Application of a radioreceptor assay to the screening and characterisation of compounds from marine organisms with activity at the phorbol ester binding site of protein kinase C

Abstract

Protein kinase C is a ubiquitous enzyme with a key role in cellular function, making it an attractive drug target. Utilising a competitive binding assay for the phorbol ester binding site of the enzyme in a rat brain membrane preparation, screening was undertaken on 686 marine macroorganisms representing a broad range of taxa and environments from throughout Australasia. Of these, extracts from 28 organisms significantly inhibited [ 3H]phorbol dibutyrate binding, while two samples enhanced binding. Sponges and echinoderms were particularly well represented in the active specimens. A combination of taxonomic and elution information for individual leads provided a rationale for dereplication and prioritisation. Utilising assay-guided purification, the identity of active compounds from the sponge Agelas axifera was examined in detail. The previously described compounds, the agelasines, were identified. The screening and characterisation methods described provide a method for readily identifying novel probes for protein kinase C.