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1
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Maki J, Oshimura A, Shiotani Y, Yamanaka M, Okuda S, Yanagita RC, Kitani S, Igarashi Y, Saito Y, Sakakibara Y, Tsukano C, Irie K. Validation of machine learning-assisted screening of PKC ligands: PKC binding affinity and activation. Biosci Biotechnol Biochem 2025; 89:668-679. [PMID: 39863420 DOI: 10.1093/bbb/zbaf008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2024] [Accepted: 01/22/2025] [Indexed: 01/27/2025]
Abstract
Protein kinase C (PKC) is a family of serine/threonine kinases, and PKC ligands have the potential to be therapeutic seeds for cancer, Alzheimer's disease, and human immunodeficiency virus infection. However, in addition to desired therapeutic effects, most PKC ligands also exhibit undesirable pro-inflammatory effects. The discovery of new scaffolds for PKC ligands is important for developing less inflammatory PKC ligands, such as bryostatins. We previously reported that machine learning combined with our knowledge of the pharmacophore yielded 15 PKC ligand candidates, but we did not evaluate their PKC binding affinities fully. In this paper, PKC binding affinities of four candidates were examined to assess their potential as PKC ligands and to validate machine learning-assisted screening. Although compound 3' did not bind to PKC C1 domains, 1a, 2', and 4a exhibited moderate PKC binding affinities, suggesting that machine learning-assisted screening is advantageous in identifying new PKC ligand scaffolds.
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Affiliation(s)
- Jumpei Maki
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Asami Oshimura
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Yudai Shiotani
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Maki Yamanaka
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Sogen Okuda
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Ryo C Yanagita
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan
| | - Shigeru Kitani
- College of Science and Engineering, Aoyama Gakuin University, Kanagawa, Japan
| | - Yasuhiro Igarashi
- Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, Toyama, Japan
| | - Yutaka Saito
- Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan
- AIST-Waseda University Computational Bio Big-Data Open Innovation Laboratory (CBBD-OIL), Shinjuku-ku, Tokyo, Japan
| | | | - Chihiro Tsukano
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
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2
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Gentry ZO, McAteer OD, Hamad JL, Moran JA, Kim JT, Marsden MD, Zack JA, Wender PA. Synthesis and preclinical evaluation of tigilanol tiglate analogs as latency-reversing agents for the eradication of HIV. SCIENCE ADVANCES 2025; 11:eads1911. [PMID: 39854456 PMCID: PMC11778240 DOI: 10.1126/sciadv.ads1911] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/02/2024] [Accepted: 12/20/2024] [Indexed: 01/26/2025]
Abstract
Tigilanol tiglate (EBC-46) is a selective modulator of protein kinase C (PKC) isoforms that is Food and Drug Administration (FDA) approved for the treatment of mast cell tumors in canines with up to an 88% cure rate. Recently, it has been FDA approved for the treatment of soft tissue sarcomas in humans. The role of EBC-46 and, especially, its analogs in efforts to eradicate HIV, treat neurological and cardiovascular disorders, or enhance antigen density in antigen-targeted chimeric antigen receptor-T cell and chimeric antigen receptor-natural killer cell immunotherapies has not been reported. Enabled by our previously reported scalable synthesis of EBC-46, we report herein the systematic design, synthesis, and evaluation of EBC-46 analogs, including those inaccessible from the natural source and their PKC affinities, ability to translocate PKC, nuclear factor κB activity, and efficacy in reversing HIV latency in Jurkat-Latency cells. Leading analogs show exceptional PKC affinities, isoform selectivities, and functional activities, serving as promising candidates for therapeutic applications.
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Affiliation(s)
- Zachary O. Gentry
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
| | - Owen D. McAteer
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
| | - Jennifer L. Hamad
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
- Department of Biology, Stanford University, Stanford, CA 94305, USA
| | - Jose A. Moran
- Department of Microbiology & Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA 92697, USA
| | - Jocelyn T. Kim
- Department of Medicine, Division of Infectious Diseases, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Matthew D. Marsden
- Department of Microbiology & Molecular Genetics, School of Medicine, University of California Irvine, Irvine, CA 92697, USA
- Department of Medicine (Division of Infectious Diseases), School of Medicine, University of California Irvine, Irvine, CA 92697, USA
| | - Jerome A. Zack
- Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA
- Department of Medicine, Division of Hematology and Oncology, University of California Los Angeles, Los Angeles, CA 90095, USA
| | - Paul A. Wender
- Department of Chemistry, Stanford University, Stanford, CA 94305, USA
- Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
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3
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Overall SA, Hartmann SJ, Luu-Nguyen QH, Judge P, Pinotsi D, Marti L, Sigurdsson ST, Wender PA, Barnes AB. Topological Heterogeneity of Protein Kinase C Modulators in Human T-Cells Resolved with In-Cell Dynamic Nuclear Polarization NMR Spectroscopy. J Am Chem Soc 2024; 146:27362-27372. [PMID: 39322225 PMCID: PMC11468733 DOI: 10.1021/jacs.4c05704] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Revised: 08/29/2024] [Accepted: 09/03/2024] [Indexed: 09/27/2024]
Abstract
Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of 13C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing 13C-13C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct 13C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of 13C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.
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Affiliation(s)
- Sarah A. Overall
- Institute
of Molecular Physical Science, ETH Zurich, 8093 Zurich, Switzerland
| | - Sina J. Hartmann
- Institute
of Molecular Physical Science, ETH Zurich, 8093 Zurich, Switzerland
| | - Quang H. Luu-Nguyen
- Department
of Chemistry, Stanford University, Stanford, California 94305-5080, United
States
| | - Patrick Judge
- Department
of Biochemistry, Biophysics, & Structural Biology, Washington University in St. Louis, St. Louis, Missouri 63110, United States
| | - Dorothea Pinotsi
- Scientific
Center for Optical and Electron Microscopy, ETH Zurich, 8093 Zurich, Switzerland
| | - Lea Marti
- Institute
of Molecular Physical Science, ETH Zurich, 8093 Zurich, Switzerland
| | | | - Paul A. Wender
- Department
of Chemistry, Stanford University, Stanford, California 94305-5080, United
States
| | - Alexander B. Barnes
- Institute
of Molecular Physical Science, ETH Zurich, 8093 Zurich, Switzerland
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4
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Maki J, Hanaki Y, Yanagita RC, Kikumori M, Kovba A, Washizaki A, Tsukano C, Akari H, Irie K. Biological evaluation of a phosphate ester prodrug of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, as a possible latency-reversing agent for HIV reactivation. Biosci Biotechnol Biochem 2023; 87:1453-1461. [PMID: 37682524 DOI: 10.1093/bbb/zbad128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 08/31/2023] [Indexed: 09/09/2023]
Abstract
10-Methyl-aplog-1 (10MA-1), a simplified analog of aplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes with minimal tumor-promoting and pro-inflammatory activities. A recent study suggests that 10MA-1 could reactivate latent human immunodeficiency virus (HIV) in vitro for HIV eradication strategy. However, further in vivo studies were abandoned by a dose limit caused by the minimal water solubility of 10MA-1. To overcome this problem, we synthesized a phosphate ester of 10MA-1, 18-O-phospho-10-methyl-aplog-1 (phos-10MA-1), to improve water solubility for in vivo studies. The solubility, PKC binding affinity, and biological activity of phos-10MA-1 were examined in vitro, and the biological activity was comparable with 10MA-1. The pharmacokinetic studies in vivo were also examined, which suggest that further optimization for improving metabolic stability is required in the future.
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Affiliation(s)
- Jumpei Maki
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Yusuke Hanaki
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan
| | - Ryo C Yanagita
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan
| | - Masayuki Kikumori
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Anastasiia Kovba
- Center for the Evolutionary Origins of Human Behavior, Kyoto University, Aichi, Japan
| | - Ayaka Washizaki
- Center for the Evolutionary Origins of Human Behavior, Kyoto University, Aichi, Japan
| | - Chihiro Tsukano
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Hirofumi Akari
- Center for the Evolutionary Origins of Human Behavior, Kyoto University, Aichi, Japan
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
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5
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Insertion Depth Modulates Protein Kinase C-δ-C1b Domain Interactions with Membrane Cholesterol as Revealed by MD Simulations. Int J Mol Sci 2023; 24:ijms24054598. [PMID: 36902029 PMCID: PMC10002858 DOI: 10.3390/ijms24054598] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Revised: 02/22/2023] [Accepted: 02/23/2023] [Indexed: 03/02/2023] Open
Abstract
Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.
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6
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Okuda S, Tsukano C, Irie K. Synthesis of Stereoisomeric Simplified Analogs of Alotaketals toward the Elucidation of the Structural Requirements of Protein Kinase C Isozyme-Selective Binding. Org Lett 2023; 25:805-809. [PMID: 36715604 DOI: 10.1021/acs.orglett.2c04328] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
A set of four stereoisomeric compounds were designed and synthesized as ligands of protein kinase C (PKC). The compounds were simplified analogs of the alotaketals, a class of natural products that were predicted to be ligands of PKC by computational screening. Bioassays revealed that the orientation of the alkyl side chain of the analogs was important for PKC binding and that the stereochemistry of the fused ring moiety influenced the PKC isozyme selectivity.
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Affiliation(s)
- Sogen Okuda
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
| | - Chihiro Tsukano
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
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7
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A simplified analog of debromoaplysiatoxin lacking the B-ring of spiroketal moiety retains protein kinase C-binding and antiproliferative activities. Bioorg Med Chem 2022; 73:116988. [DOI: 10.1016/j.bmc.2022.116988] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Revised: 08/23/2022] [Accepted: 08/24/2022] [Indexed: 11/20/2022]
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8
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Yanagita RC, Otani M, Hatanaka S, Nishi H, Miyake S, Hanaki Y, Sato M, Kawanami Y, Irie K. Analysis of binding mode of vibsanin A with protein kinase C C1 domains: An experimental and molecular dynamics simulation study. J Mol Struct 2022. [DOI: 10.1016/j.molstruc.2022.132866] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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9
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Miao LN, Pan D, Shi J, Du JP, Chen PF, Gao J, Yu Y, Shi DZ, Guo M. Role and Mechanism of PKC-δ for Cardiovascular Disease: Current Status and Perspective. Front Cardiovasc Med 2022; 9:816369. [PMID: 35242825 PMCID: PMC8885814 DOI: 10.3389/fcvm.2022.816369] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Accepted: 01/11/2022] [Indexed: 12/18/2022] Open
Abstract
Protein kinase C (PKC) is a protein kinase with important cellular functions. PKC-δ, a member of the novel PKC subfamily, has been well-documented over the years. Activation of PKC-δ plays an important regulatory role in myocardial ischemia/reperfusion (IRI) injury and myocardial fibrosis, and its activity and expression levels can regulate pathological cardiovascular diseases such as atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. This article aims to review the structure and function of PKC-δ, summarize the current research regarding its activation mechanism and its role in cardiovascular disease, and provide novel insight into further research on the role of PKC-δ in cardiovascular diseases.
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Affiliation(s)
- Li-na Miao
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- Department of Graduate School, Beijing University of Chinese Medicine, Beijing, China
| | - Deng Pan
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- Department of Graduate School, Beijing University of Chinese Medicine, Beijing, China
| | - Junhe Shi
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Jian-peng Du
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- China Heart Institute of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China
| | - Peng-fei Chen
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Jie Gao
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- China Heart Institute of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China
| | - Yanqiao Yu
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- Department of Graduate School, Beijing University of Chinese Medicine, Beijing, China
| | - Da-Zhuo Shi
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- China Heart Institute of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China
- *Correspondence: Da-Zhuo Shi
| | - Ming Guo
- Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China
- China Heart Institute of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China
- Ming Guo
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10
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Maki J, Oshimura A, Tsukano C, Yanagita RC, Saito Y, Sakakibara Y, Irie K. AI and computational chemistry-accelerated development of an alotaketal analogue with conventional PKC selectivity. Chem Commun (Camb) 2022; 58:6693-6696. [DOI: 10.1039/d2cc01759h] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The protein kinase C (PKC) family consists of ten isozymes and is a potential target for treating cancer, Alzheimer’s disease, and HIV infection. Since known natural PKC agonists have little...
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11
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Araki Y, Hanaki Y, Kita M, Hayakawa K, Irie K, Nokura Y, Nakazaki A, Nishikawa T. Total synthesis and biological evaluation of oscillatoxins D, E, and F. Biosci Biotechnol Biochem 2021; 85:1371-1382. [PMID: 33851985 DOI: 10.1093/bbb/zbab042] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Accepted: 03/07/2021] [Indexed: 11/14/2022]
Abstract
Oscillatoxins (OTXs) and aplysiatoxins are biosynthetically related polyketides produced by marine cyanobacteria. We previously developed a synthetic route to phenolic O-methyl analogs of OTX-D and 30-methyl-OTX-D during collective synthesis of these natural products. According to our synthetic strategy, we achieved total synthesis of OTX-D, 30-methyl-OTX-D, OTX-E, and OTX-F by deprotecting the O-methyl group in an earlier intermediate, and determined their biological activities. Although OTX-D and 30-methyl-OTX-D have been reported to show antileukemic activity against L1210 cell line, we found that their cytotoxicity in vitro against this cell line is relatively weak (IC50: 29-52 µm). In contrast, OTX-F demonstrated cell line-selective antiproliferative activity against DMS-114 lung cancer cells, which implies that OTXs target as yet unknown target molecules as part of this unique activity.
