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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 R-134450 phosphoramidase activity of candidate genes. Candidate genes (SMPDL3A, HINT1, HINT2, HINT3, and APTX) and the negative control (EGFP) were expressed in the human cell line, the recombinant proteins were purified using fused FLAG-tag, and the R-134450 phosphoramidase activity was tested. The purified proteins were quantified by Western blotting, and the specific activity was calculated. Four to seven independent experiments were conducted (4 for HINT1, HINT2, HINT3, and APTX, and 7 for EGFP and SMPDL3A). The results are expressed as the means ± SD (three technical replicates from a single experiment). EGFP, enhanced green fluorescent protein.
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fig07: Specific R-134450 phosphoramidase activity of candidate genes. Candidate genes (SMPDL3A, HINT1, HINT2, HINT3, and APTX) and the negative control (EGFP) were expressed in the human cell line, the recombinant proteins were purified using fused FLAG-tag, and the R-134450 phosphoramidase activity was tested. The purified proteins were quantified by Western blotting, and the specific activity was calculated. Four to seven independent experiments were conducted (4 for HINT1, HINT2, HINT3, and APTX, and 7 for EGFP and SMPDL3A). The results are expressed as the means ± SD (three technical replicates from a single experiment). EGFP, enhanced green fluorescent protein.

Mentions: We cloned the human genes of SMPDL3A, HINT1, HINT2, HINT3, and APTX, produced recombinant proteins in human cells, purified them using FLAG-tag fused with the protein, and tested their R-134450 phosphoramidase activity. As shown in Figure7, SMPDL3A showed remarkable R-134450 phosphoramidase activity while HINT1, HINT2, and APTX had much weaker specific activity than 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)

Specific R-134450 phosphoramidase activity of candidate genes. Candidate genes (SMPDL3A, HINT1, HINT2, HINT3, and APTX) and the negative control (EGFP) were expressed in the human cell line, the recombinant proteins were purified using fused FLAG-tag, and the R-134450 phosphoramidase activity was tested. The purified proteins were quantified by Western blotting, and the specific activity was calculated. Four to seven independent experiments were conducted (4 for HINT1, HINT2, HINT3, and APTX, and 7 for EGFP and SMPDL3A). The results are expressed as the means ± SD (three technical replicates from a single experiment). EGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Specific R-134450 phosphoramidase activity of candidate genes. Candidate genes (SMPDL3A, HINT1, HINT2, HINT3, and APTX) and the negative control (EGFP) were expressed in the human cell line, the recombinant proteins were purified using fused FLAG-tag, and the R-134450 phosphoramidase activity was tested. The purified proteins were quantified by Western blotting, and the specific activity was calculated. Four to seven independent experiments were conducted (4 for HINT1, HINT2, HINT3, and APTX, and 7 for EGFP and SMPDL3A). The results are expressed as the means ± SD (three technical replicates from a single experiment). EGFP, enhanced green fluorescent protein.
Mentions: We cloned the human genes of SMPDL3A, HINT1, HINT2, HINT3, and APTX, produced recombinant proteins in human cells, purified them using FLAG-tag fused with the protein, and tested their R-134450 phosphoramidase activity. As shown in Figure7, SMPDL3A showed remarkable R-134450 phosphoramidase activity while HINT1, HINT2, and APTX had much weaker specific activity than 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