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 ␣, ␤, ␥, ␦, and ⑀, with apparent K i values ranging from 340 nM for PKC␣ to 29 nM for PKC⑀. When binding to isolated C1 domains of PKC␣ and -␦, 130C037 showed good affinity (K i ‫؍‬ 1.78 nM) only for ␦C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of ␦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⑀ and PKC␣. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED 50 ‫؍‬ 286 nM), partial reduction of PKC⑀ (ED 50 > 10 M), and no effect on PKC␣. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC⑀, and no translocation of PKC␣. 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. Diacylglycerol (DAG)1 is a lipid second messenger, produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate following the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). Most but not all effects of DAG reflect its interaction with proteins containing C1 domains, resulting in their activation and/or driving their membrane translocation. Reflecting the importance and diversity of its downstream effectors, DAG is involved in signal transduction of numerous physiological and pathological processes, including proliferation, differentiation, apoptosis, angiogenesis, and drug resistance (2). These functions have focused attention on C1 domain-containing proteins as molecular targets for cancer chemotherapy (3).The interaction between DAG and its receptors is typically mediated by a DAG-responsive motif called a "C1 domain" (4). The highly conserved C1 domain (ϳ50 amino acids) is a cysteine-rich zinc finger structure (5) that was first identified in protein kinase C (PKC) as the interaction site for DAG and the phorbol esters (6). The PKC family of serine/threonine protein kinases comprises the best studied mediators of DAG signaling. 8 of its 11 family members have DAG-responsive C1 domains: (i) the conventional PKCs (␣, ␤I, ␤II, and ␥) and (ii) the novel PKCs (␦, ⑀, , and ). Both the classic and novel PKCs conta...

Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have termed “chemical zip codes”, are able to transform a relatively small chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these “chemical zip codes”. It is postulated that after binding to protein kinase C (PKC) isozymes or other non-kinase target proteins that contain diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCα to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses.