Chembiomolecular science at the frontier of chemistry and biology

Chembiomolecular Science wwwwwwwwwwww Masakatsu Shibasaki Masamitsu Iino Hiroyuki Osada Editors Chembiomolecular Science At the Frontier of Chemistry and Biology Editors Masakatsu Shibasaki Director Institute of Microbial Chemistry 3-14-23 Kamiosaki, Shinagawa-ku Tokyo 141-0021, Japan Hiroyuki Osada Director Antibiotics Laboratory Chemical Biology Core Faculty, RIKEN Advanced Science Institute 2-1 Hirosawa, Wako Saitama 351-0198, Japan Masamitsu Iino Professor Department of Pharmacology Graduate School of Medicine The University of Tokyo 7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan ISBN 978-4-431-54037-3 ISBN 978-4-431-54038-0 (eBook) DOI 10.1007/978-4-431-54038-0 Springer Tokyo Heidelberg New York Dordrecht London Library of Congress Control Number: 2012948879 © Springer Japan 2013 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface To understand biological functions at the molecular level and create new pharmaceuticals that can contribute to improving human health, the integration of both chemical and biological approaches is indispensable Chemical biology, taking advantage of the creativity of chemistry to explore biology, is currently a very important stream in life science Here we propose “chembiomolecular science” as a further advancement in the field of life science through the integration of chemical biology with molecular-level biological studies Chembiomolecular science will facilitate the elucidation of new biological mechanisms as potential drug targets and will enhance the creation of new drug leads This new field will promote worldclass life science research in Japan to the international scientific community In 2009, the Uehara Memorial Foundation announced a 3-year research program focused on chembiomolecular science To date, 20 research groups in Japan have been funded under this program The aim of the symposium was to bring together leading scientists in the field of chembiomolecular science to discuss their latest research The main topics to be addressed in the symposium were: Chembiomolecular chemistry Chembiomolecular biology Chembiomolecular medicinal chemistry The explicit aims of this symposium were to contribute to understanding the fundamentals of life science based on chemical and biological approaches, and the development of novel strategies for discovering new drug leads We are very pleased to be able to publish the proceedings of this exciting symposium Tokyo, Japan Masakatsu Shibasaki v wwwwwwwwwwww Contents Part I Chembiomolecular Chemistry Chemistry of Mycolactones, the Causative Toxins of Buruli Ulcer Yoshito Kishi Practical Synthesis of Tamiflu and Beyond Motomu Kanai An Approach Toward Identification of Target Proteins of Maitotoxin Based on Organic Synthesis Tohru Oishi, Keiichi Konoki, Rie Tamate, Kohei Torikai, Futoshi Hasegawa, Takeharu Nakashima, Nobuaki Matsumori, and Michio Murata 15 23 Inhibitors of Fatty Acid Amide Hydrolase Dale L Boger 37 Small Molecule Tools for Cell Biology and Cell Therapy Motonari Uesugi 51 Toward the Discovery of Small Molecules Affecting RNA Function Shiori Umemoto, Changfeng Hong, Jinhua Zhang, Takeo Fukuzumi, Asako Murata, Masaki Hagihara, and Kazuhiko Nakatani New Insights from a Focused Library Approach Aiming at Development of Inhibitors of Dual-Specificity Protein Phosphatases Go Hirai, Ayako Tsuchiya, and Mikiko Sodeoka The Deep Oceans as a Source for New Treatments for Cancer William Fenical, James J La Clair, Chambers C Hughes, Paul R Jensen, Susana P Gaudêncio, and John B MacMillan 59 69 83 vii viii Contents Search for New Medicinal Seeds from Marine Organisms Motomasa Kobayashi, Naoyuki Kotoku, and Masayoshi Arai 93 Identification of Protein–Small Molecule Interactions by Chemical Array 103 Hiroyuki Osada and Siro Simizu Part II Chembiomolecular Biology Small Molecule-Induced Proximity 115 Fu-Sen Liang and Gerald R Crabtree High-Throughput Screening for Small Molecule Modulators of FGFR2-IIIb Pre-mRNA Splicing 127 Erik S Anderson, Peter Stoilov, Robert Damoiseaux, and Douglas L Black Identification of Signaling Pathways That Mediate Dietary Restriction-Induced Longevity in Caenorhabditis elegans 139 Masaharu Uno, Sakiko Honjoh, and Eisuke Nishida Roles for the Stress-Responsive Kinases ASK1 and ASK2 in Tumorigenesis 145 Miki Kamiyama, Takehiro Sato, Kohsuke Takeda, and Hidenori Ichijo Tailored Synthetic Surfaces to Control Human Pluripotent Stem Cell Self-Renewal 155 Laura L Kiessling Cell-Surface Glycoconjugates Controlling Human T-Lymphocyte Homing: Implications for Bronchial Asthma and Atopic Dermatitis 167 Reiji Kannagi, Keiichiro Sakuma, and Katsuyuki Ohmori Establishment of a Novel System for Studying the Syk Function in B Cells 177 Tomohiro Kurosaki and Clifford A Lowell Visual Screening for the Natural Compounds That Affect the Formation of Nuclear Structures Kaya Shigaki, Kazuaki Tokunaga, Yuki Mihara, Yota Matsuo, Yamato Kojimoto, Hiroaki Yagi, Masayuki Igarashi, and Tokio Tani 183 Versatile Orphan Nuclear Receptor NR4A2 as a Promising Molecular Target for Multiple Sclerosis and Other Autoimmune Diseases 193 Shinji Oki, Benjamin J.E Raveney, Yoshimitsu Doi, and Takashi Yamamura Contents ix Antiviral MicroRNA 