rd
23 Congress of the International Union for Biochemistry and Molecular Biology
th
44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
th
th
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
ANTILEUKEMIA EFFECT OF PHARMACOLOGICAL INHIBITION OF THE SPLICING
REGULATORY SRPKs
Siqueira, R.P.1; Silva, M.R.1; Barbosa, E.A.A.1; Polêto, M.D.1; Righetto, G.L.2;
Seraphim, T.V.3; Salgado, R.L.1; Ferreira, J.G.1; Barros, M.V.A.4; Almeida, M.R.1;
Júnior, A.S.5; Fietto, J.L.R.1; Kobarg, J.6; Oliveira, E.B.7; Teixeira, R.R.4; Borges, J.C.3;
Yunes, J.A.8; Bressan, G.C.1
1
Universidade Federal de Viçosa, Departamento de Bioquímica e Biologia Molecular,
Minas Gerais, Brasil; 2Universidade Estadual de Campinas, Instituto de Biologia, São
Paulo, Brasil; 3Universidade de São Paulo, Instituto de Química, São Paulo, Brasil;
4
Universidade Federal de Viçosa, Departamento de Química, Minas Gerais, Brasil;
5
Universidade Federal de Viçosa, Departamento de Veterinária, Minas Gerais, Brasil;
6
Centro Nacional de Pesquisa em Energia e Materiais, Laboratório Nacional de
Biociências, São Paulo, Brasil; 7Universidade Federal de Viçosa, Departamento de
Engenharia de Alimentos, Minas Gerais, Brasil; 8Centro Infantil de Investigações
Hematológicas Dr Domingos A Boldrini, Laboratório de Biologia Molecular, São Paulo,
Brasil.
INTRODUCTION AND OBJECTIVES: Pre-mRNA splicing dysregulation has been
related to the genesis of several diseases, including cancer. The serine/arginine-rich
protein kinases family (SRPKs) plays a critical role in regulating pre-mRNA splicing
through the phosphorylation of serine/arginine-rich proteins (SR proteins). SRPKs have
been found highly expressed in several cancer types, suggesting that theey would serve
as targets for developing novel antitumor strategies. Here we aimed to evaluate the
antileukemia potential of a selective SRPKs inhibitor, the compound N-(2-(piperidin-1yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340). Also, we sought for in silico
and in vitro structural information that would explain the SRPIN340 inhibitory
activity. MATERIALS AND METHODS: MTT assays were performed to evaluate the
compound cytotoxicity and to estimate its IC50 in a panel of leukemia cell lineages.
Annexin V/PI double staining was approached to investigate cell death. Western
blotting, RT-qPCR and RT-PCR were carried out to investigate SRPK1 and SRPK2
gene expression and to access the impact of treatments on SRPKs cellular
activity. Finally, analysis of intrinsic tryptophan fluorescence emission, docking and
molecular dynamics were performed in order to gain structural information on SRPKsSRPIN340 complex. RESULTS AND CONCLUSIONS: SRPK1 and SRPK2 were found
overexpressed in several leukemia cell lineages but not in non transformed
PBMCs. Along with significant cytotoxic activity, the impact of SRPIN340 treatments on
SR proteins phosphorylation and expression of MAP2K genes were observed. Also, the
treatments triggered early and late events of apoptosis. The in vitro structural data
obtained with a recombinant SRPK2 form together with the analysis of docking and
molecular dynamics provided plausible mechanisms about SRPIN340 inhibitory
activity. These data point that SRPKs pharmacological inhibition should be considered
as a alternative antileukemia strategy. Moreover, the structural information on SRPKligand complex obtained may pave the way toward the development of novel
compounds with improved drug-like properties. ACKNOWLEDGEMENTS: CAPES,
CNPq, FAPEMIG, FUNARBE/FUNARPEX.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)
rd
23 Congress of the International Union for Biochemistry and Molecular Biology
th
44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
th
th
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
KeyWords: Serine/arginine-rich protein kinases; pre-mRNA splicing; Leukemia.
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)