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Affiliation(s)
- Yusuke Araki
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
| | - Yusuke Hanaki
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
| | - Masaki Kita
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
| | | | - Kazuhiro Irie
- Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Yoshihiko Nokura
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
| | - Atsuo Nakazaki
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
| | - Toshio Nishikawa
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
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12
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Gonda A, Takada K, Yanagita RC, Dan S, Irie K. Effects of side chain length of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, on PKC binding, anti-proliferative, and pro-inflammatory activities. Biosci Biotechnol Biochem 2021; 85:168-180. [PMID: 33577665 DOI: 10.1093/bbb/zbaa024] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Accepted: 08/22/2020] [Indexed: 02/07/2023]
Abstract
10-Methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes and potent antiproliferative activity against several cancer cells with few adverse effects. A recent study has suggested that its phenol group in the side chain is involved in hydrogen bonding and CH/π interactions with the binding cleft-forming loops in the PKCδ-C1B domain. To clarify the effects of the side chain length on these interactions, four analogs of 1 with various lengths of side chains (2-5) were prepared. The maximal PKC binding affinity and antiproliferative activity were observed in 1. Remarkably, the introduction of a bromine atom into the phenol group of 2 increased not only these activities but also proinflammatory activity. These results indicated that 1 has the optimal side chain length as an anticancer seed. This conclusion was supported by docking simulations of 1-5 to the PKCδ-C1B domain.
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Affiliation(s)
- Atsuko Gonda
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Koji Takada
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
| | - Ryo C Yanagita
- Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa, Japan
| | - Shingo Dan
- Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
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13
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Synthesis and biological activities of simplified aplysiatoxin analogs focused on the CH/π interaction. Bioorg Med Chem Lett 2020; 30:127657. [PMID: 33130291 DOI: 10.1016/j.bmcl.2020.127657] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Revised: 10/22/2020] [Accepted: 10/25/2020] [Indexed: 11/23/2022]
Abstract
Debromoaplysiatoxin (DAT) is a potent protein kinase C (PKC) activator with tumor-promoting and pro-inflammatory activities. Irie and colleagues have found that 10-methyl-aplog-1 (1), a simplified analog of DAT, has strong anti-proliferative activity against several cancer cell lines with few adverse effects. Therefore, 1 is a potential lead compound for cancer therapy. We synthesized a new derivative 2 which has a naphthalene ring at the side chain terminal position instead of a benzene ring, to increase CH/π interactions with Pro-241 of the PKCδ-C1B domain. Based on the synthetic route of 1, 2 was convergently synthesized in 26 linear steps from 6-hydroxy-1-naphthoic acid with an overall yield of 0.18%. Although the anti-proliferative activity of 2 was more potent than that of 1, the binding potency of 2 to the PKCδ-C1B domain did not exceed that of 1. Molecular dynamics simulation indicated the capability of 2 to simultaneously form hydrogen bonds and CH/π interactions with the PKCδ-C1B domain. Focusing on the hydrogen bonds, their geometry in the binding modes involving the CH/π interactions seemed to be sub-optimal, which may explain the slightly lower affinity of 2 compared to 1. This study could be of help in optimizing such interactions and synthesizing a promising lead cancer compound.
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14
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C. Yanagita R, Ashida Y, Kawanami Y, Okamura M, Dan S, Irie K. Synthesis, Conformation, and Biological Activities of a Des-A-Ring Analog of 18-Deoxy-Aplog-1, a Simplified Analog of Debromoaplysiatoxin. HETEROCYCLES 2019. [DOI: 10.3987/com-18-s(f)60] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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15
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Czikora A, Pany S, You Y, Saini AS, Lewin NE, Mitchell GA, Abramovitz A, Kedei N, Blumberg PM, Das J. Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2018; 1860:1046-1056. [PMID: 29317197 DOI: 10.1016/j.bbamem.2018.01.007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 12/06/2017] [Accepted: 01/04/2018] [Indexed: 12/31/2022]
Abstract
The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.
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Affiliation(s)
- Agnes Czikora
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Satyabrata Pany
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, United States
| | - Youngki You
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, United States
| | - Amandeep S Saini
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Nancy E Lewin
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Gary A Mitchell
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Adelle Abramovitz
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Noemi Kedei
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States
| | - Peter M Blumberg
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, United States.
| | - Joydip Das
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, United States.
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16
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Irie K, Hayakawa K, Hanaki Y, Tokuda H, C. Yanagita R, Nakagawa Y, Okamura M, Dan S. Synthesis and Biological Activities of Acetal Analogs at Position 3 of 10-Methyl-Aplog-1, a Potential Anti-Cancer Lead Derived from Debromoaplysiatoxin. HETEROCYCLES 2018. [DOI: 10.3987/com-18-s(t)37] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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17
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Iguchi N, Dönmez Mİ, Malykhina AP, Carrasco A, Wilcox DT. Preventative effects of a HIF inhibitor, 17-DMAG, on partial bladder outlet obstruction-induced bladder dysfunction. Am J Physiol Renal Physiol 2017; 313:F1149-F1160. [PMID: 28768664 DOI: 10.1152/ajprenal.00240.2017] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2017] [Revised: 07/11/2017] [Accepted: 07/24/2017] [Indexed: 01/11/2023] Open
Abstract
Posterior urethral valves are the most common cause of partial bladder outlet obstruction (PBOO) in the pediatric population. Pathological changes in the bladder developed during PBOO are responsible for long-lasting voiding dysfunction in this population despite early surgical interventions. Increasing evidence showed PBOO induces an upregulation of hypoxia-inducible factors (HIFs) and their transcriptional target genes, and they play a role in pathophysiological changes in the obstructed bladders. We hypothesized that blocking HIF pathways can prevent PBOO-induced bladder dysfunction. PBOO was surgically created by ligation of the bladder neck in male C57BL/6J mice for 2 wk. PBOO mice received intraperitoneal injection of either saline or 17-DMAG (alvespimycin, 3 mg/kg) every 48 h starting from day 1 postsurgery. Sham-operated animals received injection of saline on the same schedule as PBOO mice and served as controls. The bladders were harvested after 2 wk, and basal activity and evoked contractility of the detrusor smooth muscle (DSM) were evaluated in vitro. Bladder function was assessed in vivo by void spot assay and cystometry in conscious, unrestrained mice. Results indicated the 17-DMAG treatment preserved DSM contractility and partially prevented the development of detrusor over activity in obstructed bladders. In addition, PBOO caused a significant increase in the frequency of micturition, which was significantly reduced by 17-DMAG treatment. The 17-DMAG treatment improved urodynamic parameters, including increases in the bladder pressure at micturition and nonvoid contractions observed in PBOO mice. These results demonstrate that treatment with 17-DMAG, a HIF inhibitor, significantly alleviated PBOO-induced bladder pathology in vivo.
