Structure prediction and molecular docking studies of aromatic hydrocarbon sensing proteins TbuT, HbpR and PhnR to detect priority pollutants.

On-line detection of aromatic hydrocarbon pollutants in aqueous environments can be achieved by biosensing strains having fusion of gene responsible for pollutant sensing protein with a reporter gene. Regulatory proteins TbuT, HbpR and PhnR are such proteins for recognizing one-, two-and three-ring aromatic hydrocarbon pollutants respectively, for which the structure is not known till date. Aim of the present study was to predict the structure of proteins and to determine their in-silico interaction with array of pollutants. Structure prediction of proteins was performed using I-TASSER and Phyre2 and refined with ModRefiner and 3DRefine. Total 14 models were obtained for each protein and the best model had more than 95% coverage in Ramachandran plot region. After successful structure prediction, molecular interaction of proteins with respective aromatic hydrocarbon pollutants categorized by United States Environmental Protection Agency was studied using AutoDockVina where the binding energy was found to fall in range of -4.6 to -8.4 kcal/mol. The types of protein-pollutant interaction were analyzed by LigPlus and Discovery Studio 2017 R2 Client which were found to be similar for standard and pollutant compounds. This study enables us to predict the range of pollutants possible to be detected using these regulatory protein-based biosensors.

Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design.

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors and anticancer agents. We have designed 2-,4,-6-, and/or 7-substituted quinoline derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on 45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity. Designed compounds were docked into the binding site of hDHODH. Designed compounds which showed good predictive activity, no toxicity, and good docking score were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines (HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC50 value of 1.56 µM and 1.22 µM, against hDHODH, respectively, and these are our lead compounds for the development of new hDHODH inhibitors and anticancer agents.

L. donovani XPRT: Molecular characterization and evaluation of inhibitors.

Among all PRT enzymes of purine salvage pathway in Leishmania, XPRT (Xanthine phosphoribosyl transferase) is unique in its substrate specificity and their non-existence in human. It is an interesting protein not only for drug designing but also to understand the molecular determinants of its substrate specificity. Analysis of the 3D model of L. donovani XPRT (Ld-XPRT) revealed that Ile 209, Glu 215 and Tyr 208 may be responsible for the altered substrate specificity of Ld-XPRT. Comparisons with it's nearest homologue in humans, revealed significant differences between the two. A 28 residue long unique motif was identified in Ld-XPRT, which showed highest fluctuation upon substrate binding during MD simulations. In kinetic analysis, Ld-XPRT could phosphoribosylate xanthine, hypoxanthine and guanine with Km values of 7.27, 8.13, 8.48µM and kcat values of 2.24, 1.82, 1.19min-1 respectively. Out of 159 compounds from docking studies, six compounds were characterized further by fluorescence spectroscopy, CD spectroscopy and enzyme inhibition studies. Fluorescence quenching experiment was performed to study the binding of inhibitors with Ld-XPRT and dissociation constants were calculated. Four compounds are bi-substrate analogues and show competitive inhibition with both the substrates (Xanthine and PRPP) of Ld-XPRT. The CD spectral analysis revealed that the binding of inhibitors to Ld-XPRT induce change in its tertiary structure, where as its secondary structure pattern remains unchanged. Two Ld-XPRT inhibitors (dGDP and cGMP), which also have ability to inhibit Leishmanial HGPRT, are predicted as potential drug candidates as it can inhibit both the important enzymes of the purine salvage pathway.

Orange G is a potential inhibitor of human insulin amyloid fibrillation and can be used as a probe to study mechanism of amyloid fibrillation and its inhibition.

The extracellular insoluble deposits of highly ordered cross-ß-structure-containing amyloid fibrils form the pathological basis for protein misfolding diseases. As amyloid fibrils are cytotoxic, inhibition of the process is a therapeutic strategy. Several small molecules have been identified and used as fibrillation inhibitors in the recent past. In this work, we investigate the effect of Orange G on insulin amyloid formation using fluorescence-based assays and negative-stain electron microscopy (EM). We show that Orange G effectively attenuates nucleation, thereby inhibiting amyloid fibrillation in a dose-dependent manner. Fluorescence quenching titrations of Orange G showed a reasonably strong binding affinity to native insulin. Binding isotherm measurements revealed the binding of Orange G to pre-formed insulin fibrils too, indicating that Orange G likely binds and stabilizes the mature fibrils and prevents the release of toxic oligomers which could be potential nuclei or templates for further fibrillation. Molecular docking of Orange G with native insulin and amyloid-like peptide structures were also carried out to analyse the contributing interactions and binding free energy. The findings of our study emphasize the use of Orange G as a molecular probe to identify and design inhibitors of amyloid fibrillation and to investigate the structural and toxic mechanisms underlying amyloid formation.

