Chelerythrine inhibits the sarco/endoplasmic reticulum Ca(2+)-ATPase and results in cell Ca(2+) imbalance.

The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2 µM and Ki2=26 µM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca(2+)-dependent pNPPase was activated up to ∼5 µM chelerythrine with inhibition thereafter. Ca(2+)-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 µM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 µM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5-1.5 µM resulted in an increase of intracellular Ca(2+) on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca(2+) homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid's effects on SERCA.

Investigation of thrombin activity with PAR 1-based fluorogenic peptides.

Thrombin, a highly specific protease of blood coagulation, has two exosites that modulate its specificity. We designed two sets of synthetic substrate FRET peptides with 25- or 11- amino acids (aa) each, based on the PAR 1 sequence, to characterize the effect of exosite 1 engagement on substrate catalysis and preference. The 25-aa set encompassed a sequence binding to exosite 1, and structural modeling showed that binding to thrombin did not differ significantly from that of PAR 1 peptide. Modification at the P3´position of the 25 or 11-aa peptides resulted in small effect on kinetic parameters. Ionic strength higher than physiologic depressed thrombin action on the 25-aa peptides. Addition of ligands of the exosite 1 negatively modulated the catalysis of 25-aa substrates. In conclusion, we succeeded to mimic and study in real time, using these synthetic peptides, the influence of ligand binding to exosite 1 on thrombin activity.