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Affiliation(s)
- Nao Iguchi
- Division of Urology, Department of Surgery, University of Colorado Denver School of Medicine, Aurora, Colorado; and
| | - M İrfan Dönmez
- Division of Urology, Department of Surgery, University of Colorado Denver School of Medicine, Aurora, Colorado; and
| | - Anna P Malykhina
- Division of Urology, Department of Surgery, University of Colorado Denver School of Medicine, Aurora, Colorado; and
| | | | - Duncan T Wilcox
- Division of Urology, Department of Surgery, University of Colorado Denver School of Medicine, Aurora, Colorado; and .,Children's Hospital Colorado, Aurora, Colorado
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18
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Ohashi N, Kobayashi R, Nomura W, Kobayakawa T, Czikora A, Herold BK, Lewin NE, Blumberg PM, Tamamura H. Synthesis and Evaluation of Dimeric Derivatives of Diacylglycerol-Lactones as Protein Kinase C Ligands. Bioconjug Chem 2017; 28:2135-2144. [PMID: 28671468 DOI: 10.1021/acs.bioconjchem.7b00299] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Protein kinase C (PKC) mediates a central cellular signal transduction pathway involved in disorders such as cancer and Alzheimer's disease. PKC is regulated by binding of the second messenger sn-1,2-diacylglycerol (DAG) to its tandem C1 domains, designated C1a and C1b, leading both to PKC activation and to its translocation to the plasma membrane and to internal organelles. Depending on the isoform, there may be differences in the ligand selectivity of the C1a and C1b domains, and there is different spacing between the C1 domains of the conventional and novel PKCs. Bivalent ligands have the potential to exploit these differences between isoforms, yielding isoform selectivity. In the present study, we describe the synthesis of a series of dimeric derivatives of conformationally constrained diacylglycerol (DAG) analogs (DAG-lactones). We characterize the derivatives in vitro for their binding affinities, both to a single C1 domain (the C1b domain of PKCδ) as well as to the conventional PKCα isoform and the novel PKCδ isoform, and we measure their abilities to cause translocation of PKCδ and PKCε in intact cells. The dimeric compound with the 10-carbon linker was modestly more effective for the isolated PKCδ C1b domain than was the monomeric compound. For the intact PKCα and PKCδ, the shortest DAG-lactone dimer had similar affinity to the monomer and affinity decreased progressively up to the 16-carbon linker. The dimeric derivatives did not cause the Golgi accumulation of PKCδ. The present results provide important insights into the development of new chemical tools for biological studies on PKC.
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Affiliation(s)
- Nami Ohashi
- Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
| | - Ryosuke Kobayashi
- Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
| | - Wataru Nomura
- Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
| | - Takuya Kobayakawa
- Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
| | - Agnes Czikora
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States
| | - Brienna K Herold
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States
| | - Nancy E Lewin
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States
| | - Peter M Blumberg
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States
| | - Hirokazu Tamamura
- Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University , 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
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19
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Loss of the Phenolic Hydroxyl Group and Aromaticity from the Side Chain of Anti-Proliferative 10-Methyl-aplog-1, a Simplified Analog of Aplysiatoxin, Enhances Its Tumor-Promoting and Proinflammatory Activities. Molecules 2017; 22:molecules22040631. [PMID: 28406454 PMCID: PMC6153940 DOI: 10.3390/molecules22040631] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2017] [Revised: 04/05/2017] [Accepted: 04/11/2017] [Indexed: 01/29/2023] Open
Abstract
Aplysiatoxin (ATX) is a protein kinase C (PKC) activator with potent tumor-promoting activity. In contrast, 10-methyl-aplog-1 (1), a simplified analog of ATX, was anti-proliferative towards several cancer cell lines without significant tumor-promoting and proinflammatory activities. To determine the effects of the phenolic group on the biological activities of 1, we synthesized new derivatives (2, 3) that lack the phenolic hydroxyl group and/or the aromatic ring. Compound 2, like 1, showed potent anti-proliferative activity against several cancer cell lines, but little with respect to tumor-promoting and proinflammatory activities. In contrast, 3 exhibited weaker growth inhibitory activity, and promoted inflammation and tumorigenesis. The binding affinity of 3 for PKCδ, which is involved in growth inhibition and apoptosis, was several times lower than those of 1 and 2, possibly due to the absence of the hydrogen bond and CH/π interaction between its side chain and either Met-239 or Pro-241 in the PKCδ-C1B domain. These results suggest that both the aromatic ring and phenolic hydroxyl group can suppress the proinflammatory and tumor-promoting activities of 1 and, therefore, at least the aromatic ring in the side chain of 1 is indispensable for developing anti-cancer leads with potent anti-proliferative activity and limited side effects. In accordance with the binding affinity, the concentration of 3 necessary to induce PKCδ-GFP translocation to the plasma membrane and perinuclear regions in HEK293 cells was higher than that of 1 and 2. However, the translocation profiles for PKCδ-GFP due to induction by 1–3 were similar.
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20
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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. Bioorg Med Chem 2016; 24:4218-4227. [DOI: 10.1016/j.bmc.2016.07.011] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2016] [Revised: 07/05/2016] [Accepted: 07/06/2016] [Indexed: 01/27/2023]
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21
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Czikora A, Lundberg DJ, Abramovitz A, Lewin NE, Kedei N, Peach ML, Zhou X, Merritt RC, Craft EA, Braun DC, Blumberg PM. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity. J Biol Chem 2016; 291:11133-47. [PMID: 27022025 PMCID: PMC4900263 DOI: 10.1074/jbc.m116.725333] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Revised: 03/28/2016] [Indexed: 11/06/2022] Open
Abstract
The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.
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Affiliation(s)
- Agnes Czikora
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
| | - Daniel J Lundberg
- Department of Science, Technology, and Mathematics, Gallaudet University, Washington, D. C. 20002, and
| | - Adelle Abramovitz
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
| | - Nancy E Lewin
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
| | - Noemi Kedei
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
| | - Megan L Peach
- Basic Science Program, Leidos Biomedical Research, Inc., Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702
| | - Xiaoling Zhou
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
| | - Raymond C Merritt
- Department of Science, Technology, and Mathematics, Gallaudet University, Washington, D. C. 20002, and
| | - Elizabeth A Craft
- Department of Science, Technology, and Mathematics, Gallaudet University, Washington, D. C. 20002, and
| | - Derek C Braun
- Department of Science, Technology, and Mathematics, Gallaudet University, Washington, D. C. 20002, and
| | - Peter M Blumberg
- From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892,
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22
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Kikumori M, Yanagita RC, Tokuda H, Suenaga K, Nagai H, Irie K. Structural optimization of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, as an anticancer lead. Biosci Biotechnol Biochem 2015; 80:221-31. [PMID: 26452398 DOI: 10.1080/09168451.2015.1091718] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (1) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (2, 3) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (2) had stronger inhibitory effects on the growth of several human cancer cell lines than 1 and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (3) displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of 1 and DAT. Compound 2 would be the optimized seed for anticancer drugs among the simplified analogs of DAT.
