Identification of novel angiogenesis inhibitors

Vascular endothelial growth factor (VEGF) is a key stimulant of angiogenesis, which is the process of generating new capillary blood vessels. Inhibition of the vascular endothelial growth factor receptor (VEGFR) kinase is known to result in blockage of angiogenesis. A pharmacophore was developed based on the binding of ATP to the hinge region of the kinase domain of VEGFR and a database search of 18,000 compounds was conducted. Selected hits were assessed for their ability to limit the induction of web-like network of capillary tubes by the human umbilical vascular endothelial cells. Two compounds (1 and 4) showed good inhibitory ability to prevent sprouting and closed polygon formation of the tubular networks, promising them to be lead compounds. Compound 4 showed 60% inhibition at 0.05 microM.     

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.     

Differential activation of eIF2 kinases in response to cellular stresses in Schizosaccharomyces pombe

Phosphorylation of eukaryotic initiation factor-2 (eIF2) is an important mechanism mitigating cellular injury in response to diverse environmental stresses. While all eukaryotic organisms characterized to date contain an eIF2 kinase stress response pathway, the composition of eIF2 kinases differs, with mammals containing four distinct family members and the well-studied lower eukaryote Saccharomyces cerevisiae expressing only a single eIF2 kinase. We are interested in the mechanisms by which multiple eIF2 kinases interface with complex stress signals and elicit response pathways. In this report we find that in addition to two previously described eIF2 kinases related to mammalian HRI, designated Hri1p and Hri2p, the yeast Schizosaccharomyces pombe expresses a third eIF2 kinase, a Gcn2p ortholog. To delineate the roles of each eIF2 kinase, we constructed S. pombe strains expressing only a single eIF2 kinase gene or deleted for the entire eIF2 kinase family. We find that Hri2p is the primary activated eIF2 kinase in response to exposure to heat shock, arsenite, or cadmium. Gcn2p serves as the primary eIF2 kinase induced during a nutrient downshift, treatment with the amino acid biosynthetic inhibitor 3-aminotriazole, or upon exposure to high concentrations of sodium chloride. In one stress example, exposure to H₂O₂, there is early tandem activation of both Hri2p and Gcn2p. Interestingly, with extended stress conditions there is activation of alternative secondary eIF2 kinases, suggesting that eukaryotes have mechanisms of coordinate activation of eIF2 kinase in their stress remediation responses. Deletion of these eIF2 kinases renders S. pombe more sensitive to many of these stress conditions.     

Effect of auxins on berberine synthesis in cell suspension culture of Coscinium fenestratum (Gaertn.) Colebr--a critically endangered medicinal liana of Western Ghats

Cell suspension culture of critically endangered Coscinium fenestratum was established from young leaf segments on WPM supplemented with auxins. Effect of 2,4-D, IAA, IBA and NAA was examined on cell growth and berberine production. Berberine was synthesized and released continuously into the liquid medium. Presence of 2,4-D stimulated cell growth, but was not inhibitory on berberine synthesis. On the contrary, NAA stimulated berberine biosynthesis, but was not favourable for cell growth. Among the auxins tested, highest yield of berberine (5.79 mg/30 ml; 4.14 times to that of control) was obtained with 4 mg/l of NAA, while the best cell growth (214.43 mg dry wt., 1.96 times to that of control) was observed in the presence of 2 mg/l of 2,4-D. IAA and IBA were not favourable for cell growth and berberine synthesis.     

Ventricular Dyssynchrony Patterns in Left Bundle Branch Block,With and Without Heart Failure

Background

Assessment of ventricular dyssynchrony in patients with heart failure is used for selecting candidates for cardiac resynchronization therapy (CRT). The patterns of regional distribution of dyssynchrony in a population with LBBB with and without heart failure have not been well delineated. This aspect forms the object of the study.

Methods

Tissue Doppler Imaging (TDI) data of consecutive patients with heart failure and LBBB (Group A) was compared with those with LBBB and normal LV function (Group B). All patients had standard 2D-echocardigraphic examination and TDI. Tissue velocity curves obtained by placing sample volumes in opposing basal and mid segments of septal, lateral, inferior, anterior and posterior walls were analyzed. Inter ventricular dyssynchrony (IVD) was assessed by the difference between aortic and pulmonary pre ejection intervals. LV dyssynchrony (LVD) was assessed by the difference in times to peak velocity. A delay of ≥ 40 msec was considered significant for presence of IVD and LVD.

Results

There were 103 patients in Group A and 25 in Group B. The mean QRS duration and PR intervals respectively were 146 ± 25 vs. 152±20 msec and 182± 47 vs. 165±36 msec. (p=NS) LVEF in the 2 groups were (32 ± 6 % vs. 61± 11%; p< 0.01). Prevalence of dyssynchrony in the HF group compared to Group B was 72% vs. 16%, (P< 0.01). Lateral wall dyssynchrony in the 2 groups was 37% vs. 0% (p< 0.01) while septal dyssynchrony was 16% vs. 16% (p- NS).

Conclusions

72% of heart failure patients with LBBB have documented dyssynchrony on TDI, which has a heterogeneous regional distribution. Dyssynchrony may be seen in LBBB and normal hearts but it is does not involve the lateral wall. Septal dyssynchrony in heart failure patients may not have the same significance as lateral wall delay.     

T-iDT : tool for identification of drug target in bacteria and validation by Mycobacterium tuberculosis

With the completion of the Human Genome Project in 2003, many new projects to sequence bacterial genomes were started and soon many complete bacterial genome sequences were available. The sequenced genomes of pathogenic bacteria provide useful information for understanding host-pathogen interactions. These data prove to be a new weapon in fighting against pathogenic bacteria by providing information about potential drug targets. But the limitation of computational tools for finding potential drug targets has hindered the process and further experimental analysis. There are many in silico approaches proposed for finding drug targets but only few have been automated. One such approach finds essential genes in bacterial genomes with no human homologue and predicts these as potential drug targets. The same approach is used in our tool. T-iDT, a tool for the identification of drug targets, finds essential genes by comparing a bacterial gene set against DEG (Database of Essential Genes) and excludes homologue genes by comparing against a human protein database. The tool predicts both the set of essential genes as well as potential target genes for the given genome. The tool was tested with Mycobacterium tuberculosis and results were validated. With default parameters, the tool predicted 236 essential genes and 52 genes to encode potential drug targets. A pathway-based approach was used to validate these potential drug target genes. The pathway in which the products of these genes are involved was determined. Our analysis shows that almost all these pathways are very essential for the bacterial survival and hence these genes encode possible drug targets. Our tool provides a fast method for finding possible drug targets in bacterial genomes with varying stringency level. The tool will be helpful in finding possible drug targets in various pathogenic organisms and can be used for further analysis in novel therapeutic drug development. The tool can be downloaded from http://www.milser.co.in/research.htm and http://www.srmbioinformatics.edu.in/ forum.htm.