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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

Two-step activation of bisamidate prodrug CS-917 to the active form, R-125338. CS-917 is activated into the active form (R-125338) by a two-step enzyme-catalyzed reaction after oral administration. In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450) in the small intestine and the liver. In the second step, the P–N bond of the R-134450 is hydrolyzed by a phosphoramidase to generate R-125338 in the liver. The first step is observed to be fast and the second step is observed to be slow and rate-limiting.
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fig01: Two-step activation of bisamidate prodrug CS-917 to the active form, R-125338. CS-917 is activated into the active form (R-125338) by a two-step enzyme-catalyzed reaction after oral administration. In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450) in the small intestine and the liver. In the second step, the P–N bond of the R-134450 is hydrolyzed by a phosphoramidase to generate R-125338 in the liver. The first step is observed to be fast and the second step is observed to be slow and rate-limiting.

Mentions: After oral administration, CS-917 is converted into the active form, R-125338, by a two-step enzyme-catalyzed reaction (Fig.1) in rat hepatocytes (Dang et al. 2007) and primary human hepatocytes (Erion et al. 2005). In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450), mainly in the first-pass metabolism in the small intestine and liver. Then in the second step, the P–N bond of the intermediate is hydrolyzed by a phosphoramidase to generate the active form, R-125338, mainly in the liver. Investigation of the conversion rate using rat, monkey, and human liver homogenates demonstrated that the first esterase-catalyzed step was fast and that the second phosphoramidase-catalyzed step was slow and rate-limiting, analogous to other phosphoramidase prodrugs (Saboulard et al. 1999; Beltran et al. 2001).


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)

Two-step activation of bisamidate prodrug CS-917 to the active form, R-125338. CS-917 is activated into the active form (R-125338) by a two-step enzyme-catalyzed reaction after oral administration. In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450) in the small intestine and the liver. In the second step, the P–N bond of the R-134450 is hydrolyzed by a phosphoramidase to generate R-125338 in the liver. The first step is observed to be fast and the second step is observed to be slow and rate-limiting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Two-step activation of bisamidate prodrug CS-917 to the active form, R-125338. CS-917 is activated into the active form (R-125338) by a two-step enzyme-catalyzed reaction after oral administration. In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450) in the small intestine and the liver. In the second step, the P–N bond of the R-134450 is hydrolyzed by a phosphoramidase to generate R-125338 in the liver. The first step is observed to be fast and the second step is observed to be slow and rate-limiting.
Mentions: After oral administration, CS-917 is converted into the active form, R-125338, by a two-step enzyme-catalyzed reaction (Fig.1) in rat hepatocytes (Dang et al. 2007) and primary human hepatocytes (Erion et al. 2005). In the first step, CS-917 is hydrolyzed by an esterase and the resultant monoester is spontaneously hydrolyzed to form a monoamidate intermediate (R-134450), mainly in the first-pass metabolism in the small intestine and liver. Then in the second step, the P–N bond of the intermediate is hydrolyzed by a phosphoramidase to generate the active form, R-125338, mainly in the liver. Investigation of the conversion rate using rat, monkey, and human liver homogenates demonstrated that the first esterase-catalyzed step was fast and that the second phosphoramidase-catalyzed step was slow and rate-limiting, analogous to other phosphoramidase prodrugs (Saboulard et al. 1999; Beltran et al. 2001).

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