201 Ryota Ouda and Takashi Fujita Synaptic Function Monitored Using Chemobiomolecular Indicators 207 Masamitsu Iino Part III Chembiomolecular Medicinal Chemistry Practical Catalytic Asymmetric Synthesis of a Promising Drug Candidate 219 Masakatsu Shibasaki Hunting the Targets of Natural Product-Inspired Compounds 229 Slava Ziegler and Herbert Waldmann Chemical Approaches for Understanding and Controlling Infectious Diseases 239 Hirokazu Arimoto Nongenomic Mechanism-Mediated Renal Fibrosis-Decreasing Activity of a Series of PPAR-g Agonists 249 Hiroyuki Miyachi Novel Carbohydrate-Based Inhibitors That Target Influenza A Virus Sialidase 261 Mark von Itzstein Multidrug Efflux Pumps and Development of Therapeutic Strategies to Control Infectious Diseases 269 Kunihiko Nishino Enzymes as Chemotherapeutic Agents 281 Ronald T Raines Mechanism of Action of New Antiinfectious Agents from Microorganisms 293 Nobuhiro Koyama and Hiroshi Tomoda Correction of RNA Splicing with Antisense Oligonucleotides as a Therapeutic Strategy for a Neurodegenerative Disease 301 Yimin Hua, Kentaro Sahashi, Frank Rigo, Gene Hung, C Frank Bennett, and Adrian R Krainer Modulation of Pre-mRNA Splicing Patterns with Synthetic Chemicals and Their Clinical Applications 315 Masatoshi Hagiwara Index 321 wwwwwwwwwwww Antisense Splicing Modulation for SMA Therapy 309 chemistry has many desirable properties, including high Tm, high metabolic stability, broad distribution, and efficient cellular uptake in many tissues, without a requirement for lipid formulations or other carriers Importantly, MOE-PS/RNA duplexes are not substrates for RNase H or the RNAi pathway In addition, MOE-PS ASOs have already been used in clinical trials for other diseases, providing important precedents about chemistry-related PK/PD, safety, and tolerability 2¢-O-methyl ASOs targeted to ISS-N1 also promote exon inclusion, but in our hands this earlier-generation chemistry was not effective in some mouse tissues, and it elicited neuroinflammation [31] 2¢F modification instead of MOE unexpectedly caused efficient exon skipping because of recruitment of RNA-binding proteins to the duplex that interfered with splicing [32] Therefore, some but not all ASO chemistries will be appropriate as therapeutics for SMA Mechanism of Action ISIS-SMNRx is one of several overlapping ASOs with potent activity and clustered in the ISS-N1 region To determine the mechanism of action, we used site-directed mutagenesis to uncover specific splicing-regulatory elements within this silencer region [33] Point mutations uncovered a bipartite motif, and RNA pulldowns with HeLa cell nuclear extract and short intronic RNA immobilized on beads showed that the wild-type sequence is bound by the splicing repressors hnRNP A1 and A2 (Fig 6) The two motifs have synergistic effects on binding and on repression of exon Overexpression of hnRNP A1 causes further exon skipping in minigene co-transfection experiments, which is abrogated by mutating both parts of the bipartite ISS element [33] In addition, ISIS-SMNRx blocks hnRNP A1 binding in the RNA pulldowns, and as a result, binding of U1 snRNP to the 5¢ splice site of exon increases, confirming the proposed mechanism of action Preclinical Testing in Mice We have tested the optimized ASO in type III mice or in transgenic mice heterozygous at the mouse Smn locus, so that we could measure the ASO distribution and stability in tissues over time, in healthy animals, independently of any therapeutic effect [33] For heterozygous animals, transgene expression can be detected by RT-PCR with human-specific primers, and by immunoblotting using an SMN monoclonal antibody that does not cross-react with the mouse protein After intravenous or intraperitoneal administration of ASOs in adult mice, we observed efficient, dose-dependent correction of SMN2 splicing in liver and kidney, to a lesser extent in muscle, and no effect in spinal cord and brain [33] The latter was expected, because of the lack of distribution of ASOs across the blood– brain barrier (BBB) [34] We therefore tested direct delivery of the ASO to the 310 Y Hua et al Fig Correction of SMN2 splicing in the CNS by ISIS-SMNRx Mice with four copies of an SMN2 transgene, and either homozygous-null or heterozygous at the mouse Smn locus, were given 0–150 mg/day ASO dissolved in saline, for days, using intracerebroventricular (ICV) infusion with a surgically implanted micro-osmotic pump The mice were killed 2–3 days later and tissues were collected for RNA and protein analysis (a) Radioactive RT-PCR assay using RNA from L1–L2 lumbar spinal cord segments and primers positioned in exons and 8, specific for the human transgene (b) Immunoblotting of samples from the 50 mg/day treatment, using a human SMN-specific monoclonal antibody, and tubulin antibody as a loading control Detection was by infrared imaging CNS by several methods One method was intracerebroventricular (ICV) infusion with a stereotaxically implanted micro-osmotic pump for controlled delivery to cerebrospinal fluid for week (Fig 8); this resulted in very efficient exon splicing in the brain and spinal cord, and expression of SMN protein in ChAT-positive motor neurons Remarkably, the ASOs persisted for several months in central