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

Siqueira RP, Barbosa Éde A, Polêto MD, Righetto GL, Seraphim TV, Salgado RL, Ferreira JG, Barros MV, de Oliveira LL, Laranjeira AB, Almeida MR, Júnior AS, Fietto JL, Kobarg J, de Oliveira EB, Teixeira RR, Borges JC, Yunes JA, Bressan GC - PLoS ONE (2015)

Bottom Line: 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.Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Analysis of SRPK1 and SRPK2 expression in leukemia cell lines.The expression of SRPK1 and SRPK2 were analyzed by (A) Western blotting and (B) RT-qPCR assays in different leukemia cell lines derived from chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (ALL-T), and B-cell acute lymphoblastic leukemia (ALL-B). (A) The histogram (below) represents the ratio of the band intensities of SRPK1 and SRPK2 normalized to the actin signal for each lineage. Densitometry analysis of the band intensity was performed using ImageJ software. Error bars represent means ± standard deviation from triplicate experiments. Because SRPK1 or SRPK2 signals in the PBMC samples could not be detected during the WB assays, even when higher amounts of material were used (data not shown), they were not considered for the densitometry analysis. Although we found that actin expression varied between leukemia cells and our PBMC samples (S1 Fig), actin was detected here to qualitatively control the presence of protein material. (B) Expression of SRPK1 and SRPK2 transcripts by relative quantification. Amplification of beta-2-microglobulin mRNA (B2M) was used as an endogenous control. B2M was equally expressed among all of the leukemia lineages evaluated (S1 Fig and data not shown). SRPK1 and SRPK2 mRNA quantification in PBMC are discussed in S1 Fig. All primers used are detailed in S1 Table.
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pone.0134882.g001: Analysis of SRPK1 and SRPK2 expression in leukemia cell lines.The expression of SRPK1 and SRPK2 were analyzed by (A) Western blotting and (B) RT-qPCR assays in different leukemia cell lines derived from chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (ALL-T), and B-cell acute lymphoblastic leukemia (ALL-B). (A) The histogram (below) represents the ratio of the band intensities of SRPK1 and SRPK2 normalized to the actin signal for each lineage. Densitometry analysis of the band intensity was performed using ImageJ software. Error bars represent means ± standard deviation from triplicate experiments. Because SRPK1 or SRPK2 signals in the PBMC samples could not be detected during the WB assays, even when higher amounts of material were used (data not shown), they were not considered for the densitometry analysis. Although we found that actin expression varied between leukemia cells and our PBMC samples (S1 Fig), actin was detected here to qualitatively control the presence of protein material. (B) Expression of SRPK1 and SRPK2 transcripts by relative quantification. Amplification of beta-2-microglobulin mRNA (B2M) was used as an endogenous control. B2M was equally expressed among all of the leukemia lineages evaluated (S1 Fig and data not shown). SRPK1 and SRPK2 mRNA quantification in PBMC are discussed in S1 Fig. All primers used are detailed in S1 Table.

Mentions: Whereas no large differences in SRPK1 protein expression among the lineages were observed, at the protein level (Fig 1A), SRPK2 presented a distinct elevated expression in lineages of lymphoid origin, such as Molt4, TALL, Jurkat (ALL-T) and RS4 (ALL-B). Additionally, Molt4, TALL, Jurkat and RS4 cells presented a higher SRPK2 protein expression compared to SRPK1. Further RT-qPCR experiments revealed that among the nine lineages studied, only Molt4 and Jurkat presented correlations between mRNA and protein levels, indicating that additional layers of complexity involving the gene expression control of these kinases might exist in these cells, which deserves additional investigation in future studies (Fig 1B). We also used human PBMC as a control for non-transformed cells in Western blotting and RT-qPCR experiments. However, SRPK1 and SRPK2 were barely expressed (S1 Fig) or were even undetected in these assays (Fig 1A).


