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Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

Kubota K, Inaba S, Nakano R, Watanabe M, Sakurai H, Fukushima Y, Ichikawa K, Takahashi T, Izumi T, Shinagawa A - Pharmacol Res Perspect (2015)

Bottom Line: Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes.Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level.This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.

ABSTRACT
CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

No MeSH data available.


Related in: MedlinePlus

CS-917 esterase activity in the small intestine from different monkeys. The CS-917 esterase activity precipitated by ammonium sulfate from monkeys other than monkey #1 was subjected to a hydrophobic interaction chromatography column. In all of the cases, the activity formed a single active peak by a linear gradient elution of 1–0 mol/L ammonium sulfate. The activity was tested in the presence or absence of 1 mmol/L PMSF, a protease inhibitor for serine proteases including elastases. (A) Monkey #2, (B) monkey #3, (C) monkey #4 and (D) monkey #5. The data are shown as the average of technical duplicates from a single experiment with error bars representing the higher value. PMSF, phenylmethylsulfonyl fluoride.
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fig04: CS-917 esterase activity in the small intestine from different monkeys. The CS-917 esterase activity precipitated by ammonium sulfate from monkeys other than monkey #1 was subjected to a hydrophobic interaction chromatography column. In all of the cases, the activity formed a single active peak by a linear gradient elution of 1–0 mol/L ammonium sulfate. The activity was tested in the presence or absence of 1 mmol/L PMSF, a protease inhibitor for serine proteases including elastases. (A) Monkey #2, (B) monkey #3, (C) monkey #4 and (D) monkey #5. The data are shown as the average of technical duplicates from a single experiment with error bars representing the higher value. PMSF, phenylmethylsulfonyl fluoride.

Mentions: All experiments were independently conducted more than once to confirm reproducibility except Figure 4, where we show all of the data from individual animals. A representation of those independent experiments is shown in each figure and table, and the number of independent experiments is described in each figure and table legend. Therefore, replicates in the Figures were ‘used only to test the precision of n = 1’ in this study.


Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

Kubota K, Inaba S, Nakano R, Watanabe M, Sakurai H, Fukushima Y, Ichikawa K, Takahashi T, Izumi T, Shinagawa A - Pharmacol Res Perspect (2015)

CS-917 esterase activity in the small intestine from different monkeys. The CS-917 esterase activity precipitated by ammonium sulfate from monkeys other than monkey #1 was subjected to a hydrophobic interaction chromatography column. In all of the cases, the activity formed a single active peak by a linear gradient elution of 1–0 mol/L ammonium sulfate. The activity was tested in the presence or absence of 1 mmol/L PMSF, a protease inhibitor for serine proteases including elastases. (A) Monkey #2, (B) monkey #3, (C) monkey #4 and (D) monkey #5. The data are shown as the average of technical duplicates from a single experiment with error bars representing the higher value. PMSF, phenylmethylsulfonyl fluoride.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492754&req=5

fig04: CS-917 esterase activity in the small intestine from different monkeys. The CS-917 esterase activity precipitated by ammonium sulfate from monkeys other than monkey #1 was subjected to a hydrophobic interaction chromatography column. In all of the cases, the activity formed a single active peak by a linear gradient elution of 1–0 mol/L ammonium sulfate. The activity was tested in the presence or absence of 1 mmol/L PMSF, a protease inhibitor for serine proteases including elastases. (A) Monkey #2, (B) monkey #3, (C) monkey #4 and (D) monkey #5. The data are shown as the average of technical duplicates from a single experiment with error bars representing the higher value. PMSF, phenylmethylsulfonyl fluoride.
Mentions: All experiments were independently conducted more than once to confirm reproducibility except Figure 4, where we show all of the data from individual animals. A representation of those independent experiments is shown in each figure and table, and the number of independent experiments is described in each figure and table legend. Therefore, replicates in the Figures were ‘used only to test the precision of n = 1’ in this study.

Bottom Line: Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes.Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level.This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

View Article: PubMed Central - PubMed

Affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.

ABSTRACT
CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

No MeSH data available.


Related in: MedlinePlus