Sukhamoy Gorai

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.

Physicochemical characterization of diacyltetrol-based lipids consisting of both diacylglycerol and phospholipid headgroups

We describe the synthesis of diacyltetrol-based hybrid lipids in which one of the hydroxymethyl groups is modified with an anionic phospholipid headgroup. The hybrid lipids form a monolayer at the air–water interface. In aqueous solution, these lipids form stable liposomes that exhibit a negative surface potential across a wide pH range. The liposomes aggregate in the presence of Ca2+ ions and release encapsulated cationic reporter rhodamine 6G (R6G) at a faster rate than anionic reporter carboxyfluorescein (CF). The hybrid lipids strongly interact with the C1b subdomain of the protein kinase C (PKC)-θ isoform. These new lipids structurally mimic diacylglycerol and conventional phospholipids, and provide an opportunity to explore their physicochemical properties.

Insights into the inhibitory mechanism of triazole-based small molecules on phosphatidylinositol-4,5-bisphosphate binding pleckstrin homology domain

Background Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P2 through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P2/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways. Methods Surface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used. Results In this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P2/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC50 ranges from 53 to 159 nM for PI(4,5)P2/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], PI(4,5)P2 specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells. Conclusions Overall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment. General significance These results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.

Bilayer interaction and protein kinase C-C1 domain binding studies of kojic acid esters

Development of protein kinase C (PKC) regulators has been considered as an attractive therapeutic strategy for the treatment of cancer and other diseases. Extensive efforts are underway to synthesize PKC regulators targeted to the DAG-responsive C1 domain. Investigation of physicochemical properties of the synthesized molecules also is essential for the development of PKC-C1 domain ligands. To develop PKC regulators, we conveniently synthesized kojic acid esters targeted to the DAG/phorbol ester binding site within the C1 domain. Physicochemical studies showed that the kojic acid esters aggregate in aqueous solution at reasonably lower concentration. The results also showed that the compounds strongly interact with the lipid bilayer and the hydrophilic part of the compound localize at the bilayer/water interface. In vitro protein binding studies and molecular docking analysis revealed that the hydroxymethyl group, carbonyl groups and acyl chain length are important for their interaction with the C1 domain. The potent compound showed more than 10-fold stronger binding affinity for the C1 domain than DAG. In addition to the diverse application of kojic acid esters in food, cosmetic and skin-health industries, these findings reveal that ester analogues represent an attractive group of C1-domain ligands that can be further structurally modified to improve their binding and activity.

Evidence of PKC Binding and Translocation to Explain the Anticancer Mechanism of Chlorogenic Acid in Breast Cancer Cells

Chlorogenic acid (CGA) exhibits potentials towards liver, breast and skin cancer. Cancer cells stimulated with CGA exhibits differential expression of transcriptional factors and regulatory molecules but the molecular target of the molecule is not known. Superposition of biophoric elements of CGA with Curcumin gives maximum common substructure score of 0.90. Molecular modeling studies further suggest that CGA fits into the C1b domain of PKC with extensive interaction with residues lining binding site. It binds PKC in a concentration dependent manner with dissociation constant KD, 28.84±3.95 μM. PKC-CGA complex is stable with minimal distortion to the 3-D structure and maintains the hydrogen bonding between ligand and receptor during simulation period. Cells stimulated with CGA causes 12.1 ± 0.56% PKC translocation from the cytosol to the plasma membrane. It disturbs the cell cycle and arrest the cancer cell at the G1 phase with a reduction in S-phase. Chlorogenic acid exhibits killing of cancer cells in a dose-dependent manner with an IC50 of 75.88 ± 4.54μg/ml and 52.5 ± 4.72μg/ml towards MDAMB-231 and MCF-7 cells respectively. It induces apoptosis in cancer cells as evident by AO/EtBr staining and degradation of genomic DNA to give a laddering pattern. Apoptosis in cancer cells involves mitochondrial pathway as supported by a reduction in mitochondrial potentials and release of cyt-C into the cytosol. Hence, the current study has established PKC as an important signaling molecule to the observed anti-cancer effects of CGA and provides the impetus to design better CGA analogs for improved anti-cancer potential against the malignant tumor.

Inhibition of phosphatidylinositol-3,4,5-trisphosphate binding to the AKT pleckstrin homology domain by 4-amino-1,2,5-oxadiazole derivatives

Aberrant regulation of phosphatidylinositol-3-kinase (PI3K)-dependent cell signalling pathways is directly linked with different human cancers. AKT is a phosphatidylinositol (PIP) binding serine/threonine kinase enzyme and a key component of PI3K signalling pathways. Several efforts have been made to perturb the activities of AKT enzymes; however, the selectivity issue remains a key challenging point. The pleckstrin homology (PH) domain of AKT strongly interacts with the membrane-localized PIPs and activates the enzyme. Several attempts are underway for the development of small molecules, which can specifically inhibit the PIP/PH domain interaction to ascertain an alternative approach for the development of drug(s) for PI3K–AKT signalling pathways. This proposed approach is highly beneficial because of easy synthesis of small molecules and low side effects. In this study, we used a series of small molecule antagonists (PIs) for PI(3,4,5)P3/PH domain interaction and determined their inhibitory effect by performing competitive surface plasmon resonance (SPR) analysis (IC50 ranges from 1.85 to 16.35 μM for the PI(3,4,5)P3/AKT1-PH domain binding assay). To elucidate their binding selectivity, we also used PI(3,4,5)P3, PI(3,4)P2, and PI(4,5)P2 specific PH domains. To further understand their PH domain inhibition mechanism, we also performed various physicochemical analyses. The results showed that these water-soluble compounds do not significantly interact with the model membranes. The oxadiazole and N′-hydroxy moieties of the compounds are essential for their exothermic interaction with the PH domains and their binding does not alter the secondary structure of the PH domain. Potent compounds show a moderate effect on the phosphothreonine (308) level of the AKT enzyme. Overall, our studies demonstrate that these compounds could interact with the PIP-binding PH domains and inhibit their membrane recruitment. These studies also illustrate the feasibility of further development of 4-amino-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide moiety-based small molecule antagonists for PI3K signalling pathways.

Biochemical Studies and Virtual Screening of Phytochemical Reservoir from Northeastern Indian Plants to Identify Anti-Cancer Agents

Abstract In the present study, medicinal phytochemicals found in plants of north-eastern India were virtually screened against the C1b regulatory domain of Protein Kinase C (PKC), an important enzyme in cancer cell-biology. Amongst the few top hits, the molecules Gallic acid (GA) and Epigallocatechin gallate (EGCG) were identified through an agonist competition assay. Both phytochemical are non-toxic and non-mutagenic. An in vitro PKC binding assay confirmed that GA and EGCG were strong ligands of PKC. Both the phytochemicals formed numerous hydrogen bonding and hydrophobic interactions with the amino acids lining the ligand binding pocket of the C1b domain of PKC. They were able to induce translocation of PKC to the plasma membrane in MDAMB-231 breast cancer cells. Furthermore, GA and EGCG dose-dependently reduced the viability of breast cancer cells by arresting the cell-cycle. Eventually, mitochondrial pathway of apoptosis was identified as the mechanism of loss of breast cancer cellular viability. Thus, our study has revealed that tea phytochemicals GA and EGCG exhibit anti-cancer activity by acting as strong ligands of PKC. This study encourages the further identification of newer novel and even more potent such PKC-directed phytochemicals from natural herbs & beverages as a part of herbal anti-cancer therapy.