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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

Molecular Dynamic Analysis.(A) Occupied space of SRPIN340 during simulations from poses A (blue) and B (green). Average protein structures are shown superposed in light blue for simulation A and in light green for simulation B. The ATP analogue ANP (shown in orange sticks) occupies the same region as SRPIN340 during both simulations. (B) Snapshot from pose A (upper) and pose B (bottom) simulations highlighting the possible SRPIN340 (orange sticks) interaction network. (C) Solvent accessible surface area (SASA) for tryptophan residues in the presence of the ligand, blue and green for poses A and B, respectively. The pose A simulation shows a signal increase at 175 ns, indicating a major exposure of residues to the polar solvent from this point of simulation. This is in agreement with the fluorescence spectroscopy analysis shown in Fig 6.
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pone.0134882.g005: Molecular Dynamic Analysis.(A) Occupied space of SRPIN340 during simulations from poses A (blue) and B (green). Average protein structures are shown superposed in light blue for simulation A and in light green for simulation B. The ATP analogue ANP (shown in orange sticks) occupies the same region as SRPIN340 during both simulations. (B) Snapshot from pose A (upper) and pose B (bottom) simulations highlighting the possible SRPIN340 (orange sticks) interaction network. (C) Solvent accessible surface area (SASA) for tryptophan residues in the presence of the ligand, blue and green for poses A and B, respectively. The pose A simulation shows a signal increase at 175 ns, indicating a major exposure of residues to the polar solvent from this point of simulation. This is in agreement with the fluorescence spectroscopy analysis shown in Fig 6.

Mentions: Once we observed the pharmacological effect of SRPIN340 in leukemia cells, we sought to investigate possible molecular interactions underlying its binding to SRPKs. While high-resolution crystallographic structures of the complex SRPIN340/SRPKs have not been solved to date, computational approaches were used to infer possible binding sites into SRPK structures currently available in PDB. A comparison of amino acid sequences revealed that both kinases are highly similar in their kinase domains [47] (S3 Fig), explaining why SRPIN340 can inhibit both SRPK1 and SRPK2 [25]. Considering this structural similarity, the position of the ATP analog, previously co-crystallized with SRPK1 [38], allowed the prediction of its binding site into SRPK2 (Fig 5A), the paralog chosen to be studied herein, because its mechanisms in leukemia tumorigenesis have been previously well determined [15].


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)

Molecular Dynamic Analysis.(A) Occupied space of SRPIN340 during simulations from poses A (blue) and B (green). Average protein structures are shown superposed in light blue for simulation A and in light green for simulation B. The ATP analogue ANP (shown in orange sticks) occupies the same region as SRPIN340 during both simulations. (B) Snapshot from pose A (upper) and pose B (bottom) simulations highlighting the possible SRPIN340 (orange sticks) interaction network. (C) Solvent accessible surface area (SASA) for tryptophan residues in the presence of the ligand, blue and green for poses A and B, respectively. The pose A simulation shows a signal increase at 175 ns, indicating a major exposure of residues to the polar solvent from this point of simulation. This is in agreement with the fluorescence spectroscopy analysis shown in Fig 6.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134882.g005: Molecular Dynamic Analysis.(A) Occupied space of SRPIN340 during simulations from poses A (blue) and B (green). Average protein structures are shown superposed in light blue for simulation A and in light green for simulation B. The ATP analogue ANP (shown in orange sticks) occupies the same region as SRPIN340 during both simulations. (B) Snapshot from pose A (upper) and pose B (bottom) simulations highlighting the possible SRPIN340 (orange sticks) interaction network. (C) Solvent accessible surface area (SASA) for tryptophan residues in the presence of the ligand, blue and green for poses A and B, respectively. The pose A simulation shows a signal increase at 175 ns, indicating a major exposure of residues to the polar solvent from this point of simulation. This is in agreement with the fluorescence spectroscopy analysis shown in Fig 6.
Mentions: Once we observed the pharmacological effect of SRPIN340 in leukemia cells, we sought to investigate possible molecular interactions underlying its binding to SRPKs. While high-resolution crystallographic structures of the complex SRPIN340/SRPKs have not been solved to date, computational approaches were used to infer possible binding sites into SRPK structures currently available in PDB. A comparison of amino acid sequences revealed that both kinases are highly similar in their kinase domains [47] (S3 Fig), explaining why SRPIN340 can inhibit both SRPK1 and SRPK2 [25]. Considering this structural similarity, the position of the ATP analog, previously co-crystallized with SRPK1 [38], allowed the prediction of its binding site into SRPK2 (Fig 5A), the paralog chosen to be studied herein, because its mechanisms in leukemia tumorigenesis have been previously well determined [15].

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