FBPase Inhibitors. 1. Purine Phosphonic Acids
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2897
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Enzyme Assays. FBPase activity and its inhibition were
measured spectrophotometrically in reactions that coupled the
production of fructose-6-phosphate to the reduction of NADP+.32
AMP-activated protein kinase (rat liver, Upstate Biotechnology,
Rochester, NY) was assayed using the “SAMS” peptide substrate
according to the supplier’s instructions. Adenosine kinase (human
recombinant) was obtained, purified, and assayed as described.33
AMP deaminase (porcine heart) was purified and assayed as
described.25 Glycogen phosphorylase (rabbit muscle), phosphof-
ructokinase (rabbit liver), and adenylate kinase (rabbit muscle) were
obtained from Sigma Chemical Company (St. Louis, MO) and
assayed as described.34-36
Gluconeogenesis Inhibition in Rat Hepatocytes. Hepatocytes
were prepared from 24 h fasted, male Sprague-Dawley or freely
feeding, male ZDF rats (Genetics Models Inc., Indianapolis, IN or
Charles River Laborabories, Indianopolis, IN) according to the
procedure of Berry and Friend37 as modified by Groen et al.38
Hepatocytes (10-60 mg/mL) were preincubated with each com-
pound in suspension culture for 15-30 min prior to a 30-60 min
incubation with gluconeogenic substrate (dihydroxyacetone was
used as the substrate, which feeds into the GNG pathway at a step
just prior to FBPase). Reactions were terminated by centrifugation.
Glucose in cell supernatants was assayed by means of a glucose
oxidase kit (Sigma Chemical Company, St. Louis, MO).
Determination of Fructose-1,6-bisphosphate Levels. Snap-
frozen liver or hepatocyte samples were extracted in 10% per-
choloric acid, neutralized, and then analyzed for fructose-1,6-
bisphosphate levels by means of an enzyme-coupled spectrophoto-
metric assay as previously described.39 Coupling enzymes for these
determinations were obtained from Roche Diagnostics (Indianapolis,
IN) and Boehringer Mannheim (Ridgefield, CT).
Glucose Lowering in Sprague-Dawley Rats. Rats were housed
under standard vivarium conditions (12 h light/dark cycle) with
free access to Purina 5008 (ZDF rats) or standard chow (Sprague-
Dawley rats) and water unless otherwise indicated. All studies were
conducted in accordance with the Institutional Animal Care and
Use Committee guidelines. Prior to each study, rats (∼250 g) were
fasted overnight and then administered test compound or vehicle
(PEG-400). Blood samples were obtained from the tail vein at
baseline and at regular time intervals for up to 3 h thereafter. Blood
glucose was determined by means of a HemoCue analyzer
(HemoCue Inc., Mission Viejo, CA).
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L. R.; Schulte, G. K.; Soeller, W. C.; Treadway, J. L.; Wang, I. K.;
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Potter, S. C.; Fujitaki, J. M.; van Poelje, P. D.; Huang, J.; Lipscomb,
W. N.; Erion, M. D. Discovery of Potent and Specific Fructose-1,6-
Bisphosphatase Inhibitors and a Series of Orally Bioavailable Phos-
phoramidase-Sensitive Prodrugs for the Treatment of Type 2 Diabetes.
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differences for fructose 1,6-bisphosphatase inhibitors using the
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MB06322 (CS-917): a potent and selective inhibitor of fructose-1,6-
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Acknowledgment. We thank Dr. Raja K. Reddy, Dr. Kurt
Metzner, Jay DaRe, and Kevin Fan for their contribution to this
project, and Dr. James M. Fujitaki and Timothy J. Colby for
characterization of the compounds in the biological assays. We
also thank Dr. Jingwei Huang and Professor William N.
Lipscomb of Harvard University for providing the X-ray
structural information for compound 1.6.
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Supporting Information Available: Elemental analysis data for
all final compounds. This material is available free of charge via
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