Iridals are a novel class of ligands for phorbol ester receptors with modest selectivity for the RasGRP receptor subfamily

Since 1990, the National Cancer Institute has performed extensive in vitro screening of compounds for anticancer activity. To date, more than 70 000 c ompounds have been screened for their antiproliferation activities against a panel of 60 human cancer cell lines. We probed this database to identify novel structural classes with a pattern of biological activity on these cel l lines similar to that of the phorbol esters. The iridals form such a stru ctural class. Using the program Autodock, we show that the iridals dock to the same position on the C1b domain of protein kinase C delta as do the pho rbol esters, with the primary hydroxyl group of the iridal at the C3 positi on forming two hydrogen bonds with the amide group of Thr12 and with the ca rbonyl group of Leu 21 and the aldehyde oxygen of the iridal forming a hydr ogen bond with the amide group of Gly23. Biological analysis of two iridals , NSC 631939 and NSC 631941, revealed that they bound to protein kinase C a lpha with K-i values of 75.6 +/- 1.3 and 83.6 +/- 1.5 nM, respectively. Pro tein kinase C is now recognized to represent only one of five families of p roteins with C1 domains capable of high-affinity binding of diacylglycerol and the phorbol esters. NSC 631939 and NSC 631941 bound to RasGRP3, a phorb ol ester receptor that directly links diacylglycerol/phorbol ester signalin g with Ras activation, with Ki values of 15.5 +/- 2.3 and 41.7 +/- 6.5 nM, respectively. Relative to phorbol 12,13-dibutyrate, they showed 15- and 6-f old selectivity for RasGRP3. Both compounds caused translocation of green f luorescent protein tagged RasGRP3 expressed in HEK293 cells, and both compo unds induced phosphorylation of ERK1/2, a downstream indicator of Ras activ ation, in a RasGRP3-dependent fashion. We conclude that the iridals represe nt a promising structural motif for design of ligands for phorbol ester rec eptor family members.