A novel diacylglycerol-lactone shows marked selectivity in vitro among C1 domains of protein kinase C (PKC) isoforms α and δ as well as selectivity for RasGRP compared with PKCα

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent K-i values ranging from 340 nM for PKC alpha to 29 nM for PKC epsilon. When binding to isolated C1 domains of PKC alpha and -delta, 130C037 showed good affinity (K-i = 1.78 nM) only for delta C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of delta C1b. 130C037 bound intact RasGRP1 and RasGRP3 with K-i values of 3.5 and 3.8 nM, respectively, reflecting 8- and 90-fold selectivity relative to PKC epsilon and PKC alpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nM), partial reduction of PKC epsilon (ED50 > 10 mu M), and no effect on PKC alpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC epsilon, and no translocation of PKC alpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.