C1 Domain Of Protein Kinase C Research Articles

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists.

Protein kinase C (PKC) modulators hold therapeutic potential for various diseases, including cancer, heart failure, and Alzheimer's disease. Targeting the C1 domain of PKC represents a promising strategy; the available protein structures warrant the design of PKC-targeted ligands via a structure-based approach. However, the PKC C1 domain penetrates the lipid membrane during binding, complicating the design of drug candidates. The standard docking-scoring approach for PKC lacks information regarding the dynamics and the membrane environment. Molecular dynamics (MD) simulations with PKC, ligands, and membranes have been used to address these shortcomings. Previously, we observed that less computationally intensive simulations of just ligand-membrane interactions may help elucidate C1 domain-binding prospects. Here, we present the design, synthesis, and biological evaluation of new pyridine-based PKC agonists implementing an enhanced workflow with ligand-membrane MD simulations. This workflow holds promise to expand the approach in drug design for ligands targeted to weakly membrane-associated proteins.

Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.

Analysis of binding mode of vibsanin A with protein kinase C C1 domains: An experimental and molecular dynamics simulation study

Vibsanin A (VibA), isolated from Viburnum odoratissimum, binds to and activates protein kinase C (PKC) isozymes to induce leukemia cell differentiation. VibA is a promising seed compound for developing PKC-activating drugs because it exhibits anti-proinflammatory activity, atypical to PKC activators. However, the role of hydrogen bond-forming functional groups and the precise binding mode with C1 domains in PKC isozymes remains unknown. In this study, we evaluated the PKC-binding ability of natural vibsanins and synthetic 1′-desoxo-VibA (1) and performed molecular dynamics simulation of membrane/C1 domain-bound VibA to predict the binding mode of VibA with C1 domains. Experimental and simulation results indicated that the ester group in VibA is involved in CH–O hydrogen bonds with PKC C1 domains. Alternatively, a growth-inhibition assay against leukemia cells revealed that the ester group of VibA negatively affects antiproliferative activity.

Chiral 2-phenyl-3-hydroxypropyl esters as PKC-alpha modulators: HPLC enantioseparation, NMR absolute configuration assignment, and molecular docking studies.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. In our previous work, we identified in racemate 1-2, based on the 2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα and PKCδ ligands, targeting the C1 domain of these two kinases. Herein, we report the resolution of the racemates by enantioselective semi-preparative HPLC. The attribution of the absolute configuration (AC) of homochirals 1 was performed by NMR, via methoxy-α-trifluoromethyl-α-phenylacetic acid derivatization (MTPA or Mosher's acid). Moreover, the match between the experimental and predicted electronic circular dichroism (ECD) spectra confirmed the assigned AC. These results proved that Mosher's esters can be properly exploited for the determination of the AC also for chiral primary alcohols. Lastly, homochiral 1 and 2 were assessed for binding affinity and functional activity against PKCα. No significative differences in the Ki of the enantiopure compounds was observed, thus suggesting that chirality does not seem to play a significant role in targeting PKC C1 domain. These results are in accordance with the molecular docking studies performed using a new homology model for the human PKCαC1B domain.

Rigorous Computational Study Reveals What Docking Overlooks: Double Trouble from Membrane Association in Protein Kinase C Modulators

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-1gP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membrane-associated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Docking and Virtual Screening to Identify PKC Agonists: Potentials in Anticancer Therapeutics

Protein kinase C (PKC) is down-stream to most of the G-protein coupled receptor or tyrosine kinase receptors mediated signaling events from the cell surface. PKC C1 domain has a hydrophobic region with a polar groove to facilitate 1,2-diacyl-glycerol (DAG) binding or other agonist molecule for PKC activation. Post activation, a partial or complete blocking of hydrophilic groove makes the DAG binding site completely hydrophobic and facilitates easier penetration of the PKC into the membrane. Phorbol ester, a strong PKC agonist, uses this mechanism to induce tumor formation. A total of 300 heterocyclic compounds with 70% similarity to phorbol 12-myristate 13-acetate (PMA) were selected, and virtual docking was performed with PKC-α as target. An initial screening indicated that most of the molecules fit well into the C1 domain and had better binding energy than PMA. Further analysis in a PMA competition experiment identified five molecules, Zc 67913417, Zc 68601770, Zc 25726447, Zc 35376386 and Zc 49785214 as potent PKC agonists. In addition, as these compounds showed better binding than PMA, more interaction with PKC residues (hydrogen bonding and hydrophobic), and the top five hit molecules was potent enough to abolish carcinogenic effects of PMA. Searching the top heterocyclic compounds into the drug database gave a number of approved drugs. Testing two candidate drugs, nandrolone decanoate and budesonide, reduced cellular viability of HT1080 in a dose-dependent manner with an IC50 values of 96.8 nM and 200nM respectively. An in silico toxicity analysis indicated that top hit molecules are non-toxic, non-mutagenic in cellular and bacterial system, and have no tumorigenic potentials in a single cell or animal model. Hence, a virtual screening, agonist competition assay, and in silico toxicity assessment allowed us to identify five new PKC agonist molecules for future drug discovery against cancer.

