Determining test substances that inhibit protease, involves incubating cells expressing fusion protein having substrate with cleavage site for protease and reporter, measuring cleaved reporter and comparing with standard

摘要

Determining test substances (S) for inhibiting proteases (I) that cleave membrane-bound substrates, comprises incubating cultivated cells with protease activity and expressing membrane-associated recombinant fusion protein having a substrate with a specific cleavage site for (I) and a reporter, with (S), measuring amount of cleaved reporter and comparing with a value obtained in absence of (S). Independent claims are also included for the following: (1) a substance (S) determined by the above method, which specifically inhibits the proteolytic cleavage of gamma -secretase substrate or presenilinase substrate, and (2) a pharmaceutical formulation comprising (S). - ACTIVITY : Nootropic; neuroprotective. - MECHANISM OF ACTION : Proteolytic cleavage of gamma -secretase and presenilinase inhibitor. The cell clones Abeta -KKK-ER/40, 59 and 65 (substrate Abeta -KKK/Gal4 with ER retention signal) and the cell clone Abeta -KKK/52 (substance Abeta -KKK/Gal4 without ER retention signal) were used to test the inhibitory effect of substance A. Confluent cells were incubated overnight with different concentrations of substance A. The luciferase assay was carried out. In all cell clones substance A inhibited Abeta formation in a concentration-dependent manner, as indicated by the decrease in the luciferase signal.