Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

[1]  Omar Abdel-Wahab,et al.  Therapeutic targeting of splicing in cancer , 2016, Nature Medicine.

[2]  E. Eyras,et al.  The translational landscape of the splicing factor SRSF1 and its role in mitosis , 2014, eLife.

[3]  Dario Neri,et al.  DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries. , 2014, Accounts of Chemical Research.

[4]  Asli N. Goktug,et al.  High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor , 2014, PloS one.

[5]  Stefan Schmitt,et al.  Stratified High-Throughput Screening Sets Enable Flexible Screening Strategies from a Single Plated Collection , 2014, Journal of biomolecular screening.

[6]  Taosheng Chen,et al.  Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery. , 2013, Bioorganic & medicinal chemistry.

[7]  Anne Mai Wassermann,et al.  Biodiversity of small molecules--a new perspective in screening set selection. , 2013, Drug discovery today.

[8]  Juan J. Marugan,et al.  Induction and Reversal of Myotonic Dystrophy Type 1 Pre-mRNA Splicing Defects by Small Molecules , 2013, Nature Communications.

[9]  M. Lohse,et al.  Time-resolved fluorescence ligand binding for G protein–coupled receptors , 2013, Nature Protocols.

[10]  Michael R. Taylor,et al.  Identification of Small Molecule Activators of BMP Signaling , 2013, PloS one.

[11]  J. Quinn.  Modeling Taylor dispersion injections: determination of kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing. , 2012, Analytical biochemistry.

[12]  J. Quinn.  Evaluation of Taylor dispersion injections: determining kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing. , 2012, Analytical biochemistry.

[13]  G. Laco.  Evaluation of Two Models for Human Topoisomerase I Interaction with dsDNA and Camptothecin Derivatives , 2011, PloS one.

[14]  J. Turchi,et al.  Identification of novel small molecule inhibitors of the XPA protein using in silico based screening. , 2010, ACS chemical biology.

[15]  C. Rock,et al.  Modulation of pantothenate kinase 3 activity by small molecules that interact with the substrate/allosteric regulatory domain. , 2010, Chemistry & biology.

[16]  D. Myszka,et al.  Exploring minimal biotinylation conditions for biosensor analysis using capture chips. , 2010, Analytical biochemistry.

[17]  Teresa Carlomagno,et al.  Structure of the K-turn U4 RNA: a combined NMR and SANS study , 2010, Nucleic acids research.

[18]  Paul J Hergenrother,et al.  A general method for discovering inhibitors of protein-DNA interactions using photonic crystal biosensors. , 2008, ACS chemical biology.

[19]  P. Flynn,et al.  Functional implications for a prototypical K-turn binding protein from structural and dynamical studies of 15.5K. , 2007, Biochemistry.

[20]  P. Flynn,et al.  NMR assignment of the human spliceosomal 15.5K protein , 2007, Journal of biomolecular NMR.

[21]  Ping Li,et al.  Binding of the Human Prp31 Nop Domain to a Composite RNA-Protein Platform in U4 snRNP , 2007, Science.

[22]  Y. Zu,et al.  Review camptothecin: current perspectives. , 2006, Current medicinal chemistry.

[23]  S. Hecht,et al.  Camptothecin: current perspectives. , 2004, Bioorganic & medicinal chemistry.

[24]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[25]  Lance Stewart,et al.  The mechanism of topoisomerase I poisoning by a camptothecin analog , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  I. Eperon,et al.  A double reporter assay for detecting changes in the ratio of spliced and unspliced mRNA in mammalian cells. , 2002, Nucleic acids research.

[27]  A. Lamond,et al.  In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles , 2002, The Journal of cell biology.

[28]  John Crosby,et al.  Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site. , 2002, Biochemistry.

[29]  K. Hartmuth,et al.  Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. , 2000, Molecular cell.

[30]  H. Urlaub,et al.  Functional interaction of a novel 15.5kD[U4/U6·U5] tri‐snRNP protein with the5′ stem–loop of U4 snRNA , 1999, The EMBO journal.

[31]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[32]  A. W. Czarnik,et al.  Inhibitors of protein-RNA complexation that target the RNA: specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules. , 1998, Biochemistry.

[33]  J. Beijnen,et al.  Clinical Pharmacokinetics of Topotecan , 1996, Clinical pharmacokinetics.

[34]  G. Bemis,et al.  The properties of known drugs. 1. Molecular frameworks. , 1996, Journal of medicinal chemistry.

[35]  K. Kohn,et al.  Differential stabilization of eukaryotic DNA topoisomerase I cleavable complexes by camptothecin derivatives. , 1995, Biochemistry.

[36]  L. Liu,et al.  Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. , 1988, Cancer research.

[37]  Stefan Wetzel,et al.  The Scaffold Tree - Visualization of the Scaffold Universe by Hierarchical Scaffold Classification , 2007, J. Chem. Inf. Model..

[38]  Y. Nakamura,et al.  Cloning and mapping of a human novel cDNA (NHP2L1) that encodes a protein highly homologous to yeast nuclear protein NHP2. , 1996, Cytogenetics and cell genetics.