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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

Specific CS-917 esterase activity of candidate genes. Specific activities of human candidate genes as CS-917 esterase were compared. Carboxylesterases (CES1 and CES2) were cloned, expressed in human cells, and purified from the lysate. Activated ELA2 purified from human leukocytes was commercially available. Pancreatic elastases (ELA2A, ELA2B, ELA3A, and ELA3B) were cloned, expressed in human cells, purified from the conditioned medium, and activated with trypsin. Purified CTSA expressed in E. coli. was obtained from a commercial source, and activated with cathepsin L. Two to five independent experiments were conducted (2 for ELA2, 3 for CES1, CES2, ELA2A, and ELA3B, and 5 for ELA3A, ELA3B, and CTSA), and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; ELA, elastase.
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fig03: Specific CS-917 esterase activity of candidate genes. Specific activities of human candidate genes as CS-917 esterase were compared. Carboxylesterases (CES1 and CES2) were cloned, expressed in human cells, and purified from the lysate. Activated ELA2 purified from human leukocytes was commercially available. Pancreatic elastases (ELA2A, ELA2B, ELA3A, and ELA3B) were cloned, expressed in human cells, purified from the conditioned medium, and activated with trypsin. Purified CTSA expressed in E. coli. was obtained from a commercial source, and activated with cathepsin L. Two to five independent experiments were conducted (2 for ELA2, 3 for CES1, CES2, ELA2A, and ELA3B, and 5 for ELA3A, ELA3B, and CTSA), and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; ELA, elastase.

Mentions: As shown in Figure3, CTSA demonstrated the highest specific activity as CS-917 esterase while ELA2 and ELA3B exhibited moderate specific activity, and ELA3A showed weak specific activity. ELA2A and ELA2B did not hydrolase CS-917. In parallel, we tested ELA2A and ELA2B’s elastase activity using synthetic peptide substrates. The results revealed ELA2A had elastase activity but ELA2B did not (Fig. S3), indicating that our ELA2A was certainly active but did not have CS-917 esterase activity. With regard to ELA2B, a recent report showed ELA2B was devoid of proteolytic activity (Szepessy and Sahin Toth 2006). Thus, our ELA2B was supposed to be inactive due to its intrinsic character.


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)

Specific CS-917 esterase activity of candidate genes. Specific activities of human candidate genes as CS-917 esterase were compared. Carboxylesterases (CES1 and CES2) were cloned, expressed in human cells, and purified from the lysate. Activated ELA2 purified from human leukocytes was commercially available. Pancreatic elastases (ELA2A, ELA2B, ELA3A, and ELA3B) were cloned, expressed in human cells, purified from the conditioned medium, and activated with trypsin. Purified CTSA expressed in E. coli. was obtained from a commercial source, and activated with cathepsin L. Two to five independent experiments were conducted (2 for ELA2, 3 for CES1, CES2, ELA2A, and ELA3B, and 5 for ELA3A, ELA3B, and CTSA), and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; ELA, elastase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Specific CS-917 esterase activity of candidate genes. Specific activities of human candidate genes as CS-917 esterase were compared. Carboxylesterases (CES1 and CES2) were cloned, expressed in human cells, and purified from the lysate. Activated ELA2 purified from human leukocytes was commercially available. Pancreatic elastases (ELA2A, ELA2B, ELA3A, and ELA3B) were cloned, expressed in human cells, purified from the conditioned medium, and activated with trypsin. Purified CTSA expressed in E. coli. was obtained from a commercial source, and activated with cathepsin L. Two to five independent experiments were conducted (2 for ELA2, 3 for CES1, CES2, ELA2A, and ELA3B, and 5 for ELA3A, ELA3B, and CTSA), and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; ELA, elastase.
Mentions: As shown in Figure3, CTSA demonstrated the highest specific activity as CS-917 esterase while ELA2 and ELA3B exhibited moderate specific activity, and ELA3A showed weak specific activity. ELA2A and ELA2B did not hydrolase CS-917. In parallel, we tested ELA2A and ELA2B’s elastase activity using synthetic peptide substrates. The results revealed ELA2A had elastase activity but ELA2B did not (Fig. S3), indicating that our ELA2A was certainly active but did not have CS-917 esterase activity. With regard to ELA2B, a recent report showed ELA2B was devoid of proteolytic activity (Szepessy and Sahin Toth 2006). Thus, our ELA2B was supposed to be inactive due to its intrinsic character.

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