nervous system (CNS) tissues, driving efficient SMN2 splicing for more than months [31] We also tested the effect of single ICV injections of ASO in embryonic day 15 (E15) embryos or neonate mice [31] Again, we observed efficient correction of SMN2 splicing in CNS tissues, and moreover, this treatment was sufficient to significantly delay the onset of tail and ear necrosis In parallel experiments conducted at Genzyme Pharmaceuticals, the same ASO was tested by single ICV injections in postnatal day (P0) pups of the severe D7 model [35] Remarkably, this treatment resulted in extended survival, weight gain, preservation of motor neuron counts, and improved muscle function and coordination However, the survival gain was limited, that is, compared to the results of gene therapy with the same mouse strain [35] This limitation reflects in part the fact that the ASO half-life is shorter in the neonate mouse CNS than in the adult CNS [35]; in addition, as the animals grow, the ASO becomes effectively diluted We have done similar experiments with a very severe type I mouse strain and again observed a significant, albeit limited, effect in survival [36] We then compared the therapeutic effects of systemic versus CNS restoration of SMN, or the combination of both Surprisingly, subcutaneous ASO administration shortly after Antisense Splicing Modulation for SMA Therapy 311 birth had a much greater effect on survival than ICV administration alone The combined treatment had a further effect on survival Systemic administration alone allowed some mice to survive longer than year, compared to a mean survival of about 10 days in the controls The systemically treated mice performed very well in muscle strength and coordination tests The treatment also corrected the severe cardiac abnormalities Analysis of SMN2 expression in different tissues showed that systemic administration results in only slight effects in the CNS, as a result of some BBB permeability in newborn mice or retrograde transport of ASO from muscle However, the extent of splicing correction in the CNS is much smaller than observed by direct ICV administration These findings challenge the traditional view that increasing SMN in motor neurons is both necessary and sufficient for therapeutic benefit Thus, the genetic defect that causes motor neuron degeneration appears not to be (entirely) cell autonomous Future Directions The striking results obtained by systemic ASO administration in mouse models suggest that peripheral tissues play a critical role in SMA, indirectly influencing motor neuron health It will be important to determine which organs and cell types dominate this response to SMN2 splicing correction by the therapeutic ASO and how this then translates into preservation of motor neurons and increased survival Some of the findings made with SMA mouse models could reflect peculiarities of mouse development and physiology, or the extreme phenotypic severity of the type I models, and may not be directly relevant to the clinic The temporal window for ASO rescue appears to be within just a couple of days postnatally, but this is likely because the untreated mice survive only 10 days, with overt decline already apparent by P5 After ASO administration, some time is needed for the drug to accumulate in the target tissues, and for the resulting changes in splicing to lead to a sufficient increase in SMN protein, and to whatever downstream changes that elicits in turn The temporal window for treatment will likely be greater in less severe models Thus, there is currently an unmet need for mouse models with intermediate phenotypic severity, but that are still based on defective splicing of SMN2 Direct administration of ISIS-SMNRx to the CNS is on a clear path toward clinical trials The MOE-PS ASO does not cause inflammation in the mouse CNS, and experiments in nonhuman primates (NHP) show that intrathecal bolus injection is a simple and effective way to administer the ASO to CNS tissues [35] A full safety and tolerability study in NHP is in progress Whether systemic administration is also warranted in a clinical setting will need to be addressed by additional preclinical experiments in mice and appropriate safety and tolerability studies in NHP We are optimistic that ISIS-SMNRx may prove to be an effective drug for this devastating disease 312 Y Hua et al References Munsat TL, Davies KE (1992) International SMA consortium meeting (26–28 June 1992, Bonn, Germany) Neuromuscul Disord 2:423–428 Crawford TO (2003) Spinal muscular atrophies Butterworth-Heinemann, Philadelphia Russman BS (2007) Spinal muscular atrophy: clinical classification and disease heterogeneity J Child Neurol 22:946–951 Wang CH, Finkel RS, Bertini ES, Schroth M, Simonds A, Wong B, Aloysius A, Morrison L, Main M, Crawford TO et al (2007) Consensus statement for standard of care in spinal muscular atrophy J Child Neurol 22:1027–1049 Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M et al (1995) Identification and characterization of a spinal muscular atrophy-determining gene Cell 80:155–165 Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy Proc Natl Acad Sci USA 96:6307–6311 Cartegni L, Krainer AR (2002) Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1 Nat