Identification of glycosylated exendin-4 analogue with prolonged blood glucose-lowering activity through glycosylation scanning substitution

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 15, 2010, Pages 4631-4634

  Ueda, Taichi ^a   Ito, Takaomi ^a   Tomita, Kazuyoshi ^a   Togame, Hiroko ^a   Fumoto, Masataka ^a   Asakura, Kenji ^a   Oshima, Takeo ^a   Nishimura, Shin Ichiro ^b   Hanasaki, Kohji ^a  

^a SHIONOGI AND CO LTD   (Japan)

^b HOKKAIDO UNIVERSITY   (Japan)

Author keywords

Chemoenzymatic synthesis; Glycoconjugate; Incretin hormone; Peptide drug

Indexed keywords

ANTIDIABETIC AGENT; EXENDIN 4; EXENDIN 4 DERIVATIVE; GLUCAGON LIKE PEPTIDE 1; SIALIC ACID DERIVATIVE; SIALYL N ACETYLLACTOSAMINE; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ARTICLE; CARBOXY TERMINAL SEQUENCE; DRUG IDENTIFICATION; GLUCOSE BLOOD LEVEL; GLYCOSYLATION; IN VIVO STUDY; SIALYLATION;

AMINO ACID SEQUENCE; ANIMALS; BLOOD GLUCOSE; DIABETES MELLITUS, TYPE 2; DISEASE MODELS, ANIMAL; GLUCAGON-LIKE PEPTIDE 1; GLYCOSYLATION; HYPOGLYCEMIC AGENTS; MICE; MOLECULAR SEQUENCE DATA; PEPTIDES; VENOMS;

EID: 77955425090     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.06.002     Document Type: Article

References (19)

1. R. Göke, H. Fehmann, T. Linn, H. Schmidt, M. Krause, J. Eng, and B. Göke J. Biol. Chem. 268 1993 19650
2. L.L. Nielsen, A.A. Young, and D.G. Parkes Regul. Pept. 117 2004 77
3. C.F. Deacon Diabetes 53 2004 2181
4. J. Malone, M. Trautmann, K. Wilhelm, K. Taylor, and D.M. Kendall Expert Opin. Invest. Drugs 18 2009 359
5. L. Zhou, Z. Cai, L. Li, C. Kou, and Y. Gao Eur. J. Pharm. Biopharm. 72 2009 412
6. T. Ueda, K. Tomita, Y. Notsu, T. Ito, M. Fumoto, T. Takakura, H. Nagatome, A. Takimoto, S. Mihara, H. Togame, K. Kawamoto, T. Iwasaki, K. Asakura, T. Oshima, K. Hanasaki, S. Nishimura, and H. Kondo J. Am. Chem. Soc. 131 2009 6237
7. S. Elliott, T. Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, J. Busse, D. Chang, J. Fuller, J. Grant, N. Hernday, M. Hokum, S. Hu, A. Knudten, N. Levin, R. Komorowski, F. Martin, R. Navarro, T. Osslund, G. Rogers, N. Rogers, G. Trail, G. Rogers, G. Trail, and J. Egrie Nat. Biotechnol. 21 2003 414
8. A.M. Sinclair, and S. Elliott J. Pharm. Sci. 94 2005 1626
9. J.A. Meurer, J.R. Colca, P.S. Burton, and.P. Elhammer Metabolism 48 1999 716
10. 3GlcNAcβ)-OH. After completion of the synthesis of the peptide, the resin was treated with TFA/phenol/thioanisole/water/1,2-ethanedithiol (82.5:5:5:5:2.5) for 2 h at ambient temperature to release the peptide from the resin and for concurrent deprotections. After the mixture containing the peptide was filtered, the filtrate was precipitated from diethyl ether to give crude peptide as an amorphous solid. The crude peptide was dissolved in methanol, and NaOH aq was added. The mixture was shaken for 1 h and then neutralized with AcOH. After concentration under a stream of nitrogen, the crude peptide was dissolved in 10% AcOH aq and purified by semipreparative RP-HPLC. The column was eluted with a gradient of 0.1% TFA acetonitrile against 0.1% aq TFA. The peptide-containing fraction was collected and then lyophilized to give purified glycopeptide. GlcNAc glycosylated analogues at position 31-39 were purchased from the Peptide Institute, Inc. Synthesized glycopeptides were analyzed by LC-ESI-MS (see Supplementary data).
11. -6 M) in assay buffer containing 0.33 mM isobutylmethylxanthine and 0.67 mM RO20-1724 at rt for 1 h. The cells were lysed with 1% Triton X-100, and the cAMP formed was measured using a cAMP femtomolar kit (Cis Bio international).
12. J.W. Neidigh, R.M. Fesinmeyer, K.S. Prickett, and N.H. Andersen Biochemistry 40 2001 13188
13. S. Al-Sabah, and D. Donnelly Br. J. Pharmacol. 140 2003 339
14. S. Runge, S. Schimmer, J. Oschmann, C.B. Schiødt, S.M. Knudsen, C.B. Jeppesen, K. Madsen, J. Lau, H. Thøgersen, and R. Rudolph Biochemistry 46 2007 5830
15. 2, 0.01% Triton X-100 was incubated at 25 °C overnight. The crude peptide was purified by semipreparative RP-HPLC. The column was eluted with a gradient of 0.1% TFA acetonitrile against 0.1% aqueous TFA. The peptide-containing fraction was collected and then lyophilized to give 28N-LacNAc (3.72 mg, 89%). Then, a solution of 28N-LacNAc (1 mM) in 25 mM HEPES buffer (pH 7.5) containing 10 mM CMP-NANA, 0.15 U/mL α2,6- sialyltransferase, 0.01% Triton X-100 was incubated at 37 °C for two days. The crude peptide was purified by semipreparative RP-HPLC. The column was eluted with a gradient of acetonitrile against 25 mM ammonium acetate buffer (pH 4.0). The peptide-containing fraction was collected and then lyophilized to give 28N-sialyl LacNAc (1.15mg, 38%). Synthesized glycopeptides were analyzed by LC-ESI-MS (see Supplementary data).
16. K. Adelhorst, B.B. Hedegaard, L.B. Knudsen, and O. Kirk J. Biol. Chem. 269 1994 6275
17. db mice (13-15 weeks of age) were allowed ad libitum access to food and water until the start of the experiment. At t = -2 h, access to food was restricted, and the tip of the tail was cut. At t = 0 min, a 1 μL blood sample was collected. Immediately thereafter, each mouse was injected subcutaneously with test sample (1 nmol/kg) or vehicle, and additional blood samples were collected. The vehicle was saline containing 1% BSA. Blood glucose levels were measured with a glucose oxidase biosensor (DIAMETERR; Arkray, Inc.).
18. J. Chen, L. Yu, L. Wang, X. Fang, W. Li, and L. Li Protein Pept. Lett. 14 2007 19
19. C. Montrose-Rafizadeh, H. Yang, B.D. Rodgers, A. Beday, L.A. Pritchette, and J. Eng J. Biol. Chem. 272 1997 21201