title: Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.
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.
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Kazuishi
authors.initials: K
authors.lastname: Kubota
authors.affiliation: Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd. Tokyo, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Inaba
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Rika
authors.initials: R
authors.lastname: Nakano
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Sakurai
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Fukushima
authors.affiliation: Biologics Technology Research Laboratories, Daiichi Sankyo Co., Ltd. Tokyo, Japan.
authors.forename: Kimihisa
authors.initials: K
authors.lastname: Ichikawa
authors.affiliation: New Modality Research Laboratories, Daiichi Sankyo Co., Ltd. Tokyo, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd. Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Izumi
authors.affiliation: Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Shinagawa
keywords: Biochemical purification; CS-917; FBPase inhibitor; bisamidate prodrug; drug metabolizing enzyme; phosphoramidase