Genet 30:377–384 Kashima T, Manley JL (2003) A negative element in SMN2 exon inhibits splicing in spinal muscular atrophy Nat Genet 34:460–463 Cartegni L, Hastings ML, Calarco JA, de Stanchina E, Krainer AR (2006) Determinants of exon splicing in the spinal muscular atrophy genes, SMN1 and SMN2 Am J Hum Genet 78:63–77 10 Liu Q, Dreyfuss G (1996) A novel nuclear structure containing the survival of motor neurons protein EMBO J 15:3555–3565 11 Meister G, Buhler D, Pillai R, Lottspeich F, Fischer U (2001) A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs Nat Cell Biol 3:945–949 12 Pellizzoni L, Yong J, Dreyfuss G (2002) Essential role for the SMN complex in the specificity of snRNP assembly Science 298:1775–1779 13 Kolb SJ, Battle DJ, Dreyfuss G (2007) Molecular functions of the SMN complex J Child Neurol 22:990–994 14 Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, Kasim M, Dreyfuss G (2008) SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing Cell 133:585–600 15 Bäumer D, Lee S, Nicholson G, Davies JL, Parkinson NJ, Murray LM, Gillingwater TH, Ansorge O, Davies KE, Talbot K (2009) Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy PLoS Genet 5:e1000773 16 Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M (1997) Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos Proc Natl Acad Sci USA 94:9920–9925 17 Hsieh-Li HM, Chang JG, Jong YJ, Wu MH, Wang NM, Tsai CH, Li H (2000) A mouse model for spinal muscular atrophy Nat Genet 24:66–70 18 Monani UR, Sendtner M, Coovert DD, Parsons DW, Andreassi C, Le TT, Jablonka S, Schrank B, Rossol W, Prior TW et al (2000) The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn−/− mice and results in a mouse with spinal muscular atrophy Hum Mol Genet 9:333–339 19 Lunn MR, Wang CH (2008) Spinal muscular atrophy Lancet 371:2120–2133 20 Lim SR, Hertel KJ (2001) Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3¢ splice site pairing J Biol Chem 276:45476–45483 21 Miyajima H, Miyaso H, Okumura M, Kurisu J, Imaizumi K (2002) Identification of a cis-acting element for the regulation of SMN exon splicing J Biol Chem 277:23271–23277 22 Cartegni L, Krainer AR (2003) Correction of disease-associated exon skipping by synthetic exon-specific activators Nat Struct Biol 10:120–125 Antisense Splicing Modulation for SMA Therapy 313 23 Skordis LA, Dunckley MG, Yue B, Eperon IC, Muntoni F (2003) Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts Proc Natl Acad Sci USA 100:4114–4119 24 Singh NK, Singh NN, Androphy EJ, Singh RN (2006) Splicing of a critical exon of human survival motor neuron is regulated by a unique silencer element located in the last intron Mol Cell Biol 26:1333–1346 25 Coady TH, Shababi M, Tullis GE, Lorson CL (2007) Restoration of SMN function: delivery of a trans-splicing 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Rigo F, Hua Y, Chun SJ, Prakash TP, Krainer AR, Bennett CF (2012) Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing Nat Chem Biol 8:555–561 33 Hua Y, Vickers TA, Okunola HL, Bennett CF, Krainer AR (2008) Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice Am J Hum Genet 82:834–848 34 Geary RS, Yu RZ, Watanabe T, Henry SP, Hardee GE, Chappell A, Matson J, Sasmor H, Cummins L, Levin AA (2003) Pharmacokinetics of a tumor necrosis factor-a phosphorothioate 2¢-O-(2-methoxyethyl) modified antisense oligonucleotide: comparison across species Drug Metab Dispos 31:1419–1428 35 Passini MA, Bu J, Richards AM, Kinnecom C, Sardi SP, Stanek LM, Hua Y, Rigo F, Matson J, Hung G, Kaye EM, Shihabuddin LS, Krainer AR, Bennett CF, Cheng SH (2011) Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy Sci Transl Med 3:72ra18 36 Hua Y, Sahashi K, Rigo F, Hung G, Horev G, Bennett CF, Krainer AR (2010) Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model Nature 478:123–126 Modulation of Pre-mRNA Splicing Patterns with Synthetic Chemicals and Their Clinical Applications Masatoshi Hagiwara Introduction Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes This surprising finding underscores the importance of alternative splicing through which a single gene can generate structurally and functionally distinct protein isoforms [1] Based on genome-wide analysis, 75% of human genes are thought to encode at least two alternatively spliced isoforms [2, 3] The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis The splice sites are generally recognized by the splicing machinery, a ribonuclear protein complex known as the spliceosome Spliceosome binding is determined by competing activities of various auxiliary regulatory proteins, such as members of SR protein or heterogeneous nuclear ribonucleoprotein (hnRNP) families, which bind specific regulatory sequences and alter the binding of the spliceosome to a particular splice site [1, 4] Pre-mRNA splicing is regulated in a tissue-specific or developmental stage-specific manner [5] The selection of splice site can be altered by numerous extracellular stimuli such as hormones, immune response, neuronal depolarization, and cellular stress, through changes in synthesis/degradation, complex formation, and intracellular localization of regulatory proteins SR proteins are heavily phosphorylated in cells and involved in constitutive and alternative splicing, and the