Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 4, 2009, Pages 1177-1182

  Thomson, Stephen A ^a   Banker, Pierette ^a   Bickett, D Mark ^a   Boucheron, Joyce A ^a   Carter, H Luke ^a   Clancy, Daphne C ^a   Cooper, Joel P ^a   Dickerson, Scott H ^a   Garrido, Dulce M ^a   Nolte, Robert T ^a   Peat, Andrew J ^a   Sheckler, Lauren R ^a   Sparks, Steven M ^a   Tavares, Francis X ^a   Wang, Liping ^a   Wang, Tony Y ^a   Weiel, James E ^a  

^a GLAXOSMITHKLINE   (United States)

Author keywords

Anthranilimide; Antidiabetic; Challenge; Glucagon; Glycogen; Inhibitor; Phosphorylase

Indexed keywords

2 AMINOBENZAMIDE; ADENOSINE PHOSPHATE; ENZYME INHIBITOR; GLYCOGEN PHOSPHORYLASE; PHENYL GROUP; UREA;

ANIMAL EXPERIMENT; ANIMAL MODEL; ARTICLE; CONTROLLED STUDY; CRYSTAL STRUCTURE; DRUG BINDING; DRUG EFFICACY; DRUG POTENCY; DRUG STRUCTURE; DRUG SYNTHESIS; ENZYME INHIBITION; GLUCOSE BLOOD LEVEL; IN VIVO STUDY; MOUSE; NON INSULIN DEPENDENT DIABETES MELLITUS; NONHUMAN; RAT; X RAY CRYSTALLOGRAPHY;

ANIMALS; ANTHRANILIC ACIDS; BLOOD GLUCOSE; COMBINATORIAL CHEMISTRY TECHNIQUES; CRYSTALLOGRAPHY, X-RAY; DIABETES MELLITUS, TYPE 2; DISEASE MODELS, ANIMAL; GLYCOGEN PHOSPHORYLASE; HYPOGLYCEMIC AGENTS; MICE; MOLECULAR CONFORMATION; MOLECULAR STRUCTURE; STRUCTURE-ACTIVITY RELATIONSHIP; UREA;

EID: 59649089131     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.12.085     Document Type: Article

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20. GP protein was engineered with an N-term fusion to a hexa his-tag followed by a TEV protease site and expressed in Escherichia coli. Cells were lysed and activated to immobilized metal affinity chromatography (IMAC). Fractions containing GP were pooled, diluted and subjected to anion exchange chromatography (Q Sepharose HP). Fractions containing GP were pooled and the enzyme was activated. TEV protease was used to remove the tag and the activated cleaved GP was activated to another round of IMAC. The GP in the IMAC flow through was concentrated for crystallography. Crystals were prepared similar to the method described by Rath et al. for the active conformation of glycogen phosphorylase (see Rath, V. L.; Ammirati, M.; LeMotte, P. K.; Fennel, K. F.; Mansour, M. N.; Denley, D. E.; Hynes, T. R.; Schulte, G. K.; Wasilko, D. J.; Pandit, J. Mol. Cell 2000, 6, 139). The crystallization method was modified to include the addition of 5.0 mM caffeine and 0.1 mM ligand to the protein solution 1 h prior to setting up the crystals. Crystals were frozen in liquid nitrogen directly from the drop and X-ray diffraction data was collected at 100 K using an ADSC Q210 detector on the Advanced Photon Source Beam line 17ID at Argonne National Labs. The structure was solved by molecular replacement using the program Amore from PDB entry 1em6. Refinement was carried out using Refmac and other CCP4 tools and the final model was built using the programs O and Coot.Compound 2 cocrystals diffracted to 1.80 Å in resolution with an R-merge of 8.7%. The final model was refined to an R-factor of 17.4% with a Free R-factor of 19.8%. Compound 12 cocrystals diffracted to 1.90 Å in resolution with an R-merge of 7.8%. The final model was refined to an R-factor of 15.3%, with a Free R-factor of 18.6%. Coordinates and statistics are available from the PDB using accession code 3DDS and 3DDW, respectively.
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27. Jugular vein cannulated male CD rats (220-260 g) were received 1-2 days after cannulation, housed individually with free access to food and water for 4 days prior to the glucagon challenge (GC) studies. The rats were sorted by body weight into treatment groups (N = 4-5). Rats were dosed orally with vehicle (5% DMSO: 30% Solutol HS15: 20% PEG400: 45% 25 mM N-methylglucamine) or drug (0.1-15 ml/kg) 2 h prior to the glucagon challenge (GC). A time zero blood sample (0.4 ml) was collected for determination of glucose and the rats were dosed via the jugular vein with Sandostatin, 0.5 mg/kg, and glucagon, 10 μg/kg. Blood samples were collected after 10 and 20 min. Whole blood was allowed to sit at room temperature for 20 min and then centrifuged (3000g) to obtain serum. Serum levels of glucose were determined using an Olympus AU640™ clinical chemistry immuno-analyzer.
28. max = 536 ng/mL).
29. ob/J Stock No. 000632 (Drasher, 1955) (6-7-wk-old) were housed 3-4/cage with free access to food and water for 1-2 wk. On the day prior to analysis, the mice were sorted by blood glucose (tail snip, glucometer) and body weight into treatment groups (N = 10 for drug, N = 20 for vehicle). On the following day, the mice were moved to the study room at 6:30 am, and allowed to acclimate for ∼1 h. Basal fed glucose levels were measured by tail-nick and glucometer read and the mice were dosed with 10 ml/kg vehicle (5% DMSO: 30% Solutol HS15: 20% PEG400: 45% 25 mM N-methylglucamine) or drug, at 8 am. Blood glucose levels were measured again by tail-nick and glucometer at 11 am.