Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specifc modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic eforts had recently led to the identifcation of AJH-836, a DAG-lactone with preferential afnity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an efect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics eforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identifed major diferences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the diferential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of diferentially regulating PKC isozyme-specifc function in cellular models.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKC delta) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins. Protein kinase Cs (PKCs) define a central DAG-sensing node in intracellular phosphoinositide signaling pathways that regulate cell growth, differentiation, apoptosis, and motility. The structures of PKC C1 domain complexes with DAG and 4 agonists reveal the molecular basis of ligand recognition and capture.N

Discovery of Novel Dual Adenosine A(2A) and A(1) Receptor Antagonists with 1H-Pyrazolo[3,4-d]pyrimidin-6-amine Core Scaffold as Anti-Parkinson's Disease Agents

New compounds with 1H-pyrazolo [3,4-d]pyrimidin-6-amine core scaffolds were synthesized and characterized in vitro to determine their affinity for human A(2A) and A(1) receptors. Among the tested compounds, a few compounds displayed nanomolar binding affinities for both receptors. One particular compound, 11o, showed high binding activities (hA(2A) K-i = 13.3 nM; hA(1) K-i = 55 nM) and full antagonism (hA(2A) IC50 = 136 nM; hA(1) IC50 = 98.8 nM) toward both receptors. Further tests showed that 11o has low hepatic clearance and good pharmacokinetic properties in mice, along with high bioavailability and a high brain plasma ratio. In addition, 11o was associated with very low cardiovascular risk and mutagenic potential, and was well-tolerated in rats and dogs. When tested in an MPTP-induced mouse model of Parkinson's disease, 11o tended to improve behavior. Moreover, 11o dose-dependently reversed haloperidol-induced catalepsy in female rats, with graded ED50 of between 3 and 10 mg/kg. Taken together, these results suggest that this potent dual A(2A)/A(1) receptor antagonist, 11o, is a good candidate for the treatment of Parkinson's disease with an excellent metabolic and safety profile.N

Functional Group-Dependent Induction of Astrocytogenesis and Neurogenesis by Flavone Derivatives

Neural stem cells (NSCs) differentiate into multiple cell types, including neurons, astrocytes, and oligodendrocytes, and provide an excellent platform to screen drugs against neurodegenerative diseases. Flavonoids exert a wide range of biological functions on several cell types and affect the fate of NSCs. In the present study, we investigated whether the structure-activity relationships of flavone derivatives influence NSC differentiation. As previously reported, we observed that PD98059 (2′-amino-3′-methoxy-flavone), compound 2 (3′-methoxy-flavone) induced astrocytogenesis. In the present study, we showed that compound 3 (2′-hydroxy-3′-methoxy-flavone), containing a 3′-methoxy group, and a non-bulky group at C2′ and C4′, induced astrocytogenesis through JAK-STAT3 signaling pathway. However, compound 1 and 7–12 without the methoxy group did not show such effects. Interestingly, the compounds 4 (2′,3′-dimethoxyflavone), 5 (2′-N-phenylacetamido-3′-methoxy-flavone), and 6 (3′,4′-dimethoxyflavone) containing 3′-methoxy could not promote astrocytic differentiation, suggesting that both the methoxy groups at C3′ and non-bulky group at C2′ and C4′ are required for the induction of astrocytogenesis. Notably, compound 6 promoted neuronal differentiation, whereas its 4′-demethoxylated analog, compound 2, repressed neurogenesis, suggesting an essential role of the methoxy group at C4′ in neurogenesis. These findings revealed that subtle structural changes of flavone derivatives have pronounced effects on NSC differentiation and can guide to design and develop novel flavone chemicals targeting NSCs fate regulation

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Abstract Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models

α‑Arylidene Diacylglycerol-Lactones (DAG-Lactones) as Selective Ras Guanine-Releasing Protein 3 (RasGRP3) Ligands

