Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP

Abstract

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2 to 5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Novel transduction of nutrient stress to Notch pathway by RasGRP3 promotes malignant aggressiveness in human esophageal squamous cell carcinoma

“…In general, once Ras respond to a series of exterior and interior signals, it will subsequently amplify or turn on other factors, which ultimately regulate cell differentiation, growth, invasion, angiogenesis and survival; however, the Ras mutations are rare in tumors (15)(16)(17). Moreover, it was suggested that Ras could be constitutively overactivated by various upstream signaling elements, such as Ras guanyl nucleotide releasing peptides (RasGRPs) (18)(19)(20). Among them, RasGRP3 promotes guanine nucleotide exchange of H-Ras, R-Ras and Rap1 exerting oncogenic effects; and its overexpression or active form phospho-RasGRP3 were observed in numerous cancer types, regulating the proliferation, survival and migration of human solid tumors and several hematologic neoplasms (14,(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP

“…27 We limited our comparison to RasGRP1, since both RasGRP1 and RasGRP3 closely resembled one another in binding affinity for aryl-DAG lactones 27 and for the DAG-indololactones in our previous study. 21 In general, none of the compounds were appreciably more potent than parent compound 1 for any of the studied proteins (Table 1). Although all compounds showed equal or stronger affinity for RasGRP1 than for PKCα, the obtained values of selectivity, ranging from 1 to 6.5-fold, indicated that none of the synthesized DAG-indololactones improved the selectivity of the parent compound 1 (22-fold in parallel measurements, Table 1).…”

Regulation of the Ras-Related Signaling Pathway by Small Molecules Containing an Indole Core Scaffold: A Potential Antitumor Therapy

“…Other studies have shown that RasGRP is a target of the anticancer drug ingenol-3-angelate (Song et al, 2013). On the basis of the interaction between cell analysis and biophysical analysis with a model membrane, Jelinek and Comin's groups synthesized DAG-indololactone analogs, wherein indole rings are linked to DAG in different positions (Garcia et al, 2014;Elhalem et al, 2017). Compound 39 (Figure 9), which has an indole substituted at the R3 position, presents excellent binding affinity and selectivity to RasGRP1 as evidenced by its ClogP value of 5.0 and selectivity for PKCα, PKCε, and RasGRP1 [the binding affinities of compounds for PKCα, PKCε, and RasGRP1 were determined in vitro by competition with bound (20-3H)phorbol 12,13-dibutyrate (PDBU) in the presence of 100 µg/mL phosphatidylserine (Elhalem et al, 2017)].…”