phosphorylation states of SR proteins are altered in response to these extracellular stimuli [6] Splicing mutations located in either intronic or exonic regions frequently cause hereditary diseases, and more than 15% of mutations that cause genetic disease M Hagiwara (*) Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan e-mail: hagiwara.masatoshi.8c@kyoto-u.ac.jp M Shibasaki et al (eds.), Chembiomolecular Science: At the Frontier of Chemistry and Biology, DOI 10.1007/978-4-431-54038-0_31, © Springer Japan 2013 315 316 M Hagiwara affect pre-mRNA splicing [7] Based on a hypothetical idea that we can cure human diseases by regulating the phosphorylation state of SR proteins with synthetic inhibitors of protein kinases, we started our long voyage to challenge the development of new chemical therapeutics SRPK Inhibitor SRPIN340 for Viral Diseases and Retinopathy By extensive screening of 100,000 chemical compounds in a chemical library using in vitro phosphorylation assay, we identified several synthetic chemical compounds that inhibit SR protein kinases (SRPKs) specifically We found synthetic compounds as specific inhibitors of SRPKs and Cdc-2-like kinases (Clks) and named them SRPIN340 and TG003, respectively [8, 9] RNA processing plays crucial roles to produce the complexity of viral proteins from the limited size of the gene A virus utilizes cellular machinery for viral RNA processing, although it is still unclear how virus and cellular machinery interplay and enable achieving the virus propagation SR proteins have been shown to be involved in the alternative splicing of mRNAs encoding human immunodeficiency virus (HIV)-1 proteins We found that expression of HIV-1 triggers the dephosphorylation and diminution of SR proteins and that overexpressed SRPK2, one of the SRPKs, preserves the phosphorylation state and protein amounts of SR proteins with the concomitant enhancement of HIV-1 production [8] These results indicate that SRPKs could be a new target of AIDS therapy In reality, SRPIN340 suppresses HIV-1 replication in the T-lymphocytederived MT-4 cell line and Jurkat cells [8] SRPIN340 also suppressed propagation of other viruses including Sindbis virus, severe acute respiratory syndrome (SARS) virus, and cytomegalovirus, suggesting that they may require SRPK-dependent SR protein phosphorylation for their multiplication Recently we found that a derivative of SRPIN340 suppresses virus propagation of the Flaviviridae family, which comprises other medically important pathogens including the Japanese encephalitis, yellow fever, and hepatitis C viruses [10] In addition to SRPK inhibitors, Clk inhibitor TG003 is also reported to suppress propagation of influenza [11] Vascular endothelial growth factor (VEGF) is a key regulatory component in physiological and pathological angiogenesis Two types of VEGF isoforms, the pro-angiogenic isoforms and the anti-angiogenic isoforms, are generated by splice site choice in exon [12] Proximal splice site selection in exon generates proangiogenic isoforms such as VEGF165, and distal splice site selection results in antiangiogenic isoforms such as VEGF165b Epithelial cells treated with insulin-like growth factor (IGF)-1 increased proximal splice site selection and produced more angiogenic isoforms In contrast, SRPK1/2 inhibition promoted distal splice site selection and produced more anti-angiogenic isoforms [13] Injection of SRPIN340 reduced angiogenesis in a mouse model of retinal neovascularization [13] SRPIN340 or another SRPK inhibitor may be applicable as anticancer drugs by switching the splicing pattern and promoting production of anti-angiogenic VEGF isoforms Therapeutic Modulation of Pre-mRNA Splicing 317 Clk Inhibitor TG003 for Muscular Dystrophy Recently we found the application of Clk inhibitor TG003 for Duchenne muscular dystrophy (DMD), which is a fatal muscle wasting disease caused by a loss of the dystrophin protein We met a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein was produced in the patient’s cells Analysis of the mRNA revealed that the mutation promotes exon skipping and restores the open reading frame of dystrophin Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promoted the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increased production of the dystrophin protein in the patient’s cells (Fig 1) [14] Although more preclinical studies with animal models are needed, TG003 is the first chemical compound verified to improve dystrophin production in in vitro patient-derived myotubes The splicing of Clk1/4 mRNAs is suspended in tissues and cultured cells, and the intermediate forms retaining specific introns are abundantly pooled in the nucleus [15] Administration of TG003 increased the level of Clk1/4 mature mRNAs by promoting splicing of the intron-retaining RNAs [16] Under stress conditions, splicing of general pre-mRNAs was inhibited by dephosphorylation of SR splicing factors, but exposure to stresses, such as heat shock and osmotic stress, promoted the maturation of Clk1/4 mRNAs Clk1/4 proteins translated after heat shock catalyzed rephosphorylation of SR proteins, especially SRSF4 and SRSF10 Our experimental data indicate that Clk1/4 proteins supplied through stress-induced splicing of the intron