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.

C1 Domain-Targeted Isophthalate Derivatives Induce Cell Elongation and Cell Cycle Arrest in HeLa Cells

Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated.

Differential Regulation of Gene Expression by Protein Kinase C Isozymes as Determined by Genome-wide Expression Analysis

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCε, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCε predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

Lipid binding domains: more than simple lipid effectors

The spatial and temporal regulation of lipid molecules in cell membranes is a hallmark of cellular signaling and membrane trafficking events. Lipid-mediated targeting provides for strict control and versatility, because cell membranes harbor a large number of lipid molecules with variation in head group and acyl chain structures. Signaling and trafficking proteins contain a large number of modular domains that exhibit specific lipid binding properties and play a critical role in their localization and function. Nearly 20 years of research including structural, computational, biochemical and biophysical studies have demonstrated how these lipid-binding domains recognize their target lipid and achieve subcellular localization. The integration of this individual lipid-binding domain data in the context of the full-length proteins, macromolecular signaling complexes, and the lipidome is only beginning to be unraveled and represents a target of therapeutic development. This review brings together recent findings and classical concepts to concisely summarize the lipid-binding domain field while illustrating where the field is headed and how the gaps may be filled in with new technologies.

Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/π interaction

Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC) isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed. Focusing on our recent finding that the indole ring of IL-V is involved in the CH/π interaction with Pro-11 of the PKCδ-C1B domain, we developed new fluorescent probes ( 2– 4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substituent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent intensity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence intensity and emission maxima of 4 changed significantly when bound to the PKCδ-C1B peptide in both the presence and absence of phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with PKC C1 domains.

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.

A Single Residue in the C1 Domain Sensitizes Novel Protein Kinase C Isoforms to Cellular Diacylglycerol Production

The C1 domain mediates the diacylglycerol (DAG)-dependent translocation of conventional and novel protein kinase C (PKC) isoforms. In novel PKC isoforms (nPKCs), this domain binds membranes with sufficiently high affinity to recruit nPKCs to membranes in the absence of any other targeting mechanism. In conventional PKC (cPKC) isoforms, however, the affinity of the C1 domain for DAG is two orders of magnitude lower, necessitating the coordinated binding of the C1 domain and a Ca2+-regulated C2 domain for translocation and activation. Here we identify a single residue that tunes the affinity of the C1b domain for DAG- (but not phorbol ester-) containing membranes. This residue is invariant as Tyr in the C1b domain of cPKCs and invariant as Trp in all other PKC C1 domains. Binding studies using model membranes, as well as live cell imaging studies of yellow fluorescent protein-tagged C1 domains, reveal that Trp versus Tyr toggles the C1 domain between a species with sufficiently high affinity to respond to agonist-produced DAG to one that is unable to respond to physiological levels of DAG. In addition, we show that while Tyr at this switch position causes cytosolic localization of the C1 domain under unstimulated conditions, Trp targets these domains to the Golgi, likely due to basal levels of DAG at this region. Thus, Trp versus Tyr at this key position in the C1 domain controls both the membrane affinity and localization of PKC. The finding that a single residue controls the affinity of the C1 domain for DAG-containing membranes provides a molecular explanation for why 1) DAG alone is sufficient to activate nPKCs but not cPKCs and 2) nPKCs target to the Golgi.

Effects on Ligand Interaction and Membrane Translocation of the Positively Charged Arginine Residues Situated along the C1 Domain Binding Cleft in the Atypical Protein Kinase C Isoforms

The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKCdelta abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the deltaC1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs.

