Modulatory role of copper on hIAPP aggregation and toxicity in presence of insulin.

Aggregation of the human islets amyloid polypeptide, or hIAPP, is linked to ß-cell death in type II diabetes mellitus (T2DM). Different pancreatic ß-cell environmental variables such as pH, insulin and metal ions play a key role in controlling the hIAPP aggregation. Since insulin and hIAPP are co-secreted, it is known from numerous studies that insulin suppresses hIAPP fibrillation by preventing the initial dimerization process. On the other hand, zinc and copper each have an inhibitory impact on hIAPP fibrillation, but copper promotes the production of toxic oligomers. Interestingly, the insulin oligomeric equilibrium is controlled by the concentration of zinc ions when the effect of insulin and zinc has been tested together. Lower zinc concentrations cause the equilibrium to shift towards the monomer and dimer states of insulin, which bind to monomeric hIAPP and stop it from developing into a fibril. On the other hand, the combined effects of copper and insulin have not yet been studied. In this study, we have demonstrated how the presence of copper affects hIAPP aggregation and the toxicity of the resultant conformers with or without insulin. For this purpose, we have used a set of biophysical techniques, including NMR, fluorescence, CD etc., in combination with AFM and cell cytotoxicity assay. In the presence and/or absence of insulin, copper induces hIAPP to form structurally distinct stable toxic oligomers, deterring the fibrillation process. More specifically, the oligomers generated in the presence of insulin have slightly higher toxicity than those formed in the absence of insulin. This research will increase our understanding of the combined modulatory effect of two ß-cell environmental factors on hIAPP aggregation.

Solvent-Assisted Tyrosine-Based Dipeptide Forms Low-Molecular Weight Gel: Preparation and Its Potential Use in Dye Removal and Oil Spillage Separation from Water.

Low-molecular weight gelators (supramolecular, or simply molecular gels) are highly important molecular frameworks because of their potential application in drug delivery, catalysis, pollutant removal, sensing materials, and so forth. Herein, a small dipeptide composed of N-(tert-butoxycarbonyl)pentafluoro-l-phenylalanine and O-benzyl-l-tyrosine methyl ester was synthesized, and its gelation ability was investigated in different solvent systems. It was found that the dipeptide was unable to form gel with a single solvent, but a mixture of solvent systems was found to be suitable for the gelation of this dipeptide. Interestingly, water was found to be essential for gelation with the polar protic solvent, and long-chain hydrocarbon units such as, petroleum ether, kerosene, and diesel, were important for gelation with aromatic solvents. The structural insights of these gels were characterized by field-emission scanning electronic microscopy, atomic force microscopy, Fourier transform infrared analysis, and X-ray diffraction studies, and their mechanical strengths were characterized by rheological experiments. Both of the gels obtained from these two solvent systems were thermoreversible in nature, and these translucent gels had potential application for the treatment of waste water. The gel obtained from dipeptides with methanol-water was used to remove toxic dyes (crystal violet, Eriochrome Black T, and rhodamine B) from water. Furthermore, the gel obtained from dipeptide with assistance from toluene-petroleum ether was used as a phase-selective gelator for oil-spill recovery.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two É£ H of curcumin heptadiene chain are closely positioned to the A16-H8 and A17-H8, while G12-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.

Cα-H carries information of a hydrogen bond involving the geminal hydroxyl group: a case study with a hydrogen-bonded complex of 1,1,1,3,3,3-hexafluoro-2-propanol and tertiary amines.