retaining pre-mRNAs are essential for rapid recovery of the phosphorylation state of Fig Drug treatment of Duchenne muscular dystrophy 318 M Hagiwara SR proteins and contribute to the restart of splicing reactions after stress removal of SR proteins Therefore, we named ReSCUE (rephosphorylation of SR proteins by Clk urgent expression) for the recovery of SR protein phosphorylation after stress removal [16] Dyrk Inhibitor INDY for Down Syndrome Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) is a serine/threonine kinase that is essential for maintaining the normal development and function of the brain The physiological importance of Dyrk1A has recently been highlighted by its proposed relationship with the pathogenesis of Down syndrome (DS) [15] DS is a congenital genetic disorder, caused by a complete or partial trisomy of chromosome 21, and characterized by systemic manifestations including mental retardation, cardiovascular anomalies, craniofacial dysmorphology, malignant neoplasms such as acute myoblastic leukemia, and recurrent infections [17] Dyrk1A resides within the so-called Down syndrome critical region (DSCR) of human chromosome 21, a genomic region that plays an important role in DS manifestations The extra copy of the Dyrk1A gene in DS indeed results in a 1.5-fold increase in the Dyrk1A product at both the protein and activity levels in the brain, and the excessive activity is considered a pathogenic factor in Down syndrome [18] Therefore, we searched for the specific inhibitor of Dyrk1A, and found that a benzothiazole derivative showed a potent inhibitory effect on the kinase activity [19] X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme INDY effectively reversed the aberrant tau phosphorylation and rescued the repressed NFAT signaling induced by Dyrk1A overexpression Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo [19] Conclusion We have developed chemical inhibitors of protein kinases that are involved in splicing regulation The compounds described here have the possibility to be developed as new therapeutic drugs for neuromuscular diseases, neoplastic diseases, and viral infections by manipulating splicing patterns or transcription level of specific genes Acknowledgments The author thanks past and present Hagiwara’s laboratory members and collaborators, especially T Hosoya, A Nishida, D.G Nowak, D.O Bates, M Matsuo, A Anwar, M.A Garcia-Blanco, T Fukuhara, M Muraki, N Kataoka, Y Ogawa, and H Onogi, for their contribution to the work described here This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and research grants from the Japan Science and Technology Agency, Takeda Science Foundation, The Naito Foundation Natural Science Scholarship, and The Uehara Memorial Foundation Therapeutic Modulation of Pre-mRNA Splicing 319 References Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing Annu Rev Biochem 72:291–336 Modrek B, Lee CJ (2003) Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss Nat Genet 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18:R75–R83 18 Dowjat WK, Adayev T, Kuchna I et al (2007) Trisomy-driven overexpression of DYRK1A kinase in the brain of subjects with Down syndrome Neurosci Lett 413:77–81 19 Ogawa Y, Nonaka Y, Goto T, Ohnishi E, Hiramatsu T, Kii I, Yoshida M, Ikura T, Onogi H, Shibuya H, Hosoya T, Ito N, Hagiwara M (2010) Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A Nat Commun 1:86 Index A ABI, 123 Abscisic acid (ABA), 121–123 Actinomycetes, 184–187 Active sites, 71, 73, 74, 76–78 Acyl protein thioesterase (APT1), 234–236 Adhesamine, 53–57 Affinity chromatography, 18 matrix-based proteomics, 230 probes, 230, 232, 233 purification, 230–231 Alkane thiols, 158, 159 Alpha-ketoheterocycle, 39, 41–46 Alternative pre-mRNA splicing, 184, 186, 188, 191 Alternative splicing, 128, 133, 136 Analgesic, 46 Anandamide, 38, 45, 46 Angiogenesis, 93 Antibacterial activity, 240–242, 244 Antibiotics, 133, 269, 270, 272–275, 277 Anticancer drugs, 133 Antisense oligonucleotide (ASO), 301–311 Anti-viral drugs, 261 Apoptosis, 145, 146, 148–151 Apoptosis signal-regulating kinase (ASK), 145–151 ASK1, 145–151 ASK2, 145–151 ASK1 signalosome, 146–148 APT1 See Acyl protein thioesterase (APT1) APT1 inhibitor, 234–236 ASO See Antisense oligonucleotide (ASO) Asymmetric catalysis, 223 Atom economy, 220 Atopic dermatitis, 167–175 Autoimmune disease model, 196 Autoimmune diseases, 193–200 Autoreactive T cells, 194, 195 B BBB See Blood brain barrier (BBB) B-cell antigen-receptor (BCR), 177, 178 Bcl3, 108, 110 BCR See B-cell antigen-receptor (BCR) Bioactive compound, 132, 136 BioMol, 132 Biotinylated peptides, 163 Blood brain barrier (BBB), 309, 311 2-Bromopalmitate, 235–236 Bronchial asthma, 167–175 Buruli ulcer, 3–11 C Calorie restriction (CR), 139–143 Cancer, 281, 283–285, 287 Cantharidin, 133, 135 Cardiotonic steroids, 133 Catalytic asymmetric Diels–Alder reaction, 16, 18 CDC25 See Cell division cycle phosphatase (CDC25) Cdc-2-like kinase (Clk), 316–318 CDK See Cyclin-dependent kinase (CDK) Cell-adhesion molecules, 167 Cell-adhesive glycans, 167 Cell culture, 156, 159, 162, 163 Cell cycle, 69, 70, 75, 78–79 Cell division cycle phosphatase (CDC25), 70, 76 Cell therapy, 51–58 M Shibasaki et al (eds.), Chembiomolecular Science: At the