Alteration of the regiospecificity of human heme oxygenase-1 by unseating of the heme but not disruption of the distal hydrogen bonding network

Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.     

Effect of sinomenine on gene expression of the IL-1 beta-activated human synovial sarcoma

Sinomenine is an alkaloid with pharmacological effects of anti-inflammation, anti-angiogenesis, anti-arthritis and immunosuppression. This study aimed to investigate the effect of sinomenine on gene expression of human synovial sarcoma cells (Hs701.T) activated by IL-1 beta. The proliferative effect of sinomenine was examined in the presence or absence of IL-1 beta by the [3H]-thymidine incorporation and MTT assay, respectively. Using DNA microarray technology and RT-PCR, the activating action of IL-1 beta and modulatory effect of sinomenine on Hs701.T were simultaneously determined. Results showed that IL-1 beta could stimulate the proliferation and gene expression of Hs701.T cells. Sinomenine could significantly inhibit proliferation of IL-1 beta-activated Hs701.T cells and suppress expression of 17 genes including IL-6, PlGF, Daxx, and HSP27. These genes were found to be important in tumor progression through the mediation of inflammation, cell adhesion, proliferation, apoptosis and angiogenesis. In conclusion, our study provides supplementary information for the further studies on the pharmacological effects of sinomenine acting on synovial sarcoma.     

Syntheses and biological activity of bisdaunorubicins

To study the length and flexibility of the linkers between two monomers of bisdaunorubicins for their activity against cancer cells, seven bisdaunorubicins were rationally designed and synthesized through click chemistry. Their cytotoxicity was tested in leukemia cells with MTS assay. The results showed that the compounds with short linkers exhibited higher activity than the compounds with long linkers, while the flexibility of the linker also contributed to their activity. These results indicated that the length and flexibility of the linkers between two monomers in bisdaunorubicins are very critical to maintain their activity against cancer cells.     

Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity

Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45-54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45-54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.     

Synthesis and biological activity of conjugates between paclitaxel and the cell delivery vector penetratin

Synthesis of paclitaxel-penetratin (pAntp) constructs, in which the 2'- or 7-position of paclitaxel was used as the attachment site for linker connecting the drug and peptide moieties, is described. Paclitaxel-2'-pAntp[43-58]-NH(2)3b and paclitaxel-2'-pAntp[52-58]-NH(2)3c showed excellent antitumour activity against human lung and breast cancer cell lines. These conjugates were highly soluble and stable with a half-life of >8h under cell culture conditions. The drug-peptide conjugates may be therapeutically useful due to improved pharmaceutical properties.