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Affiliation(s)
- Masayuki Kikumori
- a Division of Food Science and Biotechnology , Graduate School of Agriculture, Kyoto University , Kyoto , Japan
| | - Ryo C Yanagita
- b Faculty of Agriculture, Department of Applied Biological Science , Kagawa University , Miki , Japan
| | - Harukuni Tokuda
- c Department of Complementary and Alternative Medicine, Clinical R&D , Graduate School of Medical Science, Kanazawa University , Kanazawa , Japan
| | - Kiyotake Suenaga
- d Faculty of Science and Technology , Keio University , Yokohama , Japan
| | - Hiroshi Nagai
- e Department of Ocean Sciences , Tokyo University of Marine Science and Technology , Tokyo , Japan
| | - Kazuhiro Irie
- a Division of Food Science and Biotechnology , Graduate School of Agriculture, Kyoto University , Kyoto , Japan
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23
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Hanaki Y, Yanagita RC, Sugahara T, Aida M, Tokuda H, Suzuki N, Irie K. Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities. Biosci Biotechnol Biochem 2015; 79:888-95. [PMID: 25612633 DOI: 10.1080/09168451.2014.1002452] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
Aplog-1 is a simplified analog of the tumor-promoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of aplog-1. In this study, we synthesized amide-aplog-1 (3), in which the C-1 ester group was replaced with an amide group, to improve chemical stability in vivo. Unfortunately, 3 exhibited seventy-fold weaker binding affinity to the C1B domain of PKCδ than that of aplog-1, and negligible anti-proliferative and cytotoxic activities even at 10(-4) M. A conformational analysis and density functional theory calculations indicated that the stable conformation of 3 differed from that of aplog-1. Since 27-methyl and 27-methoxy derivatives (1, 2) without the ability to bind to PKC isozymes exhibited marked anti-proliferative and cytotoxic activities at 10(-4) M, 3 may be an inactive control to identify the target proteins of aplogs.
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Affiliation(s)
- Yusuke Hanaki
- a Division of Food Science and Biotechnology , Graduate School of Agriculture, Kyoto University , Kyoto , Japan
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24
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Takahashi H, Adachi N, Shirafuji T, Danno S, Ueyama T, Vendruscolo M, Shuvaev AN, Sugimoto T, Seki T, Hamada D, Irie K, Hirai H, Sakai N, Saito N. Identification and characterization of PKCγ, a kinase associated with SCA14, as an amyloidogenic protein. Hum Mol Genet 2015; 24:525-39. [PMID: 25217572 DOI: 10.1093/hmg/ddu472] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Amyloid assemblies are associated with a wide range of human disorders, including Alzheimer's and Parkinson's diseases. Here, we identify protein kinase C (PKC) γ, a serine/threonine kinase mutated in the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14), as a novel amyloidogenic protein with no previously characterized amyloid-prone domains. We found that overexpression of PKCγ in cultured cells, as well as in vitro incubation of PKCγ without heat or chemical denaturants, causes amyloid-like fibril formation of this protein. We also observed that SCA14-associated mutations in PKCγ accelerate the amyloid-like fibril formation both in cultured cells and in vitro. We show that the C1A and kinase domains of PKCγ are involved in its soluble dimer and aggregate formation and that SCA14-associated mutations in the C1 domain cause its misfolding and aggregation. Furthermore, long-term time-lapse imaging indicates that aggregates of mutant PKCγ are highly toxic to neuronal cells. Based on these findings, we propose that PKCγ could form amyloid-like fibrils in physiological and/or pathophysiological conditions such as SCA14. More generally, our results provide novel insights into the mechanism of amyloid-like fibril formation by multi-domain proteins.
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Affiliation(s)
| | - Naoko Adachi
- Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
| | | | - Sally Danno
- Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
| | - Takehiko Ueyama
- Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
| | - Michele Vendruscolo
- Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
| | - Anton N Shuvaev
- Department of Neurophysiology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan
| | - Takuya Sugimoto
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
| | - Takahiro Seki
- Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan
| | - Daizo Hamada
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan and
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
| | - Hirokazu Hirai
- Department of Neurophysiology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan
| | - Norio Sakai
- Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Science, Hiroshima University, Hiroshima 734-8551, Japan
| | - Naoaki Saito
- Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
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25
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Nakagawa Y. Structural Simplification of Natural Products Toward the Generation of Biologically and Therapeutically Valuable Molecules: Analog Design of Naturally-Occurring Protein Kinase C Activators. J SYN ORG CHEM JPN 2015. [DOI: 10.5059/yukigoseikyokaishi.73.316] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Yu Nakagawa
- Graduate School of Bioagricultural Sciences, Nagoya University
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26
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Jiang W, Tan S, Hanaki Y, Irie K, Uchida H, Watanabe R, Suzuki T, Sakamoto B, Kamio M, Nagai H. Two new lyngbyatoxin derivatives from the Cyanobacterium, Moorea producens. Mar Drugs 2014; 12:5788-800. [PMID: 25470181 PMCID: PMC4278201 DOI: 10.3390/md12125788] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2014] [Revised: 11/11/2014] [Accepted: 11/19/2014] [Indexed: 11/28/2022] Open
Abstract
The toxin-producing cyanobacterium, Moorea producens, is a known causative organism of food poisoning and seaweed dermatitis (also known as "swimmer's itch"). Two new toxic compounds were isolated and structurally elucidated from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies, as well as optical rotations and CD spectra indicated two new lyngbyatoxin derivatives, 2-oxo-3(R)-hydroxy-lyngbyatoxin A (1) and 2-oxo-3(R)-hydroxy-13-N-desmethyl-lyngbyatoxin A (2). The cytotoxicity and lethal activities of 1 and 2 were approximately 10- to 150-times less potent than lyngbyatoxin A. Additionally, the binding activities of 1 and 2 possessed 10,000-times lower affinity for the protein kinase Cδ (PKCδ)-C1B peptide when compared to lyngbyatoxin A. These findings suggest that these new lyngbyatoxin derivatives may mediate their acute toxicities through a non-PKC activation pathway.
