Characterization of the Interaction of Ingenol 3-Angelate with Protein Kinase C

Abstract

Ingenol 3-angelate (I3A) is one of the active ingredients in Euphorbia peplus, which has been used in traditional medicine. Here, we report the initial characterization of I3A as a protein kinase C (PKC) ligand. I3A bound to PKC-␣ in the presence of phosphatidylserine with high affinity; however, under these assay conditions, little PKC isoform selectivity was observed. PKC isoforms did show different sensitivity and selectivity for down-regulation by I3A and phorbol 12-myristate 13-acetate (PMA) in WEHI-231, HOP-92, and Colo-205 cells. In all of the three cell types, I3A inhibited cell proliferation with somewhat lower potency than did PMA. In intact CHO-K1 cells, I3A was able to translocate different green fluorescent protein-tagged PKC isoforms, visualized by confocal microscopy, with equal or higher potency than PMA. PKC-␦ in particular showed a different pattern of translocation in response to I3A and PMA. I3A induced a higher level of secretion of the inflammatory cytokine interleukin 6 compared with PMA in the WEHI-231 cells and displayed a marked biphasic dose-response curve for the induction. I3A was unable to cause the same extent of association of the C1b domain of PKC-␦ with lipids, compared with PMA or the physiological regulator diacylglycerol, and was able to partially block the association induced by these agents, measured by surface plasmon resonance. The in vitro kinase activity of PKC-␣ induced by I3A was lower than that induced by PMA. The novel pattern of behavior of I3A makes it of great interest for further evaluation.

Analysis by Fluorescence Resonance Energy Transfer of the Interaction between Ligands and Protein Kinase Cδ in the Intact Cell

“…Among the asymmetric ligands, ingenol 3-angelate was closest to the peak in potency predicted by the Kubinyi relationship and was more Table I. potent than PMA. Similarly, we had reported previously that ingenol 3-angelate was more potent than PMA for inducing translocation of GFP-PKC␦ expressed in CHO cells (6). DISCUSSION The published literature reports that the measured in vitro binding affinity of ligands to protein kinase C is not necessarily predictive of the potencies of those ligands to activate PKCmediated responses in intact cells.…”

“…Indeed, a number of compounds targeting protein kinase C are currently at different stages of drug development. Examples include LY333531, being evaluated for diabetic retinopathy and anti-angiogenesis (1, 2), prostratin and dPP, being evaluated for AIDS chemotherapy (3,4), and bryostatin 1 and ingenol 3-angelate, being evaluated as cancer chemotherapeutic agents (5)(6)(7).…”

Evidence for a role of MRCK in mediating HeLa cell elongation induced by the C1 domain ligand HMI-1a3

“…Various PKC-or C1 domain-targeted compounds have been and are currently being studied in clinical trials against cancer. The first PKC-targeting drug, the plant-derived C1 domain ligand ingenol-3-angelate (ingenol mebutate) (Kedei et al, 2004) has recently been approved for clinical use against actinic skin (EMA, 2012;FDA, 2012). However, most of the currently known C1 domain ligands are complex in their chemical structure and thus difficult to synthesize.…”

Antiproliferative activity of PEP005, a novel ingenol angelate that modulates PKC functions, alone and in combination with cytotoxic agents in human colon cancer cells

“…Chemically, PEP005 is structurally analogous to phorbol esters ( Figure 1) and is a potent modulator of PKC isoenzymes (Kedei et al, 2004). PEP005 is currently being developed as a topical treatment for actinic keratoses and basal cell carcinoma (www.cancertrials.gov).…”