Experimental measurement of the contribution of H-bonding to intermolecular and intramolecular interactions that provide specificity to biological complex formation is an important aspect of macromolecular chemistry and structural biology. However, there are very few viable methods available to determine the energetic contribution of an individual hydrogen bond to binding and catalysis in biological systems. Therefore, the methods that use secondary deuterium isotope effects analyzed by NMR or equilibrium or kinetic isotope effect measurements are attractive ways to gain information on the H-bonding properties of an alcohol system, particularly in a biological environment. Here, we explore the anharmonic contribution to the C-H group when the O-H group of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) forms an intermolecular H-bond with the amines by quantum mechanical calculations and by experimentally measuring the H/D effect by NMR. Within the framework of density functional theory, ab initio calculations were carried out for HFP in its two different conformational states and their H-bonded complexes with tertiary amines to determine the (13)C chemical shielding, change in their vibrational equilibrium distances, and the deuterium isotope effect on (13)C2 (secondary carbon) of HFP upon formation of complexes with tertiary amines. When C2-OH was involved in hydrogen bond formation (O-H as hydrogen donor), it weakened the geminal C2-H bond; it was reflected in the NMR chemical shift, coupling constant, and the equilibrium distances of the C-H bond. The first derivative of nuclear shielding at C2 in HFP was -48.94 and -50.73 ppm Å(-1) for anti and gauche conformations, respectively. In the complex, the values were -50.28 and -50.76 ppm Å(-1), respectively. The C-H stretching frequency was lower than the free monomer, indicating enhanced anharmonicity in the C-H bond in the complex form. In chloroform, HFP formed a complex with the amine; δC2 was 69.107 ppm for HFP-triethylamine and 68.766 ppm for HFP-d2-triethylamine and the difference in chemical shift, the ΔδC2 was 341 ppb. The enhanced anharmonicity in the hydrogen-bonded complex resulted in a larger vibrational equilibrium distance in C-H/D bonds. An analysis with the Morse potential function indicated that the enhanced anharmonicity encountered in the bond was the origin of a larger isotope effect and the equilibrium distances. Change in vibrational equilibrium distance and the deuterium isotope effect, as observed in the complex, could be used as parameters in monitoring the strength of the H-bond in small model systems with promising application in biomacromolecules.

Potent anticancer activity of cystine-based dipeptides and their interaction with serum albumins.

BACKGROUND: Cancer is a severe threat to the human society. In the scientific community worldwide cancer remains a big challenge as there are no remedies as of now. Cancer is quite complicated as it involves multiple signalling pathways and it may be caused by genetic disorders. Various natural products and synthetic molecules have been designed to prevent cell proliferation. Peptide-based anticancer drugs, however, are not explored properly. Though peptides have their inherent proteolytic instability, they could act as anticancer agents. RESULTS: In this present communication a suitably protected cystine based dipeptide and its deprotected form have been synthesized. Potent anticancer activities were confirmed by MTT assay (a laboratory test and a standard colorimetric assay, which measures changes in colour, for measuring cellular proliferation and phase contrast images. The IC50 value, a measure of the effectiveness of a compound in inhibiting biological or biochemical function, of these compounds ranges in the sub-micromolar level. The binding interactions with serum albumins (HSA and BSA) were performed with all these molecules and all of them show very strong binding at sub-micromolar concentration. CONCLUSIONS: This study suggested that the cystine-based dipeptides were potential anticancer agents. These peptides also showed very good binding with major carrier proteins of blood, the serum albumins. We are currently working on determining the detailed mechanism of anticancer activity of these molecules.

Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site.

The zinc-dependent matrix metalloproteinases (MMPs) are key enzymes associated with extracellular matrix (ECM) remodeling; they play critical roles under both physiological and pathological conditions. MMP-9 activity is linked to many pathological processes, including rheumatoid arthritis, atherosclerosis, gastric ulcer, tumor growth, and cancer metastasis. Specific inhibition of MMP-9 activity may be a promising target for therapy for diseases characterized by dysregulated ECM turnover. Potent MMP-9 inhibitors including an indole scaffold were recently reported in an X-ray crystallographic study. Herein, we addressed whether melatonin, a secretory product of pineal gland, has an inhibitory effect on MMP-9 function. Gelatin zymographic analysis showed a significant reduction in pro- and active MMP-9 activity in vitro in a dose- and time-dependent manner. In addition, a human gastric adenocarcinoma cell line (AGS) exhibited a reduced (~50%) MMP-9 expression when incubated with melatonin, supporting an inhibitory effect of melatonin on MMP-9. Atomic-level interaction between melatonin and MMP-9 was probed with computational chemistry tools. Melatonin docked into the active site cleft of MMP-9 and interacted with key catalytic site residues including the three histidines that form the coordination complex with the catalytic zinc as well as proline 421 and alanine 191. We hypothesize that under physiological conditions, tight binding of melatonin in the active site might be involved in reducing the catalytic activity of MMP-9. This finding could provide a novel approach to physical docking of biomolecules to the catalytic site of MMPs, which inhibits this protease, to arrest MMP-9-mediated inflammatory signals.