Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C

Protein kinase C (PKC) is a family of serine/threonine kinases that play a central role in cellular signal transduction. The second messenger diacylglycerol having two long carbon chains acts as the endogenous ligand for the PKCs. Polyphenol curcumin, the active constituent of Curcuma longa is an anti-cancer agent and modulates PKC activity. To develop curcumin derivatives as effective PKC activators, we synthesized several long chain derivatives of curcumin, characterized their absorption and fluorescence properties and studied their interaction with the activator binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. Curcumin (1) and its C16 long chain analog (4) quenched the intrinsic fluorescence of PKCδC1B, PKCεC1B and PKCθC1B in a manner similar to that of PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). The EC₅₀s of the curcumin derivatives for fluorescence quenching varied in the range of 4–11 μM, whereas, EC₅₀s for TPA varied in the range of 3–6 μM. Fluorescence emission maxima of 1 and 4 were blue shifted and the fluorescence anisotropy values were increased in the presence of the C1B domains in a manner similar to that shown by the fluorescent analog of TPA, sapintoxin-D, confirming that they were bound to the proteins. Molecular docking of 1 and 4 with novel PKC C1B revealed that both the molecules form hydrogen bonds with the protein residues. The present result shows that curcumin and its long chain derivatives bind to the C1B subdomain of novel PKCs and can be further modified structurally to improve its binding and activity.     

Alkyl cinnamates as regulator for the C1 domain of protein kinase C isoforms

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Natural product curcumin is known to interact with PKC isoforms through the C1 domain and modulate PKC activity. The reported results demonstrate that the symmetric curcumin molecule might act as two separate units during its recognition of C1 domains. To understand the importance of the two halves of curcumin in PKC binding and to develop effective PKC regulators, we synthesized a series of alkyl cinnamates (1-8), characterized absorption and fluorescence properties and measured binding affinities with the C1b subdomains of PKC isoforms. The binding parameters of the monomeric compounds and liposomes containing compounds confirmed their interaction with the C1b subdomains of PKCδ and PKCθ. The molecular docking analysis with PKCδ and PKCθ C1b subdomains revealed that the alkyl cinnamates form hydrogen bond with the backbone of the protein at the same binding site as that of diacylglycerol and phorbol esters. The results show that the alkyl cinnamates bind to the activator binding site of PKCs and both methoxy and hydroxyl groups play important roles in the binding process.     

Binding mode prediction of aplysiatoxin,a potent agonist of protein kinase C,through molecular simulation and structure–activity study on simplified analogs of the receptor-recognition domain

Aplysiatoxin (ATX) is a naturally occurring tumor promoter isolated from a sea hare and cyanobacteria. ATX binds to, and activates, protein kinase C (PKC) isozymes and shows anti-proliferative activity against human cancer cell lines. Recently, ATX has attracted attention as a lead compound for the development of novel anticancer drugs. In order to predict the binding mode between ATX and protein kinase Cδ (PKCδ) C1B domain, we carried out molecular docking simulation, atomistic molecular dynamics simulation in phospholipid membrane environment, and structure–activity study on a simple acyclic analog of ATX. These studies provided the binding model where the carbonyl group at position 27, the hydroxyl group at position 30, and the phenolic hydroxyl group at position 20 of ATX were involved in intermolecular hydrogen bonding with the PKCδ C1B domain, which would be useful for the rational design of ATX derivatives as anticancer lead compounds.     

Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.     

Cellular uptake and localization of fluorescent derivatives of phorbol ester tumor promoters

The synthetic fluorescent derivatives of 12-O-tetradecanoylphorbol-13-acetate (TPA), dansyl-TPA, dansyl-TPA-20-acetate and dansyl-TPA-13-desacetate, have ID50 values in the [3H]PDBu binding assay of 2nM, 30nM and 1000nM respectively; the ID50 value of TPA is 4nM. Dansyl-TPA is also equipotent with TPA as an activator of protein kinase C(PKC) producing half maximum stimulation at 2nM. Dansyl-TPA-13-desacetate is almost as potent as dansyl-TPA, while dansyl-TPA-20-acetate is completely inactive as an activator of PKC. The cellular uptake of these fluorescent TPA derivatives tends to parallel their activity in the [3H]PDBu binding assay. Treatment of C3H 10T1/2 cells with 100nM dansyl-TPA results in intense fluorescence of the entire cytoplasm, while the nucleus is virtually devoid of fluorescence. The uptake of fluorescence is quenched by an excess of TPA. Thus, dansyl-TPA rapidly enters cells and binds to specific sites distributed throughout the cytoplasm. Presumably these sites reflect the cellular localization of phorbol ester receptors and protein kinase C.     

Protein kinase cθ c2 domain is a phosphotyrosine binding module that plays a key role in its activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.     

Design, synthesis, biological evaluation and molecular docking of curcumin analogues as antioxidant, cyclooxygenase inhibitory and anti-inflammatory agents

Curcuminoids were isolated from Curcuma longa and their pyrazole and isoxazole analogues were synthesized and evaluated for antioxidant, COX-1/COX-2 inhibitory and anti-inflammatory activities. The designed analogues significantly enhance COX-2/COX-1 selectivity and possess significant anti-inflammatory activity in carrageenan induced rat paw edema assay. Pyrazole, isoxazole analogues of curcumin (4 and 7) exhibited higher antioxidant activity than trolox. Molecular docking study revealed the binding orientations of curcumin analogues in the active sites of COX and thereby helps to design novel potent inhibitors.     

