title: Identification of two novel and potent competitive inhibitors of the glucose-6-phosphatase catalytic protein.
abstract: OBJECTIVE
  In this study, we show that inhibitors of the glucose-6-phosphatase (G-6-Pase) catalytic protein could be an alternative approach to the recent G-6-Pase T1-translocase inhibitors to target this enzyme for the treatment of type 2 diabetes.
  
  
  METHODS
  The active enantiomers of 4-methoxyphenyl-[4-(4-methoxyphenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-yl]methanone (Compound A-1) and 4-methoxyphenyl-[4-(4-trifluoromethoxyphenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-5-yl]methanone (Compound B-1) were characterized as inhibitors of the G-6-Pase catalytic protein using pig and rat liver microsomes and cultured rat hepatocytes.
  
  
  RESULTS
  Both compounds were found to be potent competitive inhibitors of the G-6-Pase catalytic protein obtained from pig and rat liver microsomes. The K(i) values (microM) were calculated to be 0.61 +/- 0.02 and 0.63 +/- 0.08 for compound A-1 and B-1 on intact pig microsomes, and 0.27 +/- 0.02 and 0.29 +/- 0.06 on disrupted pig microsomes. The corresponding values for rat liver microsomes were found to be 3.3 +/- 0.6 and 4.0 +/- 1.2 for compound A-1 and B-1 on intact microsomes, and 1.54 +/- 0.1 and 1.21 +/- 0.1 on disrupted microsomes. Compound A-1 was also able to inhibit pyrophosphatase activities from both intact and disrupted microsomes with equal potency (IC50; 0.43-0.55 microm). Using cultured rat hepatocytes and glycerol as the substrate, these compounds were able to prevent glucose production up to 60% with a concomitant increase in the G-6-P content (2.3-fold) using compound A-1. No increase in glycogen levels was seen.
  
  
  CONCLUSIONS
  These data demonstrated that these compounds were more potent inhibitors on G-6-Pase obtained from pig microsomes and were able to penetrate the microsomal membrane. The hepatocyte data further support the kinetic data, and are also consistent with the evoked mechanism of action.
authors.affiliation: Department of Diabetes Biochemistry and Metabolism, Novo Nordisk A/S, DK-2760 Måløv, Denmark.
authors.forename: N
authors.initials: N
authors.lastname: Westergaard
authors.forename: P
authors.initials: P
authors.lastname: Madsen
authors.forename: J M
authors.initials: JM
authors.lastname: Lundbeck
authors.forename: P
authors.initials: P
authors.lastname: Jakobsen
authors.forename: A
authors.initials: A
authors.lastname: Varming
authors.forename: B
authors.initials: B
authors.lastname: Andersen
mesh_terms: D000818:Animals; D001577:Benzophenones / Q000737:chemistry* / Q000494:pharmacology; D001667:Binding, Competitive; D002384:Catalysis; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors*; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors; D006003:Glycogen / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D011725:Pyridines / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: C465419:4-methoxyphenyl-(4-(4-methoxyphenyl)-4,5,6,7-tetrahydrothieno(3,2-c)pyridin-5-yl)methanone; C465420:4-methoxyphenyl-(4-(4-trifluoromethoxyphenyl)-4,5,6,7-tetrahydrothieno(3,2-c)pyridin-5-yl)methanone; D001577:Benzophenones; D004791:Enzyme Inhibitors; D007773:Lactates; D011725:Pyridines; D006003:Glycogen; D005954:Glucosephosphate Dehydrogenase; D005952:Glucose-6-Phosphatase; D005947:Glucose