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Affiliation(s)
- Weina Jiang
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Satoshi Tan
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Yusuke Hanaki
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
| | - Hajime Uchida
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Ryuichi Watanabe
- National Research Institute of Fisheries Science, Yokohama 236-8648, Japan.
| | - Toshiyuki Suzuki
- National Research Institute of Fisheries Science, Yokohama 236-8648, Japan.
| | - Bryan Sakamoto
- Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202, USA.
| | - Michiya Kamio
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Hiroshi Nagai
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
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27
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Affiliation(s)
- Joydip Das
- Department of Pharmacological
and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 521 Science and Research Building 2, Houston, Texas 77204, United States
| | - Ghazi M. Rahman
- Department of Pharmacological
and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 521 Science and Research Building 2, Houston, Texas 77204, United States
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28
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Li J, Ziemba BP, Falke J, Voth GA. Interactions of protein kinase C-α C1A and C1B domains with membranes: a combined computational and experimental study. J Am Chem Soc 2014; 136:11757-66. [PMID: 25075641 PMCID: PMC4140453 DOI: 10.1021/ja505369r] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2014] [Indexed: 02/01/2023]
Abstract
Protein kinase C-α (PKCα) has been studied widely as a paradigm for conventional PKCs, with two C1 domains (C1A and C1B) being important for the regulation and function of the kinase. However, it is challenging to explore these domains in membrane-bound environments with either simulations or experiments alone. In this work, we have combined modeling, simulations, and experiments to understand the molecular basis of the PKCα C1A and C1B domain interactions with membranes. Our atomistic simulations of the PKCα C1 domains reveal the dynamic interactions of the proteins with anionic lipids, as well as the conserved hydrogen bonds and the distinct nonpolar contacts formed with lipid activators. Corroborating evidence is obtained from additional simulations and experiments in terms of lipid binding and protein diffusion. Overall, our study, for the first time, explains with atomistic detail how the PKCα C1A and C1B domains interact differently with various lipids. On the molecular level, the information provided by our study helps to shed light on PKCα regulation and activation mechanism. The combined computational/experimental approach demonstrated in this work is anticipated to enable further studies to explore the roles of C1 domains in many signaling proteins and to better understand their molecular mechanisms in normal cellular function and disease development.
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Affiliation(s)
- Jianing Li
- Department
of Chemistry, Institute for Biophysical Dynamics, James Franck Institute
and Computation Institute, The University
of Chicago, 5735 South
Ellis Avenue, Chicago, Illinois 60637, United States
| | - Brian P. Ziemba
- Department
of Chemistry and Biochemistry and the Molecular Biophysics Program, University of Colorado at Boulder, Boulder, Colorado 80309, United States
| | - Joseph
J. Falke
- Department
of Chemistry and Biochemistry and the Molecular Biophysics Program, University of Colorado at Boulder, Boulder, Colorado 80309, United States
| | - Gregory A. Voth
- Department
of Chemistry, Institute for Biophysical Dynamics, James Franck Institute
and Computation Institute, The University
of Chicago, 5735 South
Ellis Avenue, Chicago, Illinois 60637, United States
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29
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Identification and Biological Activities of Bryostatins from Japanese Bryozoan. Biosci Biotechnol Biochem 2014; 76:1041-3. [DOI: 10.1271/bbb.120026] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
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30
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Jiang W, Zhou W, Uchida H, Kikumori M, Irie K, Watanabe R, Suzuki T, Sakamoto B, Kamio M, Nagai H. A new lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens. Mar Drugs 2014; 12:2748-59. [PMID: 24824022 PMCID: PMC4052313 DOI: 10.3390/md12052748] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Revised: 04/06/2014] [Accepted: 04/08/2014] [Indexed: 01/04/2023] Open
Abstract
Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of “swimmer’s itch” with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.
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Affiliation(s)
- Weina Jiang
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Wei Zhou
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Hajime Uchida
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Masayuki Kikumori
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
| | - Kazuhiro Irie
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
| | - Ryuichi Watanabe
- National Research Institute of Fisheries Science, Yokohama 236-8648, Japan.
| | - Toshiyuki Suzuki
- National Research Institute of Fisheries Science, Yokohama 236-8648, Japan.
| | - Bryan Sakamoto
- Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202, USA.
| | - Michiya Kamio
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
| | - Hiroshi Nagai
- Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
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31
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Rahman GM, Das J. Modeling studies on the structural determinants for the DAG/phorbol ester binding to C1 domain. J Biomol Struct Dyn 2014; 33:219-32. [DOI: 10.1080/07391102.2014.895679] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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32
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Irie K, Yanagita RC. Synthesis and Biological Activities of Simplified Analogs of the Natural PKC Ligands, Bryostatin-1 and Aplysiatoxin. CHEM REC 2014; 14:251-67. [DOI: 10.1002/tcr.201300036] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2013] [Indexed: 11/06/2022]
Affiliation(s)
- Kazuhiro Irie
- Division of Food Science and Biotechnology; Graduate School of Agriculture; Kyoto University; Kyoto 606-8502 Japan
| | - Ryo C. Yanagita
- Department of Applied Biological Science; Faculty of Agriculture, Kagawa University; Kagawa 761-0795 Japan
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33
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Structure–activity studies at position 27 of aplog-1, a simplified analog of debromoaplysiatoxin with anti-proliferative activity. Tetrahedron 2013. [DOI: 10.1016/j.tet.2013.02.008] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
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Yanagita RC, Kamachi H, Kikumori M, Tokuda H, Suzuki N, Suenaga K, Nagai H, Irie K. Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities. Bioorg Med Chem Lett 2013; 23:4319-23. [PMID: 23803585 DOI: 10.1016/j.bmcl.2013.05.096] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Revised: 05/25/2013] [Accepted: 05/30/2013] [Indexed: 11/15/2022]
Abstract
Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.
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Affiliation(s)
- Ryo C Yanagita
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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35
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Kamachi H, Tanaka K, Yanagita RC, Murakami A, Murakami K, Tokuda H, Suzuki N, Nakagawa Y, Irie K. Structure–activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity. Bioorg Med Chem 2013; 21:2695-702. [DOI: 10.1016/j.bmc.2013.03.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2013] [Revised: 03/13/2013] [Accepted: 03/14/2013] [Indexed: 11/25/2022]
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Abstract
Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumour promoter phorbol ester in 1982. Although initial therapeutic efforts focused on cancer, additional indications--including diabetic complications, heart failure, myocardial infarction, pain and bipolar disorder--were targeted as researchers developed a better understanding of the roles of eight conventional and novel PKC isozymes in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This Review provides a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs.
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37
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Kikumori M, Yanagita RC, Tokuda H, Suzuki N, Nagai H, Suenaga K, Irie K. Structure–Activity Studies on the Spiroketal Moiety of a Simplified Analogue of Debromoaplysiatoxin with Antiproliferative Activity. J Med Chem 2012; 55:5614-26. [DOI: 10.1021/jm300566h] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Masayuki Kikumori
- Division of
Food Science and
Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
| | - Ryo C. Yanagita
- Division of
Food Science and
Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
- Department of Applied Biological
Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan
| | - Harukuni Tokuda
- Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan
| | - Nobutaka Suzuki
- Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan
| | - Hiroshi Nagai
- Department
of Ocean Sciences, Tokyo University of Marine Science and Technology,
Tokyo 108-8477, Japan
| | - Kiyotake Suenaga
- Faculty
of Science and Technology, Keio University, Yokohama 223-8522, Japan
| | - Kazuhiro Irie
- Division of
Food Science and
Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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38
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Miralem T, Lerner-Marmarosh N, Gibbs PEM, Tudor C, Hagen FK, Maines MD. The human biliverdin reductase-based peptide fragments and biliverdin regulate protein kinase Cδ activity: the peptides are inhibitors or substrate for the protein kinase C. J Biol Chem 2012; 287:24698-712. [PMID: 22584576 DOI: 10.1074/jbc.m111.326504] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCδ. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.
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Affiliation(s)
- Tihomir Miralem
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
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39
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Irie K, C. Yanagita R, Tokuda H, Suzuki N, Shu Y. Synthesis of Antineoplastic Analogs of Aplysiatoxin with Various Side Chain Structures. HETEROCYCLES 2012. [DOI: 10.3987/com-12-s(n)8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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40
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Kawamura T, Matsubara K, Otaka H, Tashiro E, Shindo K, Yanagita RC, Irie K, Imoto M. Generation of 'Unnatural Natural Product' library and identification of a small molecule inhibitor of XIAP. Bioorg Med Chem 2011; 19:4377-85. [PMID: 21696964 DOI: 10.1016/j.bmc.2011.05.009] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2011] [Revised: 05/10/2011] [Accepted: 05/10/2011] [Indexed: 10/18/2022]
Abstract
Natural products have been utilized for drug discovery. To increase the source diversity, we generated a new chemical library consisting of chemically modified microbial metabolites termed 'Unnatural Natural Products' by chemical conversion of microbial metabolites in crude broth extracts followed by purification of reaction products with the LC-photo diode array-MS system. Using this library, we discovered an XIAP inhibitor, C38OX6, which restored XIAP-suppressed enzymatic activity of caspase-3 in vitro. Furthermore, C38OX6 sensitized cancer cells to anticancer drugs, whereas the unconverted natural product did not. These findings suggest that our library could be a useful source for drug seeds.
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Affiliation(s)
- Tatsuro Kawamura
- Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
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41
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Irie K, Yanagita RC, Nakagawa Y. Challenges to the development of bryostatin-type anticancer drugs based on the activation mechanism of protein kinase Cδ. Med Res Rev 2010; 32:518-35. [DOI: 10.1002/med.20220] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Kazuhiro Irie
- Division of Food Science and Biotechnology; Graduate School of Agriculture; Kyoto University; Kitashirakawa Oiwake-cho, Sakyo-ku Kyoto Japan
| | - Ryo C. Yanagita
- Division of Food Science and Biotechnology; Graduate School of Agriculture; Kyoto University; Kitashirakawa Oiwake-cho, Sakyo-ku Kyoto Japan
| | - Yu Nakagawa
- Synthetic Cellular Chemistry Laboratory; Advanced Science Institute; RIKEN; Wako-shi Saitama Japan
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42
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Yanagita RC, Kamachi H, Tanaka K, Murakami A, Nakagawa Y, Tokuda H, Nagai H, Irie K. Role of the phenolic hydroxyl group in the biological activities of simplified analogue of aplysiatoxin with antiproliferative activity. Bioorg Med Chem Lett 2010; 20:6064-6. [DOI: 10.1016/j.bmcl.2010.08.051] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2010] [Revised: 08/10/2010] [Accepted: 08/11/2010] [Indexed: 10/19/2022]
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43
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Nakagawa Y, Yanagita RC, Hamada N, Murakami A, Takahashi H, Saito N, Nagai H, Irie K. A simple analogue of tumor-promoting aplysiatoxin is an antineoplastic agent rather than a tumor promoter: development of a synthetically accessible protein kinase C activator with bryostatin-like activity. J Am Chem Soc 2009; 131:7573-9. [PMID: 19449873 DOI: 10.1021/ja808447r] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Protein kinase C (PKC) is widely recognized as a therapeutic target in intractable diseases such as cancer, Alzheimer's disease (AD), and acquired immune deficiency syndrome (AIDS). While inhibition of PKC is a general therapeutic strategy for the treatment of cancer, PKC activators are potential therapeutic agents for AD and AIDS. However, concerns have been raised about their therapeutic use since PKC activators such as phorbol esters exhibit potent tumor-promoting activities. Naturally occurring bryostatin 1 (bryo-1), prostratin, and 12-deoxyphorbol 13-phenylacetate (DPP) are fascinating PKC activators without tumor-promoting activities. Bryo-1 is currently in clinical trials for the treatment of cancer and is also effective against AD. Prostratin and DPP are attractive candidates for the adjunctive treatment of human immunodeficiency virus (HIV) infection. However, their limited availability from natural sources and synthetic complexity have hampered further development as therapeutic agents. We report here easy access (22 steps) to a simple analogue (1) of the tumor-promoting aplysiatoxin (ATX) as a novel PKC activator with anticancer and anti-tumor-promoting activities. Anticancer activities of 1 against several human cancer cell lines were comparable to those of bryo-1. Moreover, 1 as well as bryo-1 significantly inhibited the Epstein-Barr virus early antigen (EBV-EA) induction by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), whereas ATX strongly induced EBV-EA. This inhibitory effect is characteristic of antitumor promoters. Compound 1 as well as bryo-1 displayed significant binding and activation of PKCdelta and induced its translocation to the nuclear membrane in CHO-K1 cells. This study provides a synthetically accessible PKC activator with bryo-1-like activities, which could be another therapeutic lead for cancer, AD, and AIDS.
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Affiliation(s)
- Yu Nakagawa
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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44
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Yanagita RC, Nakagawa Y, Yamanaka N, Kashiwagi K, Saito N, Irie K. Synthesis, conformational analysis, and biological evaluation of 1-hexylindolactam-V10 as a selective activator for novel protein kinase C isozymes. J Med Chem 2007; 51:46-56. [PMID: 18072722 DOI: 10.1021/jm0706719] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Conventional and novel protein kinase C (PKC) isozymes are the main targets of tumor promoters. We developed 1-hexylindolactam-V10 ( 5) as a selective activator for novel PKC isozymes that play important roles in various cellular processes related to tumor promotion, ischemia--reperfusion injury in the heart, and Alzheimer's disease. The compound existed as a mixture of three conformers. The trans-amide restricted analogues of 5 ( 14 and 15) hardly bound to PKC isozymes, suggesting that the active conformation of 5 could be that with a cis-amide. Compound 5 selectively translocated novel PKC isozymes over conventional PKC isozymes in HeLa cells at 0.1-1 microM. These results suggest that 5 could be useful for the functional analysis of novel PKC isozymes.
