential candidates, with predicted affinity for α-amylase and α-glucosidase. These molecules have been identified via hierarchical multistep docking of small molecules database with the estimated binding free energies. A Glide XP Score cutoff −8.00 kcal/mol was implemented to filter out non potential molecules. Four molecules viz. amb22034702, amb18105639, amb17153304, and amb9760832 have been identified after an exhaustive computational study involving, evaluation of binding interactions and assessment of the pharmacokinetics and toxicity profiles. The in-depth analysis of protein– ligand interactions was performed using a 100ns molecular dynamics (MD) simulation to establish the dynamic stability. Furthermore MM-GBSA based binding free energies were computed for 1000 trajectory snapshots to ascertain the strong binding affinity of these molecules for α-amylase and αglucosidase. The identified molecules can be considered as promising candidates for further drug development through necessary experimental assessments.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Interaction of multimicrobial synthetic inhibitor 1,2-bis(2-benzimidazolyl)-1,2-ethanediol with serum albumin: spectroscopic and computational studies.

The molecule, 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol (BBE) is known to act as a selective inhibitor of poliovirus, rhinovirus, Candida albicans, several bacterial species, and is easily synthesized by Phillips reaction. The interaction of BBE with BSA and the effects of its binding on the conformation and unfolding/refolding pathways of the protein were investigated using multispectroscopic techniques and molecular modeling. The binding studies indicate that BSA has one high affinity BBE binding site with association constant 6.02±0.05×10(4) M(-1) at 298 K. By measuring binding at different temperatures, we determined the changes in enthalpy (ΔH = -15.13±2.15 kJ mol(-1)), entropy (ΔS = 40.87±7.25 J mol(-1) K(-1)) and free energy (ΔG( = )26.78±1.02) of interaction, which indicate that the binding was spontaneous and both enthalpically and entropically driven. Based on molecular modeling and thermodynamic parameters, we proposed that the complex formation involved mainly hydrophilic interaction such as hydrogen bonding between hydroxyl groups of ethane-1,2-diol fragment with Tyr410 and benzimidazole sp(2) nitrogen atom with Ser488 and hydrophobic interaction between phenyl ring of one benzimidazole of the ligand and hydrophobic residues namely, Ile387, Cys391, Phe402, Val432 and Cys437. The sequential unfolding mechanism of BSA, site-specific marker displacement experiments and molecular modeling showed that the molecule preferably binds in subdomain IIIA. The BBE binding to BSA was found to cause both secondary and tertiary structural alterations in the protein as studied by intrinsic fluorescence, near-UV and far-UV circular dichroism results. The unfolding/refolding study showed that BBE stabilized native to intermediate states (N⇌I) transition of the protein by ∼2 kJ mol(-1) without affecting the intermediate to unfolded states (I⇌U) transition and general mechanism of unfolding of BSA

Characterization of pioglitazone cyclodextrin complexes: Molecular modeling to in vivo evaluation

Aims: The objective of present study was to study the influence of different β-cyclodextrin derivatives and different methods of complexation on aqueous solubility and consequent translation in in vivo performance of Pioglitazone (PE). Material and Methods: Three cyclodextrins: β-cyclodextrin (BCD), hydroxypropyl-β-cyclodextrin (HPBCD) and Sulfobutylether-7-β-cyclodextrin (SBEBCD) were employed in preparation of 1:1 Pioglitazone complexes by three methods viz. co-grinding, kneading and co-evaporation. Complexation was confirmed by phase solubility, proton NMR, Fourier Transform Infrared spectroscopy, Differential Scanning Calorimetry (DSC) and X-Ray diffraction (XRD). Mode of complexation was investigated by molecular dynamic studies. Pharmacodynamic study of blood glucose lowering activity of PE complexes was performed in Alloxan induced diabetic rat model. Results: Aqueous solubility of PE was significantly improved in presence of cyclodextrin. Apparent solubility constants were observed to be 254.33 M–1 for BCD-PE, 737.48 M–1 for HPBCD-PE and 5959.06 M–1 for SBEBCD-PE. The in silico predictions of mode of inclusion were in close agreement with the experimental proton NMR observation. DSC and XRD demonstrated complete amorphization of crystalline PE upon inclusion. All complexes exhibited >95% dissolution within 10 min compared to drug powder that showed <40% at the same time. Marked lowering of blood glucose was recorded for all complexes. Conclusion: Complexation of PE with different BCD significantly influenced its aqueous solubility, improved in vitro dissolution and consequently translated into enhanced pharmacodynamic activity in rat

The BBE binding to BSA is specific.

<p>Particle formation by BBE in the absence and presence of 0.01% Triton X-100 as monitored by Rayleigh light scattering at 350 nm (A). Stern-Volmer plots of BBE-induced fluorescence quenching of BSA with increasing concentrations of the protein (B).</p

The effect of 1,2-<i>Bis</i>(2-Benzimidazolyl) 1,2-Ethanediol binding on unfolding transition of BSA.

<p>The effect of 1,2-<i>Bis</i>(2-Benzimidazolyl) 1,2-Ethanediol binding on unfolding transition of BSA.</p

1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol interaction and thermodynamic parameters.

<p>1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol interaction and thermodynamic parameters.</p

1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol induced conformational alteration in BSA.

<p>1,2<i>-</i>Bis(2-Benzimidazolyl) 1,2-Ethanediol induced conformational alteration in BSA.</p

Preparation and structure of 1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE).

<p>Preparation and structure of 1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE).</p

The equilibrium unfolding process and stability of BBE-BSA complex.

<p>The urea-induced unfolding profile of BSA and BBE-BSA complex at BBE/BSA molar ratios of 0∶1 and 2∶1 monitored by intrinsic fluorescence of the protein (A). The fraction denatured (f_d) versus [urea] plots for NI and IU transitions of the protein and complex. Lines represent the nonlinear regression fitting of the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053499#pone.0053499.e008" target="_blank">equation 7</a> (B). The dependence of free energy change as a function of urea concentrations for the transitions shown in Figure B (C).</p

1,2-Bis(2-Benzimidazolyl)-1,2-Ethanediol (BBE) induced fluorescence quenching mechanism of bovine serum albumin.

<p>The BBE induced intrinsic fluorescence quenching (A) and Stern-Volmer plots for fluorescence quenching data (B) of BSA at different temperatures. The inset of Figure A shows fluorescence spectra of BSA in the absence and presence of increasing BBE concentrations at 298 K. The concentration of BSA was 5 µM and the intrinsic fluorescence of the protein was measured in 60 mM sodium phosphate buffer of pH 7.4 upon excitation at 280 nm.</p