Abstract
Numerous analogues of insulin have been prepared over the past three decades for use in diabetic therapy. However, only two long-acting insulins have been approved for clinical use. One is Levemir (Novo Nordisk) and the other is Lantus (Sanofi-Aventis). Glargine (commercial name: Lantus) is characterized by a substitution of Gly in place of Asn at the C terminus of the A-chain and addition of two Arg residues to the C terminus of the B-chain. Despite the clinical advantages of glargine, it is not without concern that its increased affinity for the IGF-1 receptor may correlate with increased mitogenic activity. Recently, a systematic study of modified analogues of glargine showed that placement of an extra Arg residue at the N terminus of the A-chain conferred improved insulin:IGF-1 receptor selectivity without significant loss of pharmacological profile. However, as it is difficult to prepare such an analogue in high yield by recombinant DNA methods, we undertook its chemical assembly by our refined solid phase synthesis method. We describe herein its chemical preparation and biological activity in both insulin receptor binding assays and DNA synthesis assays. The synthetic analogue, A0:R glargine, showed slightly reduced affinity for IR-B (twofold) compared to native insulin. In stimulating DNA synthesis, A0:R glargine was slightly less potent compared to insulin or glargine. This result ultimately confirms the previous report that A0:R glargine has a lower potency in mitogenic assays compared to glargine. This glargine analogue thus could be a potential lead compound for drug design and development for the treatment of diabetes.
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References
Alvino CL, McNeil KA, Ong SC, Delaine C, Booker GW, Wallace JC, Whittaker J, Forbes BE (2009) A novel approach to identify two distinct receptor binding surfaces of insulin-like growth factor II. J Biol Chem 284:7656–7664
Bahr M, Kolter T, Seipke G, Eckel J (1997) Growth promoting and metabolic activity of the human insulin analogue [GlyA21, ArgB31, ArgB32]insulin (HOE 901) in muscle cells. Eur J Pharmacol 320:259–265
Bathgate RA, Lin F, Hanson NF, Otvos L Jr, Guidolin A, Giannakis C, Bastiras S, Layfield SL, Ferraro T, Ma S, Zhao C, Gundlach AL, Samuel CS, Tregear GW, Wade JD (2006) Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo. Biochemistry 45:1043–1053
Berti L, Kellerer M, Bossenmaier B, Seffer E, Seipke G, Haring HU (1998) The long acting human insulin analog HOE 901: characteristics of insulin signalling in comparison to Asp(B10) and regular insulin. Horm Metab Res 30:123–129
Colhoun HM (2009) Use of insulin glargine and cancer incidence in Scotland: a study from the Scottish Diabetes Research Network Epidemiology Group. Diabetologia 52:1755–1765
Currie CJ, Poole CD, Gale EA (2009) The influence of glucose-lowering therapies on cancer risk in type 2 diabetes. Diabetologia 52:1766–1777
Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW, Wallace JC (2004) Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR. Mol Endocrinol 18:2502–2512
Duckworth WC, Bennett RG, Hamel FG (1998) Insulin degradation: progress and potential. Endocr Rev 19:608–624
Hemkens LG, Grouven U, Bender R, Gunster C, Gutschmidt S, Selke GW, Sawicki PT (2009) Risk of malignancies in patients with diabetes treated with human insulin or insulin analogues: a cohort study. Diabetologia 52:1732–1744
Hossain MA, Lin F, Zhang S, Ferraro T, Bathgate RA, Tregear GW, Wade JD (2006) Regioselective Disulfide Solid Phase Synthesis, Chemical Characterization and In Vitro Receptor Binding Activity of Equine Relaxin. Int J Pept Res Ther 12:211–215