Frontier of Chemistry and Biology, DOI 10.1007/978-4-431-54038-0, © Springer Japan 2013 321 322 Central helper memory T cells, 169–172, 174 Central nervous system (CNS), 310, 311 Cerebrospinal fluid, 310 Chemical array, 103–110 Chemical library, 59, 62 Chemically induced proximity (CIP), 116, 117, 118–123 Chemical modification, 239, 246 Chemical proteomics, 230 Chronic kidney disease (CKD), 249, 250, 260 CLA See Cutaneous lymphocyte-associated antigen (CLA) Clinical trials, 304, 309, 311 Colitis-induced cancer, 150 Competition, 231 Core structure, 71–79 Cortistatin A, 93, 94, 96, 100 Curtius rearrangement, 18 Cutaneous lymphocyte-associated antigen (CLA), 171, 172 Cyclic sialyl 6-sulfo Lewis x, 170, 172–175 Cyclin-dependent kinase (CDK), 70, 78 Cyclosporin A (CsA), 116, 117, 120, 122, 123 Cyslabdan, 293, 296–298 D 2D-DIGE, 230 Depalmitoylation, 235, 236 Diazirines, 230 Dietary restriction (DR), 139–143 Differentiation, 156, 158, 159, 162, 163 DMD See Duchenne muscular dystrophy (DMD) DNA binding motif, 195 microarray analysis, 197 Down syndrome, 318 Drug efflux, 269–271, 273–278 resistance, 239 synthesis, 219–227 Dual-specificity protein phosphatase, 69–79 Dual-specificity tyrosine-(Y)-phosphorylationregulated kinase 1A (Dyrk1A), 318 Duchenne muscular dystrophy (DMD), 317 Dynamins, 231–233 E EAE See Experimental autoimmune encephalomyelitis (EAE) Effector helper memory T cells, 169–171, 174 ELISA See Enzyme-linked immunosorbent assay (ELISA) Index Endocytosis, 233, 286 Enzyme-linked immunosorbent assay (ELISA), 231 Epithelial splicing regulatory protein (ESRP), 129 Epithelial to mesenchymal transition, 128 ERK See Extracellular signal-regulated kinase (ERK) ESRP See Epithelial splicing regulatory protein (ESRP) Exon, 302–306, 308–310 skipping, 316 Experimental autoimmune encephalomyelitis (EAE), 195–198 Extracellular matrix (ECM), 156 Extracellular signal-regulated kinase (ERK), 70, 71, 78 F Fatty acid amide hydrolase (FAAH), 37–46 Fatty acid amides, 37–39, 44 FGFR2 exons, 129 Fibroblast growth factor receptor (FGFR2), 128, 130, 135 FK506, 116, 117, 120, 122, 123 FK1012, 117, 118, 120, 121 FK506 binding protein 12 (FKBP12), 116, 117, 120, 121 FKBP, 116, 117, 120, 121 FKBP rapamycin-binding (FRB), 117, 120, 121 Fluorescence lifetime imaging microscopy (FLIM), 231, 235, 236 Fluorescence polarization (FP), 231 Fluorescent indicator displacement assay, 60–63, 66 Fluorescent tags, 86 Fluorogenic TLC detection, 10 Fluorophores, 286 Focused library, 69–79 Foerster resonance energy transfer (FRET), 231, 236 FRB*, 120, 121 Frontotemporal dementia with parkinsonism linked to chromosome 17, 128 Furospinosulin-1, 94, 97–100 G Gastric cancer (GC), 150, 151 Gene library, 106, 107 Gene regulation, 127–128 Gene therapy, 304, 310 Glutamate, 207, 208, 210–214 Glycosaminoglycan (GAG), 157, 159–162 Index H Hairpin loops, 59, 63–66 Haploinsufficiency screens, 230 HEK293T, 106–109 HeLa cells, 185–189 Helper memory T cells, 168, 169, 172 Heparanase, 75, 77 Hepatocarcinoma, 151 hnRNP A1, 302, 303, 307, 309 Human embryonic stem (hES) cells, 155–157, 159–162 Hypoxia selective growth inhibitor, 94, 97–99 I IL-17, 195, 197, 198, 200 Immediate/early genes, 194 Immunoprecipitation (IP), 86, 87, 89 Induced pluripotent stem (iPS) cells, 155 INDY, 318 Inflammation, 145, 148–150 Inflammatory responses, 195 Influenza virus, 261–266 Inhibitors, 69–79, 261–266 Inositol 1,4,5-trisphosphate (IP3), 208, 212–214 In situ hybridization, 185, 189, 190 Insulin/IGF-like signaling, 140, 142 Integrins, 157, 159, 160 Interferon (IFN), 201, 202, 204 Intermittent fasting (IF), 139, 141–142 Intracellular signal transduction, 69 Intrathecal, 311 IP See Immunoprecipitation (IP) ISIS-SMNRx, 304, 307, 309–311 Isothermal titration (ITC), 231 J c-Jun N-terminal kinase (JNK), 70, 71, 78, 145, 146, 148, 151 L Lactam-ring formation, 172, 175 Ladder-shaped polyether (LSP), 23, 25, 26, 30–34 Lariatin, 293–296 Lipstatin, 234 LSP See Ladder-shaped polyether (LSP) Lymphocyte homing, 167–175 323 M Maitotoxin (MTX), 23–34 Mammalian target of rapamycin (mTOR), 140 MAPT See Microtubule-associated protein tau (MAPT) Marine microorganisms, 84 Marine organism, 93–100 Mass spectroscopy (MS), 230 Matrigel, 156, 159, 162, 163 Matrix, 230 Mechanism of action, 293–298 Melanoma, 108–110 Melophlin A, 231–233 Membrane permeability, 75 Methicilin-resistant staphylococcus aureus (MRSA), 293, 296–298 Microbial metabolites, 104 Microtubule-associated protein tau (MAPT), 128 Migration, 108–110 miR-23b, 202–204 Mitogen-activated kinase phosphatase (MKP), 70, 71 Mitogen-activated protein kinase (MAPK) cascades, 145, 146 Mitogen-activated protein (MAP) kinases, 70, 71, 78, 79 MKP See Mitogen-activated kinase phosphatase (MKP) Molecular modeling, 75, 77, 78 Motor neurons, 301–303, 310, 311 Mouse, 303, 309–311 mRNA display, 230 MRSA See Methicilin-resistant staphylococcus aureus (MRSA) mTOR See Mammalian target of rapamycin (mTOR) Multidrug resistance, 270–273, 276, 278 Multiple sclerosis, 193–200 Muscle, 301, 303, 309–311 Mycolactones, 3–11 N Natural product (NP), 229–236 Natural products depository (NPDepo), 103–108 Neurodegenerative disease, 301–311 NHS sepharose, 230 Nitric oxide (NO), 208–210, 213 Noncoding RNA (ncRNA), 59 Nonhuman primates (NHP), 311 NR4A2 