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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 or R-134450 phosphoramidase activity from monkey liver. R-134450 phosphoramidase activity was purified from monkey liver by ammonium sulfate precipitation and successive five-step chromatography. Fractions of the third (A), fourth (B) and final (C) purification step were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 50 kDa correlating well with the enzyme activity are indicated by arrowheads. The bands indicated by open arrowheads were cut and analyzed by mass spectrometry. Enzyme activity of the fractions (solid line) and the intensities of the arrowed bands in the above panel (bars) are shown in the third (D), fourth (E) and final (F) purification step. Boxed fractions were subjected to the next purification step. 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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fig06: Purification and identification or R-134450 phosphoramidase activity from monkey liver. R-134450 phosphoramidase activity was purified from monkey liver by ammonium sulfate precipitation and successive five-step chromatography. Fractions of the third (A), fourth (B) and final (C) purification step were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 50 kDa correlating well with the enzyme activity are indicated by arrowheads. The bands indicated by open arrowheads were cut and analyzed by mass spectrometry. Enzyme activity of the fractions (solid line) and the intensities of the arrowed bands in the above panel (bars) are shown in the third (D), fourth (E) and final (F) purification step. Boxed fractions were subjected to the next purification step. 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: We purified R-134450 phosphoramidase activity from monkey liver by successive five-step purification after ammonium sulfate precipitation. The enzyme activity was concentrated more than 9000-fold (Table3). The enzyme activity formed a single peak in all chromatography steps, which indicated the enzyme was single. After the third purification step, we found that bands of 50 kDa on SDS-PAGE gels were correlating well with the enzyme activity (Fig.6). We identified the protein of these 50 kDa bands by mass spectrometry as sphingomyelin phosphodiesterase, acid-like 3A (Human homologous gene symbol: SMPDL3A).


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 or R-134450 phosphoramidase activity from monkey liver. R-134450 phosphoramidase activity was purified from monkey liver by ammonium sulfate precipitation and successive five-step chromatography. Fractions of the third (A), fourth (B) and final (C) purification step were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 50 kDa correlating well with the enzyme activity are indicated by arrowheads. The bands indicated by open arrowheads were cut and analyzed by mass spectrometry. Enzyme activity of the fractions (solid line) and the intensities of the arrowed bands in the above panel (bars) are shown in the third (D), fourth (E) and final (F) purification step. Boxed fractions were subjected to the next purification step. 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

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

fig06: Purification and identification or R-134450 phosphoramidase activity from monkey liver. R-134450 phosphoramidase activity was purified from monkey liver by ammonium sulfate precipitation and successive five-step chromatography. Fractions of the third (A), fourth (B) and final (C) purification step were subjected to SDS-PAGE and the gel was stained by a fluorescent dye. Bands of 50 kDa correlating well with the enzyme activity are indicated by arrowheads. The bands indicated by open arrowheads were cut and analyzed by mass spectrometry. Enzyme activity of the fractions (solid line) and the intensities of the arrowed bands in the above panel (bars) are shown in the third (D), fourth (E) and final (F) purification step. Boxed fractions were subjected to the next purification step. 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: We purified R-134450 phosphoramidase activity from monkey liver by successive five-step purification after ammonium sulfate precipitation. The enzyme activity was concentrated more than 9000-fold (Table3). The enzyme activity formed a single peak in all chromatography steps, which indicated the enzyme was single. After the third purification step, we found that bands of 50 kDa on SDS-PAGE gels were correlating well with the enzyme activity (Fig.6). We identified the protein of these 50 kDa bands by mass spectrometry as sphingomyelin phosphodiesterase, acid-like 3A (Human homologous gene symbol: SMPDL3A).

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