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

Siqueira RP, Barbosa Éde A, Polêto MD, Righetto GL, Seraphim TV, Salgado RL, Ferreira JG, Barros MV, de Oliveira LL, Laranjeira AB, Almeida MR, Júnior AS, Fietto JL, Kobarg J, de Oliveira EB, Teixeira RR, Borges JC, Yunes JA, Bressan GC - PLoS ONE (2015)

Analysis of SRPK1 and SRPK2 expression in leukemia cell lines.The expression of SRPK1 and SRPK2 were analyzed by (A) Western blotting and (B) RT-qPCR assays in different leukemia cell lines derived from chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (ALL-T), and B-cell acute lymphoblastic leukemia (ALL-B). (A) The histogram (below) represents the ratio of the band intensities of SRPK1 and SRPK2 normalized to the actin signal for each lineage. Densitometry analysis of the band intensity was performed using ImageJ software. Error bars represent means ± standard deviation from triplicate experiments. Because SRPK1 or SRPK2 signals in the PBMC samples could not be detected during the WB assays, even when higher amounts of material were used (data not shown), they were not considered for the densitometry analysis. Although we found that actin expression varied between leukemia cells and our PBMC samples (S1 Fig), actin was detected here to qualitatively control the presence of protein material. (B) Expression of SRPK1 and SRPK2 transcripts by relative quantification. Amplification of beta-2-microglobulin mRNA (B2M) was used as an endogenous control. B2M was equally expressed among all of the leukemia lineages evaluated (S1 Fig and data not shown). SRPK1 and SRPK2 mRNA quantification in PBMC are discussed in S1 Fig. All primers used are detailed in S1 Table.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526641&req=5

pone.0134882.g001: Analysis of SRPK1 and SRPK2 expression in leukemia cell lines.The expression of SRPK1 and SRPK2 were analyzed by (A) Western blotting and (B) RT-qPCR assays in different leukemia cell lines derived from chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (ALL-T), and B-cell acute lymphoblastic leukemia (ALL-B). (A) The histogram (below) represents the ratio of the band intensities of SRPK1 and SRPK2 normalized to the actin signal for each lineage. Densitometry analysis of the band intensity was performed using ImageJ software. Error bars represent means ± standard deviation from triplicate experiments. Because SRPK1 or SRPK2 signals in the PBMC samples could not be detected during the WB assays, even when higher amounts of material were used (data not shown), they were not considered for the densitometry analysis. Although we found that actin expression varied between leukemia cells and our PBMC samples (S1 Fig), actin was detected here to qualitatively control the presence of protein material. (B) Expression of SRPK1 and SRPK2 transcripts by relative quantification. Amplification of beta-2-microglobulin mRNA (B2M) was used as an endogenous control. B2M was equally expressed among all of the leukemia lineages evaluated (S1 Fig and data not shown). SRPK1 and SRPK2 mRNA quantification in PBMC are discussed in S1 Fig. All primers used are detailed in S1 Table.
Mentions: Whereas no large differences in SRPK1 protein expression among the lineages were observed, at the protein level (Fig 1A), SRPK2 presented a distinct elevated expression in lineages of lymphoid origin, such as Molt4, TALL, Jurkat (ALL-T) and RS4 (ALL-B). Additionally, Molt4, TALL, Jurkat and RS4 cells presented a higher SRPK2 protein expression compared to SRPK1. Further RT-qPCR experiments revealed that among the nine lineages studied, only Molt4 and Jurkat presented correlations between mRNA and protein levels, indicating that additional layers of complexity involving the gene expression control of these kinases might exist in these cells, which deserves additional investigation in future studies (Fig 1B). We also used human PBMC as a control for non-transformed cells in Western blotting and RT-qPCR experiments. However, SRPK1 and SRPK2 were barely expressed (S1 Fig) or were even undetected in these assays (Fig 1A).

Bottom Line: 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.Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus