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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 of CS-917 esterase activity from the monkey liver. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey liver was subjected to a hydrophobic interaction (HIC). The activity was tested in the absence (gray bars) or presence (hatched bars) of 1 mmol/L BNPP, a CES specific inhibitor. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. The CS-917 esterase activities were separated by the final anion exchange column purified from the first (B) or the second (D) active peak. The BNPP inhibitory effect was further tested on the active fractions from the first (C) or the second (E) active peak (gray and hatched bars). Quantitation of CES1 (C) and CTSA (E) by mass spectrometry using emPAI (closed and open box lines) correlated well with the enzyme activity. Four independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; BNPP, bis(4-nitrophenyl) phosphate.
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fig05: Purification and identification of CS-917 esterase activity from the monkey liver. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey liver was subjected to a hydrophobic interaction (HIC). The activity was tested in the absence (gray bars) or presence (hatched bars) of 1 mmol/L BNPP, a CES specific inhibitor. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. The CS-917 esterase activities were separated by the final anion exchange column purified from the first (B) or the second (D) active peak. The BNPP inhibitory effect was further tested on the active fractions from the first (C) or the second (E) active peak (gray and hatched bars). Quantitation of CES1 (C) and CTSA (E) by mass spectrometry using emPAI (closed and open box lines) correlated well with the enzyme activity. Four independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; BNPP, bis(4-nitrophenyl) phosphate.

Mentions: CES is well known as an activating enzyme for many prodrugs, including bisamidate prodrugs and is supposed to be a good candidate for an additional CS-917 esterase in the liver (Imai 2006; Liederer and Borchardt 2006; Satoh and Hosokawa 2006; Murakami et al. 2010). To test this hypothesis, we investigated the effects of a CES-specific inhibitor, BNPP, on human liver S9 and observed that the high concentration of BNPP (10 mmol/L) did not show an inhibitory effect on human liver S9, but it did show significant inhibition on dialyzed human liver S9 (Fig. S6). These data indicated that BNPP could not inhibit CS-917 esterase activity in a crude sample for some reason. Therefore, we separated 20–70% ammonium sulfate precipitate of monkey liver homogenates by HIC, and tested the BNPP effect on its fractions. Considering the possibility of the purification of liver CS-917 esterase, we used cynomolgus monkey liver for the same reasons we used the monkey small intestine. As shown in Figure5A, the CS-917 esterase activity formed two active peaks and the first active peak was strongly inhibited by BNPP, which suggested that the first active peak was contributed by CES and that the second active peak was contributed by CTSA.


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 of CS-917 esterase activity from the monkey liver. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey liver was subjected to a hydrophobic interaction (HIC). The activity was tested in the absence (gray bars) or presence (hatched bars) of 1 mmol/L BNPP, a CES specific inhibitor. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. The CS-917 esterase activities were separated by the final anion exchange column purified from the first (B) or the second (D) active peak. The BNPP inhibitory effect was further tested on the active fractions from the first (C) or the second (E) active peak (gray and hatched bars). Quantitation of CES1 (C) and CTSA (E) by mass spectrometry using emPAI (closed and open box lines) correlated well with the enzyme activity. Four independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; BNPP, bis(4-nitrophenyl) phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Purification and identification of CS-917 esterase activity from the monkey liver. (A) The CS-917 esterase activity precipitated by ammonium sulfate from monkey liver was subjected to a hydrophobic interaction (HIC). The activity was tested in the absence (gray bars) or presence (hatched bars) of 1 mmol/L BNPP, a CES specific inhibitor. CS-917 esterase activity of each active peak in the HIC was separately purified by successive chromatography. The CS-917 esterase activities were separated by the final anion exchange column purified from the first (B) or the second (D) active peak. The BNPP inhibitory effect was further tested on the active fractions from the first (C) or the second (E) active peak (gray and hatched bars). Quantitation of CES1 (C) and CTSA (E) by mass spectrometry using emPAI (closed and open box lines) correlated well with the enzyme activity. Four independent experiments were conducted, and the average of technical duplicates from a representative single experiment is shown with error bars representing the higher value. CTSA, cathepsin A; BNPP, bis(4-nitrophenyl) phosphate.
Mentions: CES is well known as an activating enzyme for many prodrugs, including bisamidate prodrugs and is supposed to be a good candidate for an additional CS-917 esterase in the liver (Imai 2006; Liederer and Borchardt 2006; Satoh and Hosokawa 2006; Murakami et al. 2010). To test this hypothesis, we investigated the effects of a CES-specific inhibitor, BNPP, on human liver S9 and observed that the high concentration of BNPP (10 mmol/L) did not show an inhibitory effect on human liver S9, but it did show significant inhibition on dialyzed human liver S9 (Fig. S6). These data indicated that BNPP could not inhibit CS-917 esterase activity in a crude sample for some reason. Therefore, we separated 20–70% ammonium sulfate precipitate of monkey liver homogenates by HIC, and tested the BNPP effect on its fractions. Considering the possibility of the purification of liver CS-917 esterase, we used cynomolgus monkey liver for the same reasons we used the monkey small intestine. As shown in Figure5A, the CS-917 esterase activity formed two active peaks and the first active peak was strongly inhibited by BNPP, which suggested that the first active peak was contributed by CES and that the second active peak was contributed by CTSA.

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