Diacylglycerol-lactones have proven to be a powerful template for the design of potent ligands targeting C1 domains, the recognition motif for the cellular second messenger diacylglycerol. A major objective has been to better understand the structure activity relations distinguishing the seven families of signaling proteins that contain such domains, of which the protein kinase C (PKC) and RasGRP families are of particular interest. Here, we synthesize a series of aryl- and alkyl-substituted diacylglycerol-lactones and probe their relative selectivities for RasGRP3 versus PKC. Compound <b>96</b> showed 73-fold selectivity relative to PKCα and 45-fold selectivity relative to PKCε for in vitro binding activity. Likewise, in intact cells, compound <b>96</b> induced Ras activation, a downstream response to RasGRP stimulation, with 8–29 fold selectivity relative to PKCδ S299 phosphorylation, a measure of PKCδ stimulation

Novel Radiolabeled Vanilloid with Enhanced Specificity for Human Transient Receptor Potential Vanilloid 1 (TRPV1)

Transient receptor potential vanilloid 1 (TRPV1) has emerged as a promising therapeutic target. While radiolabeled resiniferatoxin (RTX) has provided a powerful tool for characterization of vanilloid binding to TRPV1, TRPV1 shows 20-fold weaker binding to the human TRPV1 than to the rodent TRPV1. We now describe a tritium radiolabeled synthetic vanilloid antagonist, 1-((2-(4-(methyl-[³H])­piperidin-1-yl-4-[³H])-6-(trifluoromethyl)­pyridin-3-yl)­methyl)-3-(3-oxo-3,4-dihydro-2<i>H</i>-benzo­[<i>b</i>]­[1,4]­oxazin-8-yl)­urea ([³H]­MPOU), that embodies improved absolute affinity for human TRPV1 and improved synthetic accessibility

Discovery of simplified leucyladenylate sulfamates as novel leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors

Leucyl-tRNA synthetase (LRS) has been reported to be a possible mediator of intracellular amino acids signaling to mTORC1. Given that mTORC1 is associated with cell proliferation and tumorigenesis, the LRS-mediated mTORC1 pathway may offer an alternative strategy in anticancer therapy. In this study, we developed a series of simplified analogues of leucyladenylate sulfamate (1) as LRS-targeted mTORC1 inhibitors. We replaced the adenylate group with a N-(3,4-dimethoxybenzyl)benzenesulfonamide (2a) or a N-(2-phenoxyethyl)benzenesulfonamide groups (2b) that can maintain specific binding, but has more favorable physicochemical properties such as reduced polarity and asymmetric centers. Among these simplified analogues, compound 16 and its constrained analogue 22 effectively inhibited S6K phosphorylation in a dose-dependent manner and exhibited cancer cell specific cytotoxicity against six different types of cancer cells. This result supports that LRS is a viable target for novel anticancer therapy. (C) 2017 Elsevier Ltd. All rights reserved.OAIID:RECH_ACHV_DSTSH_NO:T201730174RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A078273CITE_RATE:2.881DEPT_NM:제약학과EMAIL:[email protected]_YN:YN

Curcumin suppresses oncogenicity of human colon cancer cells by covalently modifying the cysteine 67 residue of SIRT1

SIRT1, an NAD(+)-dependent histone/protein deacetylase, has diverse physiological actions. Recent studies have demonstrated that SIRT1 is overexpressed in colorectal cancer, suggesting its oncogenic potential. However, the molecular mechanisms by which overexpressed SIRT1 induces the progression of colorectal cancer and its inhibition remain largely unknown. Curcumin (diferuloymethane), a major component of the spice turmeric derived from the plant Curcuma longa L., has been reported to exert chemopreventive and anti-carcinogenic effects on colon carcinogenesis. In the present study, we found that curcumin reduced the expression of SIRT1 protein without influencing its mRNA expression in human colon cancer cells, suggesting posttranslational regulation of SIRT1 by this phytochemical. Notably, ubiquitination and subsequent proteasomal degradation of SIRT1 were induced by curcumin treatment. Results of nano-LC-ESI-MS/MS revealed the direct binding of curcumin to cysteine 67 of SIRT1. In line with this result, the protein stability and clonogenicity of a mutant SIRT1 in which cysteine 67 was substituted by alanine were unaffected by curcumin. Taken together, these observations suggest that curcumin facilitates the proteasomal degradation of oncogenic SIRT1 through covalent modification of SIRT1 at the cysteine 67 residue