Identification and biological activities of bryostatins from Japanese bryozoan

Six bryostatins were isolated from Japanese bryozoan by evaluating their binding to the C1B domain of protein kinase Cδ (PKCδ). Structure-activity studies of bryostatins 4, 10, and 14 suggested that the ester group at C20 was not necessary for binding to and activating PKCδ. These bryostatins showed significant anti-tumor-promoting activity in induction tests with the Epstein-Barr virus early antigen.     

Computational analyses of curcuminoid analogs against kinase domain of HER2

Background

Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, β-diketone, monoketone, pyrazole, and isoxazole.

Results

In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future.

Conclusions

The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-261) contains supplementary material, which is available to authorized users.     

Chemical modifications of resveratrol for improved protein kinase C alpha activity

Resveratrol (1) is a naturally occurring phytoalexin that affects a variety of human disease models, including cardio- and neuroprotection, immune regulation, and cancer chemoprevention. One of the possible mechanisms by which resveratrol affects these disease states is by affecting the cellular signaling network involving protein kinase C alpha (PKCα). PKCα is a member of the family of serine/threonine kinases, whose activity is inhibited by resveratrol. To study the structure-activity relationship, several monoalkoxy, dialkoxy and hydroxy analogs of resveratrol have been synthesized, tested for their cytotoxic effects on HEK293 cells, measured their effects on the membrane translocation properties of PKCα in the presence and absence of the PKC activator TPA, and studied their binding with the activator binding domain of PKCα. The analogs showed less cytotoxic effects on HEK293 cells and caused higher membrane translocation (activation) than that of resveratrol. Among all the analogs, 3, 16 and 25 showed significantly higher activation than resveratrol. Resveratrol analogs, however, inhibited phorbol ester-induced membrane translocation, and the inhibition was less than that of resveratrol. Binding studies using steady state fluorescence spectroscopy indicated that resveratrol and the analogs bind to the second cysteine-rich domain of PKCα. The molecular docking studies indicated that resveratrol and the analogs interact with the protein by forming hydrogen bonds through its hydroxyl groups. These results signify that molecules developed on a resveratrol scaffold can attenuate PKCα activity and this strategy can be used to regulate various disease states involving PKCα.     

Protein kinase C-δ negatively regulates T cell receptor-induced NF-κB activation by inhibiting the assembly of CARMA1 signalosome

T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-κB and JNK activation. In this report, we identified protein kinase Cδ (PKCδ) as a CARMA1-associated protein by a biochemical affinity purification approach. PKCδ interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKCδ inhibited CARMA1-mediated NF-κB activation, whereas knockdown of PKCδ potentiated TCR-triggered NF-κB activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKCδ kinase-dead mutant indicated that the kinase activity of PKCδ was dispensable for its ability to inhibit TCR-triggered NF-κB activation. Furthermore, we found that PKCδ inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKCθ, Bcl10, or MALT1. These observations suggest that PKCδ is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome.     

The C4 hydroxyl group of phorbol esters is not necessary for protein kinase C binding

To investigate the role of the hydroxyl group at position 4 of the phorbol esters in protein kinase C (PKC) binding and function, 4beta-deoxy-phorbol-12,13-dibutyrate (4beta-deoxy-PDBu, 5a) and 4beta-deoxy-phorbol-13-acetate (6a) were synthesized from phorbol (1). The binding affinities of these 4beta-deoxy compounds (5a, 6a) to the 13 PKC isozyme C1 domains were quite similar to those of the corresponding 4beta-hydroxy compounds (4a, 4b), suggesting that the C4 hydroxyl group of phorbol esters is not necessary for PKC binding. Moreover, functional assays showed that 4beta-deoxy-PDBu (5a) exhibited biological activities (Epstein-Barr virus induction and superoxide generation) equally potent to those of PDBu (4a). These solution phase results differ from expectations based on the previously reported solid-phase structure of the complex of PKCdelta-C1B and phorbol-13-acetate (4b).     

Recent Updates in Curcumin Pyrazole and Isoxazole Derivatives: Synthesis and Biological Application

Curcumin is an admired, plant‐derived compound that has been extensively investigated for diverse range of biological activities, but the use of this polyphenol is limited due to its instability. Chemical modifications in curcumin are reported to seize this limitation; such efforts are intensively performed to discover molecules with similar but improved stability and better properties. Focal points of these reviews are synthesis of stable pyrazole and isoxazole analogs of curcumin and application in various biological systems. This review aims to emphasize the latest evidence of curcumin pyrazole analogs as a privileged scaffold in medicinal chemistry. Manifold features of curcumin pyrazole analogs will be summarized herein, including the synthesis of novel curcumin pyrazole analogs and the evaluation of their biological properties. This review is expected to be a complete, trustworthy and critical review of the curcumin pyrazole analogs template to the medicinal chemistry community.     

Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0–4.5. On the other hand, an induction test with Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.     

Curcumin suppresses the TPA-induced invasion through inhibition of PKCα-dependent MMP-expression in MCF-7 human breast cancer cells

Curcumin (diferuloylmethane) is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. Although anti-carcinogenic, anti-oxidant, anti-inflammation, and anti-angiogenic properties have been reported, the effect of curcumin on breast cancer metastasis is unknown. Matrix metalloproteinase-9 (MMP-9) is a major component in cancer cell invasion. In this study, we investigated the inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion and the molecular mechanisms involved in MCF-7 cells. Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Also, curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that curcumin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells. Curcumin may have potential value in restricting breast cancer metastasis.