Optimization of the Cterminal Sequence in Glucagon to

Optimization of the C-terminal Sequence in Glucagon to Maximize Receptor Affinity J. Levy, V. Gelfanov, and R. Di. Marchi Department of Chemistry Indiana University, Bloomington Indiana 47405 U. S. A. Abstract Experimental Design & Results Glucagon is a linear peptide of 29 amino acids that is highly conserved among vertebrates. The C-terminal -helical region is central to receptor recognition. The depth of structural analysis in this section of the hormone pales in comparison to those at the N-terminal region, where the presence of the first amino acid and the nature of the ninth are vital to receptor signaling (1). Since native glucagon constitutes a life-saving medicine with notable biophysical deficiencies, the individual contribution of each C-terminal residue to glucagon’s pharmaceutical properties should be known. We have completed an alanine scan of the C-terminal region of glucagon in the range of amino acid residues 20 -28. This portion of the molecule is highly homologous to the related hormone GLP-1. Glucagon GLP-1 Synthesis of Glucagon Analogs: Each analog was synthesized on a solid support using Boc-based in situ neutralization chemistry as described by Kent, et al. (3) on either a highly-modified Applied Biosystems 430 A peptide synthesizer or a CSBio Model CS 336 peptide synthesizer. Peptides were then cleaved from the support using HF/p-cresol, 95: 5, for 1 hr at 0°C. Following HF removal and ether precipitation, the peptides were extracted into 10% HOAc and lyophilized. Each peptide was then purified using RP-HPLC in 0. 1% TFA using a linear gradient of CH 3 CN, on a Waters Associates preparative HPLC system. Fractions containing the desired peptide were pooled and lyophilized. The identity and purity of each analog was confirmed by analytical HPLC and MS analyses. QDFVQWLMN KEFIAWLVK Biological Activity (c. AMP induction): The ability of each glucagon analog to induce c. AMP was measured in a firefly luciferase-based reporter assay. HEK 293 cells cotransfected with either glucagon- or GLP-1 receptor and luciferase gene linked to c. AMP responsive element were employed for bioassay. The cells were serum deprived by culturing 16 h in DMEM (Invitrogen, Carlsbad, CA) supplemented with 0. 25% Bovine Growth Serum (Hy. Clone, Logan, UT) and then incubated with serial dilutions of either glucagon, GLP-1 or novel Glucagon analogs for 5 h at 37 o. C, 5% CO 2 in 96 well poly-D -Lysine-coated “Biocoat” plates (BD Biosciences, San Jose, CA). At the end of the incubation 100 microliters of Luc. Lite luminescence substrate reagent (Perkin Elmer, Wellesley, MA) were added to each well. The plate was shaken briefly, incubated 10 min in the dark and light output was measured on Micro. Beta-1450 liquid scintillation HPLC Purified Glucagon(Ala-29) counter (Perkin-Elmer, Wellesley, MA). Inhibitory 50% (IC 50) and effective 50% concentrations (EC 50) were calculated by using Origin software (Origin. Lab, Northampton, MA). Vydac C-4, 4. 6 x 15 cm, 1. 5 ml/min, 220 nm Theoretical Mass = 3453. 78 Similar alanine substitution in GLP-1 demonstrated that four of these same nine amino acids and, in particular, the single Phe were vital for full potency (2). We have studied the glucagon bioactivity of each glucagon analog, as well as selectivity with the GLP-1 receptor. Our results demonstrate that glucagon behavior is highly dependent on the nature of amino acid sequence in this region of the hormone. Additional modifications with non-alanine based amino acids are in progress. Structure of Native Glucagon 1 5 0. 1%TFA in Acetonitrile Gradient His Ser Gln Gly Thr Phe Thr Ser Asp 15 Ala 20 Arg Ser Asp Tyr Leu 10 Tyr Lys Ser Met Asn Thr --COOH 25 Gln Asp Phe Val Gln Trp Leu Figure 1. Representative Glucagon Alanine Analog (Ala-29) Chromatographic & Mass Spectral Analysis Introduction We explored the structural modification of native glucagon with the intent of enhancing the physical properties with minimal change in pharmacology. As a first step in optimizing the pharmaceutical properties of glucagon we have completed an alanine scan in the C-terminal region of the hormone (residues 20 -28) to identify those residues that are central to biological function. Alanine scaning is a proven approach to initial segregation of those amino acids that contribute structurally to receptor signaling through peptide backbone conformational effect versus direct side chain interaction. In a similar study with the highly homologous peptide GLP-1 (2) it was demonstrated that in vitro bioactivity was extremely sensitive