modulators, 199, 200 Nuclear receptor NR4A2, 193–200 Nuclear speckles, 183–191 324 O Oleamide, 37–39 Organic synthesis, 23–34 Oseltamivir, 15, 18–19 P p38, 70 Palmitoylation/depalmitoylation, 234 Palmostatin B, 233–236 Peptides, 157–161, 163 Peroxisome proliferator-activated receptor (PPAR)-g, 249–260 Phage display, 230 Phenotype, 103, 104 Phospholipase A2, 70, 75, 77 Photo-cross-linking, 105, 107 Photoreactive groups, 230 Photoreactive moiety, 231 Pirin inhibitor, 108–110 Pluripotency, 160, 161, 163 Polyethylene glycol (PEG) linker, 232 Polypyrimidine tract-binding protein (PTB), 128, 129, 131, 132, 136 Polysaccharides, 160, 161 PP2C, 122, 123 Pre-mRNA splicing, 127–137, 315–318 Prestwick, 132, 134, 135 Proinflammatory cytokines and chemokines, 196 Protein de/palmitoylation, 233–236 kinases, 69, 70 microarrays, 230 phosphatase inhibitors, 133 phosphatases, 69–79 phosphorylation, 69 targets, 85–87, 89 Protein structure similarity clustering (PSSC), 234 Proteoglycans, 160–162 Proteomics, 230, 231, 236 PSSC See Protein structure similarity clustering (PSSC) PTB See Polypyrimidine tract-binding protein (PTB) PYL, 122, 123 R Rapamycin, 116–118, 120, 121, 123 Ras, 231, 232, 234, 236 Ras signaling, 231–233, 235 Index RbFox1, 129 Reactive oxygen species (ROS), 147, 148 Red fluorescent protein (RFP), 105–108 Reporter gene assay, 232, 233 Retroviral infection, 198 Reverse transcription-polymerase chain reaction (RT-PCR), 131, 132, 135 RFP See Red fluorescent protein (RFP) RFP-fused proteins, 105, 107 Rheb/TOR pathway, 142 Rhinovirus1B (RV1B), 202–204 Ribonucleases, 281–289 RIG-I-like receptors (RLR), 201, 202 RNA binding molecules, 59 splicing, 301–311 RNAi, 231, 236 ROR-gt, 196 ROS See Reactive oxygen species (ROS) RT-PCR See Reverse transcriptionpolymerase chain reaction (RT-PCR) S SAM See Self-assembled monolayers (SAM) SAR See Structure activity relationship (SAR) SC35, 183, 184, 189–191 SCONP See Structural classification of natural products (SCONP) Screen, 158–163 Selectin, 167–170, 173, 174 Self antigen, 196 Self-assembled monolayers (SAM), 158–160, 162, 163 Serine hydrolase, 39, 41–43 Sialic acid cyclization, 173–175 Sialidase, 261–266 Sialyl Lewis x, 168, 170–172, 174 Sialyl 6-sulfo Lewis x, 168–175 SILAC See Stable isotope labeling in cell culture (SILAC) siRNA See Small interfering RNA (siRNA) Sleep aid, 38 Slug, 110 SMA See Spinal muscular atrophy (SMA) Small interfering RNA (siRNA), 78, 104, 108 Small molecules, 51–58, 129, 132–134, 136, 137 compounds, 129 modulators, 194 screen, 132–133, 135, 136 SMN2 See Survival motor neuron (SMN2) Spinal cord, 301, 309, 310 Index Spinal muscular atrophy (SMA), 301–304, 308, 309, 311 Spleen tyrosine kinase (Syk), 177–180 Spliceosome, 127–129, 186, 189 Splicing, 301–311 dependent dual fluorescence vector, 131 factors, 183, 184, 186, 187, 189, 191 Splicing factor-2 (SF2), 185–189, 191 SPR See Surface plasmon resonance (SPR) SRPIN340, 316 SR protein, 315, 316, 318 SR protein kinase (SRPK), 316 Stable isotope labeling in cell culture (SILAC), 231 Staurosporine, 188–191 Stem cell niche, 156, 157 Stereochemistry, 4–5, Streptavidin, 230, 232, 233 Structural classification of natural products (SCONP), 229 Structure, 4–11 Structure activity relationship (SAR), 230, 232, 234 Substrata, 156–158, 160, 162, 163 Surface array, 158–163 Surface plasmon resonance (SPR), 231, 232 Survival motor neuron (SMN2), 301–306, 309–311 Synapses, 207, 208, 210, 212, 213 Synthetic ligand for FKBP (SLF), 117, 120, 121 T Tamiflu-resistant virus, 19–20 Target identification, 230–231 Target proteins, 23–34 Tetrahydrolipstatin, 234 TG003, 316–318 TGF-b See Transforming growth factor-beta (TGF-b) Th17 cells, 196, 197 Therapeutic, 301–311 Thioredoxin (Trx), 147 TNF receptor-associated factor (TRAF2), 147 325 TNF receptor-associated factor (TRAF6), 147 TRAF2 See TNF receptor-associated factor (TRAF2) TRAF6 See TNF receptor-associated factor (TRAF6) Transcription factor, 194–198 Transfection, 135 Transforming growth factor-beta (TGF-b), 249–254, 256–257, 259–260 Transgenic mice, 303, 309 Tuberculosis, 293, 294, 296 Two-stage skin tumorigenesis model, 148, 149 V Vaccina H-1 related (VHR) phosphatase, 70–72 Vancomycin, 239–246 Vancomycin-resistant enterococci (VRE), 239–241, 246 van Gogh-like receptor protein (VANGL1), 233, 234 Vascular endothelial growth factor (VEGF), 316 Very low density lipoprotein receptor (VLDLR), 202–204 Virulence, 269, 276–278 Vitronectin, 161 VLDLR See Very low density lipoprotein receptor (VLDLR) VRE See Vancomycin-resistant enterococci (VRE) W Wntepans, 233, 234 Wnt pathway, 233 Y Yeast three-hybrid systems, 230 Z Zymogens, 287–288 ... development of the universal NMR database approach to assign the relative and absolute configuration of unknown compounds without degradation or derivatization, and we noticed that the universal NMR database... mycolactone core The unsaturated fatty acid is prepared from the building blocks D and E via the Horner–Emmons reaction, followed by saponification The coupling of the unsaturated fatty acid with the core,... products On the other hand, a remarkable structural diversity is observed in the unsaturated fatty acid portion, including the length of the fatty acid backbone, degree of unsaturation, degree of hydroxylation,