Inhibition of insulin amyloid fibrillation by Morin hydrate.

We report here the inhibition of amyloid fibrillation of human insulin in vitro by Morin hydrate, a naturally occurring small molecule. Using spectroscopic assays and transmission electron microscopy, we found that Morin hydrate effectively inhibits insulin amyloid fibrillation in a dose dependent manner with more than 80% inhibition occurring even at only a 1:1 concentration. As suggested by fluorescence spectroscopic titration studies, Morin hydrate binds to insulin with a fairly strong affinity of -26.436kJmol-1. Circular dichroism (CD) spectroscopy was used to analyse structural changes of insulin in the presence of Morin hydrate demonstrating the ability of Morin hydrate to bind with the native monomeric protein and/or its near native state, intermediate oligomeric species and amyloid fibrils. Based on computational docking and molecular dynamics study, we propose that Morin hydrate binds to residues having greater aggregation propensity and prevent structural and/or conformational changes leading to amyloid fibrillation. Morin hydrate should also bind to fibrils by hydrogen bonding and/or hydrophobic forces throughout the surface, stabilize them and inhibit the release of oligomeric species which could be nuclei or template for further fibrillation. Overall results provide an insight into the mechanism of inhibition of insulin amyloid fibrillation by Morin hydrate.

Inhibition of amyloid fibril formation of lysozyme by ascorbic acid and a probable mechanism of action.

Amyloid fibrillation of proteins and polypeptides and their deposition in cells and tissues is associated with a number of pathological states collectively known as amyloid disorders. Inhibition of protein misfolding and aggregation is thus of utmost importance in the prevention and treatment of such diseases. There is a growing interest in identification of small molecules that can bind to native monomeric proteins or their partially unfolded states, thereby stabilizing them and preventing or delaying them from undergoing amyloid fibril formation. Here we report the inhibitory effect of ascorbic acid, an essential dietary component richly present in many natural food items, on the amyloid fibrillation of hen egg white lysozyme, a model protein for amyloid formation. The effect was dose dependent with more than 80% inhibition occurring even at only a five-fold molar excess of ascorbic acid. TEM images show complete absence of fibrils in the presence of ascorbic acid. From our spectroscopic and computational characterization of ascorbic acid binding to HEWL, we propose that ascorbic acid binds to the aggregation prone beta domain of HEWL, stabilizes the partially unfolded conformation and prevents further conformational changes leading to fibrillation. Hence ascorbic acid has a great therapeutic potential for amyloid disorders.

Combined in silico approaches for the identification of novel inhibitors of human islet amyloid polypeptide (hIAPP) fibrillation.

Human islet amyloid polypeptide (hIAPP) is a natively unfolded polypeptide hormone of glucose metabolism, which is co-secreted with insulin by the ß-cells of the pancreas. In patients with type 2 diabetes, IAPP forms amyloid fibrils because of diabetes-associated ß-cells dysfunction and increasing fibrillation, in turn, lead to failure of secretory function of ß-cells. This provides a target for the discovery of small organic molecules against protein aggregation diseases. However, the binding mechanism of these molecules with monomers, oligomers and fibrils to inhibit fibrillation is still an open question. In this work, ligand and structure-based in silico approaches were used to identify novel fibrillation inhibitors and/or fibril binding compounds. The best pharmacophore model was used as a 3D search query for virtual screening of a compound database to identify novel molecules having the potential to be therapeutic agents against protein aggregation diseases. Docking and molecular dynamics simulation studies were used to explore the interaction pattern and mechanism of the identified novel small molecules with predicted hIAPP structure, its aggregation prone conformation and fibril forming segments. We show that catechins with galloyl group and molecules having two to three planar apolar rings bind to hIAPP structures and fibril forming segments with greater affinity. The differences in binding affinities of different compounds against several fibril forming segments of the peptide suggest that a mixture of active compounds may be required for treatment of aggregation diseases.