Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340)

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

[1]  J. Oxley,et al.  SERINE ARGININE PROTEIN KINASE-1 (SRPK1) INHIBITION AS A POTENTIAL NOVEL TARGETED THERAPEUTIC STRATEGY IN PROSTATE CANCER , 2014, Oncogene.

[2]  D. Bates,et al.  Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma , 2014, British Journal of Cancer.

[3]  A. Newton,et al.  Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt. , 2014, Molecular cell.

[4]  S. Chi,et al.  Identification of a Novel Function of CX-4945 as a Splicing Regulator , 2014, PloS one.

[5]  Qianqian Wu,et al.  SRPK1 Dissimilarly Impacts on the Growth, Metastasis, Chemosensitivity and Angiogenesis of Glioma in Normoxic and Hypoxic Conditions , 2013, Journal of Cancer.

[6]  S. Knapp,et al.  Topical antiangiogenic SRPK1 inhibitors reduce choroidal neovascularization in rodent models of exudative AMD. , 2013, Investigative ophthalmology & visual science.

[7]  C. Sette,et al.  Phosphorylation-Mediated Regulation of Alternative Splicing in Cancer , 2013, International journal of cell biology.

[8]  S. Harper,et al.  SRPK1 inhibition modulates VEGF splicing to reduce pathological neovascularization in a rat model of retinopathy of prematurity. , 2013, Investigative ophthalmology & visual science.

[9]  Ryan M. Plocinik,et al.  Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases. , 2013, Journal of molecular biology.

[10]  A. Krainer,et al.  Isolated pseudo–RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition , 2013, Proceedings of the National Academy of Sciences.

[11]  Peter M. Kasson,et al.  GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit , 2013, Bioinform..

[12]  Xiang-Dong Fu,et al.  Regulation of splicing by SR proteins and SR protein-specific kinases , 2013, Chromosoma.

[13]  M. Hagiwara,et al.  Specific inhibition of serine/arginine-rich protein kinase attenuates choroidal neovascularization , 2013, Molecular vision.

[14]  K. Odunsi,et al.  Elevated Expression of the Serine-Arginine Protein Kinase 1 Gene in Ovarian Cancer and Its Role in Cisplatin Cytotoxicity In Vitro , 2012, PloS one.

[15]  E. Brambilla,et al.  Abnormal Expression of the Pre-mRNA Splicing Regulators SRSF1, SRSF2, SRPK1 and SRPK2 in Non Small Cell Lung Carcinoma , 2012, PloS one.

[16]  M. Ladomery,et al.  WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing. , 2011, Cancer cell.

[17]  G. Ghosh,et al.  The SRSF1 linker induces semi-conservative ESE binding by cooperating with the RRMs , 2011, Nucleic acids research.

[18]  M. Garcia-Blanco,et al.  The Kinase Inhibitor SFV785 Dislocates Dengue Virus Envelope Protein from the Replication Complex and Blocks Virus Assembly , 2011, PloS one.

[19]  E. Nikolakaki,et al.  Serine‐arginine protein kinases: a small protein kinase family with a large cellular presence , 2011, The FEBS journal.

[20]  Damian Szklarczyk,et al.  Specific CLK Inhibitors from a Novel Chemotype for Regulation of Alternative Splicing , 2011, Chemistry & biology.

[21]  K. Ye,et al.  The N-terminal Fragment from Caspase-cleaved Serine/Arginine Protein-specific Kinase2 (SRPK2) Translocates into the Nucleus and Promotes Apoptosis* , 2010, The Journal of Biological Chemistry.

[22]  M. Hagiwara,et al.  Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase , 2010, Antimicrobial Agents and Chemotherapy.

[23]  J. Engelman,et al.  The PI3K pathway as drug target in human cancer. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  K. Shokat,et al.  Targeting the cancer kinome through polypharmacology , 2010, Nature Reviews Cancer.

[25]  Kwok-Kin Wong,et al.  Targeting the PI3K signaling pathway in cancer. , 2010, Current opinion in genetics & development.