Conformationally Constrained Analogues of Diacylglycerol. 26. Exploring the Chemical Space Surrounding the C1 Domain of Protein Kinase C with DAG-Lactones Containing Aryl Groups at the sn-1 and sn-2 Positions

Diacylglycerol lactones (DAG-lactones) are known to operate as effective agonists of protein kinase C (PKC), surpassing in potency the activity of natural diacylglycerol (DAG). Localization of activated PKC isozymes in the cell is determined in part by the different cellular scaffolds, the lipid composition of the specific membranes, and the targeting information intrinsic to the individual isoforms bound to DAG. This multifaceted control of diversity suggests that, to develop effective DAG-lactones capable of honing in on a specific cellular target, we need to gain a better understanding of the chemical space surrounding its binding site. Seeking to augment the chemical repertoire of DAG-lactone side chains that could steer the translocation of PKC to specific cellular domains, we report herein the effects of incorporating simple or substituted phenyl residues. A combined series of n-alkyl and phenyl substitutions were used to explore the optimal location of the phenyl group on the side chains. The substantial differences in binding affinity between DAG-lactones with identical functionalized phenyl groups at either the sn-1 or sn-2 position are consistent with the proposed binding model in which the DAG-lactone binds to the C1 domain of PKC with the acyl chain oriented toward the interior of the membrane and the alpha-alkylidene or alpha-arylalkylidene chains directed to the surface of the C1 domain adjacent to the lipid interface. We conclude that DAG-lactones containing alpha-phenylalkylidene side chains at the sn-2 position represent excellent scaffolds upon which to explore further chemical diversity.

Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase Cβ

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.

Conformationally constrained analogues of diacylglycerol (DAG). 23. Hydrophobic ligand-protein interactions versus ligand-lipid interactions of DAG-lactones with protein kinase C (PK-C).

The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C (PK-C) with affinities that approximate those of phorbol esters. These branched chains not only appear to be involved in making important hydrophobic contacts with the protein (specific interactions) but also provide adequate lipophilicity to facilitate partitioning into the lipid-rich membrane environment (nonspecific interactions). With the idea of minimizing the nonspecific interactions without reducing lipophilicity, the present work explores the strategy of relocating lipophilicity from the side chain to the lactone "core". Such a transfer of lipophilicity, exemplified by compounds 1 and 3, was conceived to allow the new hydrophobic groups on the lactone to engage in specific hydrophobic contacts inside the binding pocket without any expectation of interfering with the hydrogen-bonding network of the DAG-lactone pharmacophore. Surprisingly, both (E)-3 and (Z)-3 showed a significant decrease in binding affinity. From the molecular docking studies performed with the new ligands, we conclude that the binding pocket of the C1 domain of PK-C is sterically restricted and prevents the methyl groups at the C-3 position of the lactone from engaging in productive hydrophobic contacts with the receptor.

Indolactam and benzolactam compounds as new medicinal leads with binding selectivity for C1 domains of protein kinase C isozymes.

Protein kinase C (PKC) isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta) are major receptors of tumor promoters and also play a crucial role in cellular signal transduction via the second messenger, 1,2-diacyl-sn-glycerol (DG). Each isozyme of PKC is involved in diverse biological events, indicating that it serves as a novel therapeutic target. Since PKC isozymes contain two possible binding sites of tumor promoters and DG (C1A and C1B domains), the design of agents with binding selectivity for individual PKC C1 domains is a pressing need. We developed a synthetic C1 peptide library of all PKC isozymes for high-throughput screening of new ligands with such binding selectivity. This peptide library enabled us to determine that indolactam and benzolactam compounds bound to the C1B domains of novel PKC isozymes (delta, epsilon, eta, theta) in some selective manner, unlike phorbol esters and DG. Simpler in structure and higher in stability than the other potent tumor promoters, a number of indolactam and benzolactam derivatives have been synthesized to develop new PKC isozyme modulators by several groups. We focused on the amide function of these compounds because recent investigations revealed that both the amide hydrogen and carbonyl oxygen of indolactam-V (ILV) are involved in hydrogen bonding with the C1B domains of PKCdelta. Synthesis of several conformationally fixed analogues of ILV led to the conclusion that the trans-amide restricted analogues with a hydrophobic chain at an appropriate position (2,7) are promising leads with a high binding selectivity for novel PKC isozyme C1B domains. We also developed a new lactone analogue of benzolactam-V8 (17) which shows significant binding selectivity for the C1B domains of PKCepsilon and PKCeta. Furthermore, our synthetic approach with the PKC C1 homology domains clarified that diacylglycerol kinase beta and gamma are new targets of phorbol esters.

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes: DGKγ AND DGKβ ARE NEW TARGETS OF TUMOR-PROMOTING PHORBOL ESTERS

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.