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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

Purification and identification of CS-917 esterase activity from the small intestine of monkey #1. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey #1 was subjected to a hydrophobic interaction chromatography (HIC) column. The activity was bound to a column and formed two active peaks by a linear gradient elution of 1–0 mol/L ammonium sulfate. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. (B) Fractions of the final purification step of the first active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 30, 17, and 15 kDa correlating well with the enzyme activity are indicated by arrowheads. (C) The enzyme activity of the fractions (solid line) and the intensity of arrowed bands of 30 kDa in (B) are shown as bars, respectively. (D) Fractions of the final purification step of the second active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 26 and 17 kDa correlating well with the enzyme activity are indicated by arrowheads. (E) The enzyme activity of the fractions (solid line) and the intensity of the arrowed bands of 26 kDa in (D) are shown as bars, respectively. Three independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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fig02: Purification and identification of CS-917 esterase activity from the small intestine of monkey #1. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey #1 was subjected to a hydrophobic interaction chromatography (HIC) column. The activity was bound to a column and formed two active peaks by a linear gradient elution of 1–0 mol/L ammonium sulfate. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. (B) Fractions of the final purification step of the first active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 30, 17, and 15 kDa correlating well with the enzyme activity are indicated by arrowheads. (C) The enzyme activity of the fractions (solid line) and the intensity of arrowed bands of 30 kDa in (B) are shown as bars, respectively. (D) Fractions of the final purification step of the second active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 26 and 17 kDa correlating well with the enzyme activity are indicated by arrowheads. (E) The enzyme activity of the fractions (solid line) and the intensity of the arrowed bands of 26 kDa in (D) are shown as bars, respectively. Three independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mentions: To identify the enzyme responsible for CS-917 esterase activity in the small intestine, we used cynomolgus monkeys as a purification source because of the abundant sample availability and their similarity to humans. The monkey small intestine was homogenized and separated to nuclear, mitochondrial, microsomal, and cytosolic fractions by centrifugation and the activity was found to be mostly localized in the cytosolic and soluble fraction (Fig. S1). The observed molecular weight of the activity was 60–70 kDa determined by gel filtration chromatography, and interestingly, the activity from one out of the five monkeys (monkey #1) was bound to an HIC column and formed two almost equivalent peaks (Fig.2A).


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)

Purification and identification of CS-917 esterase activity from the small intestine of monkey #1. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey #1 was subjected to a hydrophobic interaction chromatography (HIC) column. The activity was bound to a column and formed two active peaks by a linear gradient elution of 1–0 mol/L ammonium sulfate. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. (B) Fractions of the final purification step of the first active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 30, 17, and 15 kDa correlating well with the enzyme activity are indicated by arrowheads. (C) The enzyme activity of the fractions (solid line) and the intensity of arrowed bands of 30 kDa in (B) are shown as bars, respectively. (D) Fractions of the final purification step of the second active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 26 and 17 kDa correlating well with the enzyme activity are indicated by arrowheads. (E) The enzyme activity of the fractions (solid line) and the intensity of the arrowed bands of 26 kDa in (D) are shown as bars, respectively. Three independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Purification and identification of CS-917 esterase activity from the small intestine of monkey #1. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey #1 was subjected to a hydrophobic interaction chromatography (HIC) column. The activity was bound to a column and formed two active peaks by a linear gradient elution of 1–0 mol/L ammonium sulfate. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. (B) Fractions of the final purification step of the first active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 30, 17, and 15 kDa correlating well with the enzyme activity are indicated by arrowheads. (C) The enzyme activity of the fractions (solid line) and the intensity of arrowed bands of 30 kDa in (B) are shown as bars, respectively. (D) Fractions of the final purification step of the second active peak were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 26 and 17 kDa correlating well with the enzyme activity are indicated by arrowheads. (E) The enzyme activity of the fractions (solid line) and the intensity of the arrowed bands of 26 kDa in (D) are shown as bars, respectively. Three independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Mentions: To identify the enzyme responsible for CS-917 esterase activity in the small intestine, we used cynomolgus monkeys as a purification source because of the abundant sample availability and their similarity to humans. The monkey small intestine was homogenized and separated to nuclear, mitochondrial, microsomal, and cytosolic fractions by centrifugation and the activity was found to be mostly localized in the cytosolic and soluble fraction (Fig. S1). The observed molecular weight of the activity was 60–70 kDa determined by gel filtration chromatography, and interestingly, the activity from one out of the five monkeys (monkey #1) was bound to an HIC column and formed two almost equivalent peaks (Fig.2A).

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