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

doi:10.1016/j.ejmech.2014.11.025

European Journal of Medicinal Chemistry

Volume 90, 27 January 2015, Pages 332-341

https://doi.org/10.1016/j.ejmech.2014.11.025 Get rights and content

Highlights

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DAG-lactones with linoleic acid derivatives showed modest selectivity for PKCε.
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DAG-lactones with branched chain showed the best selectivity for PKCε up to 32-fold.
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In nuclear membrane, the selectivity for PKCε versus PKCα was enhanced.

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.

Graphical abstract

Introduction

Protein kinase Cs (PKCs) represent a family of serine/threonine kinases which are central signaling elements downstream of the numerous cellular receptors that are coupled to phospholipase C activation [1]. PKCs display complex regulation, with membrane phospholipids and diacylglycerol being critical regulators and elevated Ca²⁺ additionally being important for the classical PKCs. PKC isoforms divide into three groups; classical PKCs (cPKCs: PKCα, PKCβI, PKCβII and PKCγ), novel PKCs (nPKCs: PKCδ, PKCε, PKCη and PKCθ) and atypical PKCs (aPKCs: PKCξ, PKCι/λ), distinguished by their Ca²⁺ dependency and cofactors for activation [2], [3]. Additionally, individual isozymes vary in their patterns of tissue distribution, subcellular localization, substrate specificity, and biological function [4], [5]. For example, different isoforms may be growth promoting or growth inhibitory, with stimulation of one isoform being functionally antagonistic of another [6]. Although the high level of conservation of the regulatory domains of PKC isoforms poses a challenge for the development of isoform selective ligands, the complexity of their regulation also provides promising opportunities [7].

Among the PKC isozymes, particular attention has focused on protein kinase C epsilon (PKC-ε), one of the calcium-independent but phorbol ester/diacylglycerol dependent novel PKC isoforms. Structurally, it contains an N-terminal regulatory domain and C-terminal kinase domain composed on four conserved (C1–C4) and five variable (V1–V5) regions. The regulatory domain contains a pseudosubstrate domain which inhibits PKC activity before activation, a tandem repeat of C1 domain zinc finger structures that bind diacylglycerol (DAG) and phorbol ester, and a C2-like domain which contributes to membrane interaction [8], [9]. Additionally, PKC-ε possesses a unique actin-binding site [10] between the first and second cysteine rich C1-domains which promotes association with actin filaments in intact cells. Due to its specific tissue distribution and localization pattern, it has been reported that PKC-ε has critical roles in both the cardiovascular and central nervous system [11] such as cardioprotection [12], neurite outgrowth [13], synapse formation, neurotransmitter release, synaptic loss prevention [14], and sensitization to pain through TRPV1 [15] (see Fig. 1).

As an initial effort to design and develop PKC-ε selective ligands, our attention was drawn to reports that saturated and unsaturated fatty acids may influence PKC-ε activity. Recently, Nishizaki and co-workers described that a linoleic acid derivative with cyclopropane rings replacing the cis-double bonds, viz. 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA, Fig. 2. Compound 5) acted as a selective and direct activator of PKC-ε [16]. In biological studies, DCP-LA effectively prevented both deposition of amyloid plaques and loss of synaptic connections [17].

Previously, we have demonstrated that diacylglycerol lactones (DAG-lactones), which are rigidified structures derived from the endogenous PKC ligand DAG, function as DAG analogs to potently bind to the regulatory C1 domain of PKC and cause PKC activation (Fig. 1) [18]. For this binding, both the acyl (R₁) and α-alkylidene (R₂) positions are critical elements in controlling biological activity and PKC isozyme selectivity [19]. Modeling reveals that there are two different modes of binding, involving interaction between the C1 domain and either the sn-1 carbonyl or the sn-2 carbonyl. The alkyl chain which is not involved in hydrogen bonding to the binding cleft of the C1 domain contributes to the binding energy through its interactions with the C1 domain surface and the lipid bilayer.

In this study, we have evaluated whether the incorporation of linoleic and DCP-LA derivatives into the side chains of DAG-lactones would provide combined structures with enhanced selectivity for PKC-ε. The newly designed linoleic acid derivatives, incorporating cyclo-saturated (2, 5), linear-unsaturated (10, 12), linear-saturated (11), and branched alkyl chains (13–15), were alternatively attached to the carbonyl group for acyl branching (R₁) or connected to the lactone via a methylene group for α-alkylidene branching (R₂). Novel features of the study include both the specific class of DAG-lactones themselves as well as their analysis, directed at comparing selectivities for the novel PKC-ε isoform relative to that for the classical PKC-α isoform.

Section snippets

Chemistry

As illustrated in Fig. 2, three branched groups as well as linoleic acid and five different linoleic acid derivatives in which the cis-double bonds were replaced by other groups were introduced as R₁ and R₂ side chains. Commercially available linoleic acid (1), stearic acid (11), octadec-9-ynoic acid (12), and the branched compounds pivaloyl chloride (13) and isovaleraldehyde (15) were directly used for alkylation. Among the branched-substituents, 3-isobutyl-5-methylhexanoyl chloride (14) [20]

Conclusion

In conclusion, different types of alkyl chains derived from linoleic acid were introduced as substituents for the DAG-lactone and their effects on selectivity for PKC-ε relative to PKC-α was explored. Because cells contain a variety of membranes with different compositions, we also assessed the influence of membrane lipid contribution on the measured isozyme selectivity. Most of the compounds showed at least modest PKC-ε selectivity with a number approaching 5-fold in the presence of the

Chemistry

All chemical reagents were commercially available. Silica gel column chromatography was performed on silica gel 60, 230–400 mesh, Merck. Nuclear magnetic resonance (1H NMR) spectra were recorded on a JEOL JNM-LA 300 and a Bruker Avance 400 MHz FT-NMR spectrometer. Chemical shifts are reported in ppm units with Me₄Si as a reference standard. Mass spectra were recorded on a VG Trio-2 GC-MS and a 6460 Triple Quad LC/MS. Elemental analyses were performed with an EA 1110 Automatic Elemental

Acknowledgments

This research was supported by research grants from the National Research Foundation of Korea (R11-2007-107-02001-0) and by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, NCI (Project Z1A BC 005270).

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Based on the experiments conducted on isolated C1Bδ with modified DAG lactones in the presence/absence of PtdSer membranes, it is proposed that the role of unbound carbonyl group of DAG lactones (i.e. “sn-1”) could be to interact with phospholipids (Benzaria et al., 1998; Kang et al., 2005). The synthetic accessibility of DAG lactones enabled their modifications to include a variety of hydrophobic groups (Choi et al., 2003; Lee et al., 2001; Lee et al., 2004; Lee et al., 1996a; 1996b; Nomura et al., 2011; Ohashi et al., 2017), as a route to achieve isoenzyme-specificity (Ann et al., 2015; Cooke et al., 2018; Garcia-Bermejo et al., 2002; Pu et al., 2005). For instance, the synthetic DAG lactone AJH-836 (Fig. 3A) preferentially binds, activates, and selectively down-regulates novel PKC isoenzymes δ and ε (Cooke et al., 2018).
Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation. Despite significant progress, the rational design of the C1 modulators remains challenging due to difficulties associated with structure determination of the C1-ligand complexes. Given the dearth of experimental structural data, computationally derived models have been instrumental in providing atomistic insight into the interactions of the C1 domains with PKC agonists. In this review, we provide an overview of the in silico approaches for seven classes of C1 modulators and outline promising future directions.
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Characterization of AJH-836, a diacylglycerol-lactone with selectivity for novel PKC isozymes
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The differential binding of AJH-836 to PKCs may be the consequence of different scenarios, one of the obvious being the preferential association between the ligand and nPKC C1 domains dictated by specific residues interacting with the DAG-lactone backbone and/or alkyl chains in the appropriate binding modes. It is probable that specific “ternary complex” interactions that involve membrane phospholipids may contribute to AJH-836 isozyme specificity as well, a setting that is consistent with the dependence of its binding affinity on the membrane composition (26). Although dissecting the individual structural features contributing to the pattern of PKC isoform selectivity will depend on extensive structural analysis, the current data make unambiguously clear that it is possible to generate differential PKC isoform selectivity such as described here along with differential biology.
Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide–releasing proteins (RasGRPs), and Munc13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [³H]phorbol 12,13-dibutyrate ([³H]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKCδ and PKC∈ relative to classical PKCα and PKCβII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKC∈ to the plasma membrane relative to PKCα. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKCδ and PKC∈ without affecting PKCα expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 domain ligands that promote novel biological response patterns.
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Original articleDesign and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones)

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Conclusion

Chemistry

Acknowledgments

J. Biol. Chem.

Curr. Opin. Cell Biol.

J. Lipid Res.

Bioorg. Med. Lett.

Conformationally constrained analogues of diacylglycerol (DAG). 16. How much structural complexity is necessary for recognition and high binding affinity to protein kinase C?

J. Med. Chem.

Intracellular targeting of protein kinase C isozymes: functional implications

Biochem. Soc. Trans.

Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities

J. Med. Chem.

Protein kinase C and lipid signaling for sustained cellular responses

FASEB J.

Protein kinase C and other diacylglycerol effectors in Cancer

Nat. Rev. Cancer

Wealth of opportunity – the C1 domain as a target for drug development

Curr. Drug Targets

The role of C2 domains in PKC signaling

Adv. Exp. Med. Biol.

Protein kinase c-ε (PKC-ε): Its unique structure and function

J. Biochem.

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