title: A novel diacylglycerol-lactone shows marked selectivity in vitro among C1 domains of protein kinase C (PKC) isoforms alpha and delta as well as selectivity for RasGRP compared with PKCalpha.
abstract: 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 Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. 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.
authors.affiliation: Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Pu
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Perry
authors.forename: Dazhi
authors.initials: D
authors.lastname: Yang
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Lewin
authors.forename: Noemi
authors.initials: N
authors.lastname: Kedei
authors.forename: Derek C
authors.initials: DC
authors.lastname: Braun
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Choi
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Susan H
authors.initials: SH
authors.lastname: Garfield
authors.forename: James C
authors.initials: JC
authors.lastname: Stone
authors.forename: Dehui
authors.initials: D
authors.lastname: Duan
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007783:Lactones / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D021381:Protein Transport; D020663:ras Guanine Nucleotide Exchange Factors
chemical_list: C011439:1,2-diacylglycerol; D004268:DNA-Binding Proteins; D004075:Diglycerides; D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D007783:Lactones; C491790:RASGRP1 protein, human; C413212:RASGRP3 protein, human; D020663:ras Guanine Nucleotide Exchange Factors; D015240:Phorbol 12,13-Dibutyrate; C496628:PRKCA protein, human; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta