Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones)

Abstract

DAG-lactones afford a synthetically accessible, high affinity platform for probing structure activity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role of PKC isoforms in cellular signaling, along with their differential biological activities, a critical objective is the design of isoform selective ligands. Here, we report the synthesis of a series of DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives. We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid conditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold.

Characterization of AJH-836, a diacylglycerol-lactone with selectivity for novel PKC isozymes

“…Extensive synthetic efforts have generated DAG-lactones substituted with a diversity of saturated and unsaturated alkyl and aryl chains at the sn-1 and sn-2 positions on the DAGlactone (21,26). In binding studies using a lipid mixture designed to mimic nuclear membranes (23), the DAG-lactone (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5oxotetrahydrofuran-2-yl)methyl pivalate (referred to here as AJH-836; Fig.…”

“…In binding studies using a lipid mixture designed to mimic nuclear membranes (23), the DAG-lactone (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5oxotetrahydrofuran-2-yl)methyl pivalate (referred to here as AJH-836; Fig. 1A) was found to display some specificity toward the novel PKC⑀ relative to the classical PKC␣, although the assay conditions were not fully comparable (26). For comparison, no specificity was observed under these conditions for the DAG-lactone (Z)-(2-(hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl octadeca-9,11-diynoate (referred to here as AJH-1512; Fig.…”

“…It has been previously reported that natural DAGs bind with similar affinities to all cPKCs and nPKCs (25). Toward the objective of generating isozyme-specific PKC ligands, Ann et al (26) had described the incorporation of linoleic acid derivatives as well as saturated and unsaturated alkyl chains into the side chains of DAG-lactones and characterized these derivatives for their in vitro binding affinity toward PKC⑀, an oncogenic member of the PKC family, relative to PKC␣ (27)(28)(29)(30)(31)(32). Following up on this analysis, here we describe that the DAG-lactone AJH-836 showed marked selectivity for PKC⑀ and PKC␦ relative to PKC␣ in multiple cell types, unlike the typical phorbol ester PMA, and correspondingly displayed a distinct pattern of biological activity.…”

Protein kinase C in cancer: The top five unanswered questions

“…The rationale behind this approach is that the low binding affinity of DAG relative to phorbol esters was attributable to the flexibility of the glycerol backbone, thus cyclization of the structure would reduce the entropic penalty associated with DAG binding. By modifying DAG lactones through incorporation of different branched hydrophobic chains it has been possible to generate ligands with preferential affinity for discrete PKCs or “non‐PKC” phorbol ester receptors (see below) . Although DAG lactones have not yet been fully characterized in a physiological setting, the expectation is that they could be rationally designed to dissect isozyme‐specific responses.…”

“…66,67 One PKCs or "non-PKC" phorbol ester receptors (see below). [74][75][76][77] Although DAG lactones have not yet been fully characterized in a physiological setting, the expectation is that they could be rationally designed to dissect isozyme-specific responses.…”