Hossain MA, Bathgate RA, Kong CK, Shabanpoor F, Zhang S, Haugaard-Jonsson LM, Rosengren KJ, Tregear GW, Wade JD (2008a) Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5). Chembiochem 9:1816–1822
Hossain MA, Rosengren KJ, Haugaard-Jonsson LM, Zhang S, Layfield S, Ferraro T, Daly NL, Tregear GW, Wade JD, Bathgate RA (2008b) The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors. J Biol Chem 283:17287–17297
Hossain MA, Belgi A, Lin F, Zhang S, Shabanpoor F, Chan L, Belyea C, Truong HT, Blair AR, Andrikopoulos S, Tregear GW, Wade JD (2009) Use of a temporary “solubilizing” peptide tag for the Fmoc solid-phase synthesis of human insulin glargine via use of regioselective disulfide bond formation. Bioconjug Chem 20:1390–1396
Jonasson JM, Ljung R, Talback M, Haglund B, Gudbjornsdottir S, Steineck G (2009) Insulin glargine use and short-term incidence of malignancies-a population-based follow-up study in Sweden. Diabetologia 52:1745–1754
Kohn WD, Micanovic R, Myers SL, Vick AM, Kahl SD, Zhang L, Strifler BA, Li S, Shang J, Beals JM, Mayer JP, DiMarchi RD (2007) pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity. Peptides 28:935–948
Kurtzhals P, Schaffer L, Sorensen A, Kristensen C, Jonassen I, Schmid C, Trub T (2000) Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use. Diabetes 49:999–1005
Lin F, Otvos L Jr, Kumagai J, Tregear GW, Bathgate RA, Wade JD (2004) Synthetic human insulin 4 does not activate the G-protein-coupled receptors LGR7 or LGR8. J Pept Sci 10:257–264
Mayer JP, Zhang F, DiMarchi RD (2007) Insulin structure and function. Biopolymers 88:687–713
Rosenstock J, Fonseca V, McGill JB, Riddle M, Halle JP, Hramiak I, Johnston P, Davis M (2009a) Similar progression of diabetic retinopathy with insulin glargine and neutral protamine Hagedorn (NPH) insulin in patients with type 2 diabetes: a long-term, randomised, open-label study. Diabetologia 52:1778–1788
Rosenstock J, Fonseca V, McGill JB, Riddle M, Halle JP, Hramiak I, Johnston P, Davis M (2009b) Similar risk of malignancy with insulin glargine and neutral protamine Hagedorn (NPH) insulin in patients with type 2 diabetes: findings from a 5 year randomised, open-label study. Diabetologia 52:1971–1973
Ross M, Francis GL, Szabo L, Wallace JC, Ballard FJ (1989) Insulin-like growth factor (IGF)-binding proteins inhibit the biological activities of IGF-1 and IGF-2 but not des-(1–3)-IGF-1. Biochem J 258:267–272
Zhao M, Wan ZL, Whittaker L, Xu B, Phillips NB, Katsoyannis PG, Ismail-Beigi F, Whittaker J, Weiss MA (2009) Design of an insulin analog with enhanced receptor binding selectivity: rationale, structure, and therapeutic implications. J Biol Chem 284:32178–32187
Acknowledgments
This study was funded by a grant to JDW from the National Health and Medical Research Council (NHMRC) of Australia (ID no. 508995). JDW is a recipient of a Research Fellowship from the NHMRC, BEF is a recipient of a University of Adelaide Biochemistry Fellowship, while MAH is the recipient of a John T Reid Charitable Trusts Fellowship. Seon-Yeong was the recipient of a travel award from the Brain Korea 21 Program to the Howard Florey Institute, Melbourne, Australia. We would like to thank Ms Linda Chan for amino acid analysis and Ms Kerrie McNeil with assistance with the receptor binding and DNA synthesis assays.
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Kwak, SY., Forbes, B.E., Lee, YS. et al. Solid Phase Synthesis of an Analogue of Insulin, A0:R glargine, That Exhibits Decreased Mitogenic Activity. Int J Pept Res Ther 16, 153–158 (2010). https://doi.org/10.1007/s10989-010-9218-8
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DOI: https://doi.org/10.1007/s10989-010-9218-8