Synthesis and biological evaluation of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives as potential anticancer agents

A series of novel alkyl amide functionalized trifluoromethyl substituted pyrazolo[3,4-b]pyridine derivatives 5, 6 and 7 were prepared starting from 6-phenyl-4-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-3-amine 3 via selective N-alkylation, followed by reaction with different primary aliphatic amines, cyclic secondary amines or l-amino acids under different set of conditions. All the synthesized compounds 5, 6 and 7 were screened for anticancer activity against four cancer cell lines such as A549—Lung cancer (CCL-185), MCF7—Breast cancer (HTB-22), DU145—Prostate cancer (HTB-81) and HeLa—Cervical cancer (CCL-2). The compounds 5i and 6e are found to have promising bioactivity at micro molar concentration.     

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.     

Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

Protein structure prediction using mutually orthogonal Latin squares and a genetic algorithm

We combine a new, extremely fast technique to generate a library of low energy structures of an oligopeptide (by using mutually orthogonal Latin squares to sample its conformational space) with a genetic algorithm to predict protein structures. The protein sequence is divided into oligopeptides, and a structure library is generated for each. These libraries are used in a newly defined mutation operator that, together with variation, crossover, and diversity operators, is used in a modified genetic algorithm to make the prediction. Application to five small proteins has yielded near native structures.     

Effect of DNA structural flexibility on promoter strength--molecular dynamics studies of E. coli promoter sequences

To study possible correlations between promoter activity and the structural flexibility of the DNA helix, we have carried out unrestrained molecular dynamics simulations of the -10 consensus region sequence and five variants forming the -10 region of various Escherichia coli promoter sequences. Analyses of the trajectories obtained from the simulations show that the consensus sequence has a pattern of two structurally flexible nucleotide steps sandwiched between two stiff steps. In the other sequences, this pattern varies in consonance with the change in the sequence. The variations in the patterns show correlation with the promoter strength.     

Principal component analysis of DNA oligonucleotide structural data

The microstructure of a DNA helix is characterized by several base pair and base step parameters such as twist, rise, roll, propeller twist, etc., in addition to conformational parameters such as the backbone and the glycosidic torsion angles. Among these only a few, which are independent of all others and of each other, may be used to precisely characterize the helix. The problem however is to identify these independent parameters. We have used principal component analysis to identify a relatively small set of independent parameters, with which to characterize each DNA helix. We show that these principal components clearly discriminate between A and B DNA helical types. The calculations further suggest that the microstructure of a DNA helix is better characterized using dinucleotides.     

Conformational space exploration of Met- and Leu-enkephalin using the MOLS method, molecular dynamics, and Monte Carlo simulation--a comparative study

We report here a comparative study of the molecular conformational energy landscape generated using the mutually orthogonal Latin squares (MOLS) method, molecular dynamics (MD), and Monte Carlo (MC) simulation. The MOLS method, as described earlier from our laboratory, uses an experimental design technique to rapidly and exhaustively sample the low energy conformations of a molecule. MD and MC simulations have been used to perform similar tasks. In the comparison reported here, the three methods were applied to a pair of neuropeptides, namely Met- and Leu-enkephalin. A set of 1500 conformations of these enkephalins were generated using these methods with CHARMM22 force field, and the resulting samples were analyzed to determine the extent and nature of coverage of the conformational space. The results indicate that the MOLS method samples a larger number of possible conformations and identifies conformations closer to the experimental structures than the MD and MC simulations.     

MOLS sampling and its applications in structural biophysics

This review describes the MOLS method and its applications. This computational method has been developed in our laboratory primarily to explore the conformational space of small peptides and identify features of interest, particularly the minima, i.e., the low energy conformations. A systematic “brute-force” search through the vast conformational space for such features faces the insurmountable problem of combinatorial explosion, whilst other techniques, e.g., Monte Carlo searches, are somewhat limited in their region of exploration and may be considered inexhaustive. The MOLS method, on the other hand, uses a sampling technique commonly employed in experimental design theory to identify a small sample of the conformational space that nevertheless retains information about the entire space. The information is extracted using a technique that is a variant of the self-consistent mean field technique, which has been used to identify, for example, the optimal set of side-chain conformations in a protein. Applications of the MOLS method to understand peptide structure, predict the structures of loops in proteins, predict three-dimensional structures of small proteins, and arrive at the best conformation, orientation, and positions of a small molecule ligand in a protein receptor site have all yielded satisfactory results.