[26]  T. Cooper,et al.  The pathobiology of splicing , 2010, The Journal of pathology.

[27]  David S. Goodsell,et al.  AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility , 2009, J. Comput. Chem..

[28]  C. Will,et al.  The Spliceosome: Design Principles of a Dynamic RNP Machine , 2009, Cell.

[29]  Xiang-Dong Fu,et al.  Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones. , 2009, Genes & development.

[30]  Steven J. Harper,et al.  VEGF-A splicing: the key to anti-angiogenic therapeutics? , 2008, Nature Reviews Cancer.

[31]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[32]  S. Yang,et al.  Serine/arginine protein-specific kinase 2 promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1. , 2008, Cancer research.

[33]  P. Dorrestein,et al.  A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1. , 2008, Molecular cell.

[34]  K. Lynch,et al.  Regulation of Alternative Splicing: More than Just the ABCs* , 2008, Journal of Biological Chemistry.

[35]  David S. Goodsell,et al.  A semiempirical free energy force field with charge‐based desolvation , 2007, J. Comput. Chem..

[36]  L. Miller,et al.  Serine-arginine protein kinase 1 overexpression is associated with tumorigenic imbalance in mitogen-activated protein kinase pathways in breast, colonic, and pancreatic carcinomas. , 2007, Cancer research.

[37]  M. Hagiwara,et al.  Utilization of host SR protein kinases and RNA-splicing machinery during viral replication. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[38]  F. Gozzo,et al.  Spectroscopic characterization of the tumor antigen NY-REN-21 and identification of heterodimer formation with SCAND1. , 2006, Biochemical and biophysical research communications.

[39]  Laurence J Miller,et al.  Targeting the RNA splicing machinery as a novel treatment strategy for pancreatic carcinoma. , 2006, Cancer research.

[40]  T. Uchiyama,et al.  Serological identification of adult T‐cell leukaemia‐associated antigens , 2005, British journal of haematology.

[41]  Juan F Medrano,et al.  Real-time PCR for mRNA quantitation. , 2005, BioTechniques.

[42]  마사아키 스즈키,et al.  Methods for controlling SR protein phosphorylation, and antiviral agents whose active ingredients comprise agents that control SR protein activity , 2004 .

[43]  Chris Oostenbrink,et al.  A biomolecular force field based on the free enthalpy of hydration and solvation: The GROMOS force‐field parameter sets 53A5 and 53A6 , 2004, J. Comput. Chem..

[44]  A. W. Schüttelkopf,et al.  PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. , 2004, Acta crystallographica. Section D, Biological crystallography.

[45]  S. Salesse,et al.  p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells , 2004, Leukemia.

[46]  J. Adams,et al.  Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p. , 2003, Biochemistry.

[47]  S. Bustin.  Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. , 2000, Journal of molecular endocrinology.

[48]  L. Cantley,et al.  SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells , 1998, The Journal of cell biology.

[49]  Mark S. Gordon,et al.  General atomic and molecular electronic structure system , 1993, J. Comput. Chem..

[50]  M B Roth,et al.  SR proteins: a conserved family of pre-mRNA splicing factors. , 1992, Genes & development.

[51]  C. Breneman,et al.  Determining atom‐centered monopoles from molecular electrostatic potentials. The need for high sampling density in formamide conformational analysis , 1990 .

[52]  J. C. Borges,et al.  Structural and functional studies of Leishmania braziliensis Hsp90. , 2013, Biochimica et biophysica acta.

[53]  J. Cáceres,et al.  The SR protein family of splicing factors: master regulators of gene expression. , 2009, The Biochemical journal.

[54]  James Stévenin,et al.  Broad specificity of SR (serine/arginine) proteins in the regulation of alternative splicing of pre-messenger RNA. , 2004, Progress in nucleic acid research and molecular biology.

[55]  Brant C. White,et al.  United States patent , 1985 .