to substitution with alanine at the amino acids comparable to residues 22 and 23 in this region of the peptide. Our study explores changes in potency at the glucagon receptor and specificity for in vitro action at the GLP-1 receptor. GLP-1 Receptor mediated c. AMP Induction Glucagon Receptor mediated c. AMP Induction 5000 A Glucagon A 20 A 21 A 22 A 23 A-24 A-25 A 26 A-27 A-28 GLP-1 4000 3000 CPS Glucagon is a linear peptide hormone of 29 amino acids of central importance in physiology. For more than half a century it has been used as a critical care medicine in the treatment of life-threatening insulin-induced hypoglycemia. The biophysical properties of natural sequence glucagon are not conducive to formulation in a patientfriendly formulation. The hormone is poorly soluble at physiologic p. H and prone to physical aggregation to insoluble fibrils. Consequently, glucagon is commercially supplied as a lyophilized powder to be solubilized in dilute aqueous HCl immediately prior to administration. To a patient that is semi-conscious or unconscious this represents an obstacle to proper administration and could constitute a fatal flaw. EC 50, n. M n* EC 50, n. M n Glucagon 0. 16 0. 11 3 6. 48 2. 40 3 A-20 0. 22 0. 12 3 4. 96 2. 54 3 A-21 0. 23 0. 09 2 42. 43 13. 12 3 A-22 97. 65 35. 35 4 6150. 31 293. 79 3 A-23 18. 96 10. 49 4 533. 81 9. 97 3 A-24 0. 09 0. 03 2 10. 78 5. 92 3 A-25 2. 22 1. 07 3 133. 64 70. 21 3 A-26 6. 44 0. 19 2 312. 03 118. 56 3 A-27 0. 66 0. 19 2 72. 03 37. 63 3 A-28 0. 21 0. 04 2 31. 60 10. 04 3 2908. 50 357. 09 2 0. 05 0. 02 5 GLP-1 * Number of experiments Q 20 K 20 V 23 I 23 Q 24 L 26 D 21 F 22 L 26 W 25 Glucagon A 24 E 21 W 25 F 22 Figure 3 GLP-1 Results & Discussion 2000 1000 B 30000 25000 20000 15000 10000 5000 0 0 1 E-3 0. 01 0. 1 1 10 100 [Peptide], n. M 10000 1 E-4 1 E-3 0. 01 0. 1 1 10 10000 [Peptide], n. M Figure 2. Glucagon (A) and GLP-1 (B) receptor-mediated c. AMP induction by glucagon analogs CPS NH 2 -- Peptide Table 1. Bioactivity of Glucagon Alanine Analogs GLP-1 Receptor Glucagon Receptor The synthesis and purification of the nine glucagon analogs was relatively straightforward and each peptide was obtained in total yields in excess of 20% based on the weight of the starting amino acid resin. None of the peptides proved any more problematic than native glucagon in the physical handling and formulation for bioassay. A single example in the synthesis of this set of alanine-substituted glucagon analogs is shown in Figure 1. The representative chromatographic and mass spectral analyses demonstrate the integrity and purity of the analogs studied in this report. The results of the bioassay at each of the two receptors are shown in Figure 2 and Table 1. It is immediately obvious that glucagon has a number of amino acids in this Cterminal region of the peptide where the bioactivity at both receptors is extremely sensitive to substitution with alanine. Consistent with previously reported alanine scanning studies with GLP-1, residues 22 and 23 were extremely sensitive to alanine substitution (2). Residues 25 and 26 were also significantly reduced in bioactivity with alanine substitution but to a more modest degree. Most notably, the amino acids that border these four residues were relatively insensitive to substitution and support the report that this region of glucagon is prone to alpha-helix formation (4). The directional changes in bioactivity at the two receptors with each substitution studied was consistent and differed in magnitude only at positions 21 and 28 where in both instances the GLP-1 activity appeared to decrease to an appreciably larger extent. Figure 3 provides a helical wheel representation of this C-terminal region and illustrates the amphiphatic structure and high homology within these two peptide hormones, particularly in the biologically sensitive hydrophobic residues (colored in red). Of particular note on the hydrophilic side of this helix (colored in yellow) is the change of the glutamines at residues 20 and 24 in glucagon with lysine-20 and alanine-24. We believe that these two differences are the structural basis for the physical properties that render glucagon more prone to physical aggregation and formation of high molecular weight fibrils (5), since glutamine is much more supportive of secondary beta -structure than either alanine or lysine. These results in concert with prior reports (6) of structure-activity in this region of glucagon form the basis for design of more potent and physically stable glucagonists. References 1. Unson, C. G. and Merrifield, R. B. (1994) PNAS 91, 454 -458.