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Affiliation(s)
- Ryo C Yanagita
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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45
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Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/pi interaction. Bioorg Med Chem 2007; 16:650-7. [PMID: 17977729 DOI: 10.1016/j.bmc.2007.10.045] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2007] [Revised: 10/13/2007] [Accepted: 10/16/2007] [Indexed: 11/22/2022]
Abstract
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/pi interaction with Pro-11 of the PKCdelta-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 PKCdelta-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.
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46
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Hirai G, Shimizu T, Watanabe T, Ogoshi Y, Ohkubo M, Sodeoka M. Importance of Interaction between C1 Domain and Lipids in Protein Kinase Cα Activation: Hydrophobic Side Chain Direction in Isobenzofuranone Ligands Controls Enzyme Activation Level. ChemMedChem 2007; 2:1006-9. [PMID: 17492704 DOI: 10.1002/cmdc.200700080] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Go Hirai
- Synthetic Organic Chemistry Laboratory, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan
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47
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Ling M, Sunesson L, Larsson C. Comparison of the PKCalpha and the PKCepsilon C1b domains: identification of residues critical for PKCepsilon-mediated neurite induction. J Mol Biol 2007; 368:951-65. [PMID: 17382347 DOI: 10.1016/j.jmb.2007.02.073] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2007] [Revised: 02/12/2007] [Accepted: 02/16/2007] [Indexed: 11/15/2022]
Abstract
We showed earlier that over-expression of protein kinase C (PKC) epsilon induces neurite outgrowth. The effect is mediated by a region (PKCepsilonPSC1V3) encompassing the pseudosubstrate, the two C1 domains and part of the V3 region, and is independent of the catalytic activity of the enzyme. In this region, residues immediately N-terminal of the C1b domain are crucial for neurite outgrowth. However, in this study we show that the PKCepsilon C1b domain itself is necessary for neurite induction, since a mutant in which the PKCepsilon C1b domain has been replaced with the C1b domain from PKCalpha, PKCepsilonPSC1a(alphaC1b)V3 lacks neurite-inducing capacity. The molecular basis for the importance of the PKCepsilon C1b domain was investigated by mutation studies of the PKCalpha C1b domain. Point mutations were done in the PKCalpha C1b domain of the PKCepsilonPSC1a(alphaC1b)V3 construct, in which the PKCalpha residues were mutated into the corresponding residues in PKCepsilon. This highlighted residues in the C-terminal part of the primary sequence of the C1b domain, located in the base of the C1b domain, as important for neurite outgrowth. The mutations S48P, D32K and L49N all influenced neurite induction positively. Furthermore, the mutation of L49N alone was sufficient to make PKCepsilonPSC1a(alphaC1b)V3 neuritogenic in phorbol ester-stimulated cells, and mutation of this residue in full-length PKCepsilon into the corresponding residue in PKCalpha, N291L reduced the neurite-inducing effect of PKCepsilon. In conclusion, we have identified residues in the PKCepsilon C1b domain, in particular Asn49, that are essential for neurite outgrowth.
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Affiliation(s)
- Mia Ling
- Lund University, Department of Laboratory Medicine, Malmö University Hospital, Malmö, Sweden
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48
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Nakagawa Y, Irie K, Yanagita RC, Ohigashi H, Tsuda KI, Kashiwagi K, Saito N. Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta. J Med Chem 2006; 49:2681-8. [PMID: 16640328 DOI: 10.1021/jm050857c] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Conventional (alpha, betaI, betaII, gamma) and novel (delta, epsilon, eta, theta) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring, showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed 8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4 showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated PKC epsilon and eta from the cytoplasm to the plasma membrane of HeLa cells at 1 microM, whereas other PKC isozymes did not respond even at 10 microM. These results indicate that 4 could be a selective activator for PKC epsilon and eta.
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Affiliation(s)
- Yu Nakagawa
- Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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49
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Guo B, Reed K, Parissenti AM. Scanning Mutagenesis Studies Reveal Multiple Distinct Regions within the Human Protein Kinase C Alpha Regulatory Domain Important for Phorbol Ester-dependent Activation of the Enzyme. J Mol Biol 2006; 357:820-32. [PMID: 16460753 DOI: 10.1016/j.jmb.2005.12.056] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2005] [Revised: 12/14/2005] [Accepted: 12/16/2005] [Indexed: 11/22/2022]
Abstract
While phorbol ester-binding sites within protein kinase C alpha (PKCalpha) have been identified and characterized utilizing fragments of the enzyme, it remains unclear whether additional regions within the enzyme may play an important role in its ability to be activated by phorbol ester. To examine this hypothesis, we generated 20 glutathione-S-transferase-tagged, V1-deficient, human PKCalpha holoenzyme constructs in which tandem six or 12 amino acid residue stretches along the full regulatory domain were changed to alanine residues. Each protein was assessed for its ability to bind phorbol ester and to induce growth repression when its catalytic activity was activated by phorbol ester upon expression in yeast cells. Mutagenesis of residues 99-158 potently reduced phorbol binding, consistent with previously published findings on the importance of the C1b region in phorbol binding. In addition, we identified a number of regions within the PKC regulatory domain that, when mutagenized, blocked the activation of PKC-mediated growth repression by phorbol ester while actually enhancing phorbol ester binding in vitro (residues 33-62, and 75-86). This study thus helps distinguish regions important for phorbol binding from regions important for the ability of phorbol ester to activate the enzyme. Our findings also suggest that multiple regions within C2 are necessary for full activation of the enzyme by phorbol ester, in particular residues 231-254. Finally, three regions, when mutagenized, completely, blocked catalytic domain activity in vivo (residues 33-62, 75-86, and 123-146), underscoring the important role of regulatory domain sequences in influencing catalytic domain function, even in the absence of the V1 region containing the pseudosubstrate sequence. This is the first tandem mutagenesis study for PKC that assesses the importance of regions for both phorbol binding and for phorbol-dependent activation in the context of the entire holoenzyme.
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Affiliation(s)
- Baoqing Guo
- Tumour Biology Research Program, Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada
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50
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Matsubara T, Shirai Y, Miyasaka K, Murakami T, Yamaguchi Y, Ueyama T, Kai M, Sakane F, Kanoh H, Hashimoto T, Kamada S, Kikkawa U, Saito N. Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus. J Biol Chem 2006; 281:6152-64. [PMID: 16407189 DOI: 10.1074/jbc.m509873200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.
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Affiliation(s)
- Takehiro Matsubara
- Laboratory of Molecular Pharmacology and Laboratory of Biochemistry, Biosignal Research Center, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501
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