Site Specific Introduction of Unnatural Amino Acids at

Site Specific Introduction of Unnatural Amino Acids at Sites Critical to Insulin Receptor Recognition & Biological Activity B. Quan, D. Smiley, V. Gelfanov, and R. Di. Marchi Department of Chemistry Indiana University, Bloomington Indiana 47405 U. S. A Abstract Experimental Design 55 50 49 60 CH 2 3. 55 VAL LYS ARG 42 GLU 41 GLN A-CHAIN 40 GLY 39 VAL Insulin ILE GLN 38 3 VAL LEU GLU 4 S GLN 5 B-CHAIN S CONNECTING PEPTIDE CYS THR 7 8 1 PHE VAL 2 ASP 36 CYS 6 9 ILE 10 S CYS SER ASN LEU TYR GLN CYS LEU GLU ASN TYR 11 12 13 14 15 16 ASN 3 4 17 18 0. 51 19 20 0 1 E-3 0. 01 0. 1 1 10 5 LEU PRO CYS 7 8 THR S GLY SER 9 HIS 10 LEU 11 VAL 12 TYR ARG GLU ALA GLU LEU TYR LEU VAL CYS GLY 13 14 15 16 17 18 19 20 21 22 GLY 23 0. 49 1 E-3 PHE 24 0. 01 0. 1 CF 3 1 10 100 [Peptide], n. M B 24(2 -Me) B 24(3 -Me) B 24(4 -Me) 200 B 24 IC 50 n. M 80 CH 2 CH 3 18. 25 180 CH 2 22. 54 N 60 CH 2 40 Insulin 6. 78 CH 2 0. 63 S CH 3 20 CH 2 35 34 1 E-3 5. 25 0. 01 0. 1 1 10 100 140 120 80 Results & Conclusion 60 40 -20 1 E-3 [Peptide], n. M Insulin 0. 01 0. 1 1 10 100 [Peptide], n. M 29 28 27 26 Fig 4. Binding Affinity of Insulin Analogs Substituted at Position B 24 25 B 25 IC 50 n. M 140 >1000 Insulin B 25 Phe(4 -CF 3) Theoretical MW= 5875 Ubiquitin (M+H)+ average Ubiquitin (M+2 H) 2+average CH 2 13. 15 B 25(4 -Me) B 25(4 -Br) B 25(4 -CH 2 NH 2) h. Phe 25 B 25(4 -COOH) 120 100 CH 2 0. 67 CH 3 80 CH 2 60 CH 2 Insulin 1000 Nineteen different amino acids (17 non-natural) were substituted for the native phenylalanine residues at positions B 24 and B 25 in insulin. The analogs were successfully prepared by SPPS from individual A and B chains, in total yields that varied between 1 and 10%. The relative binding affinity of this group of insulin analogs for the insulin receptor was determined to differ by more than thousand-fold from the most potent to the least potent peptide studied. Consistent with previous observations predominantly employing native amino acids, we observed that insulin activity was highly dependent upon aromatic character at these two residues (1). Position B 24 was extremely restrictive to structural modification while B 25 was extremely permissive. In Figure 3 we report the extreme difference between the same substitutions at B 24 and B 25. Each of the B 25 insulin analogs were of high affinity while only one of the B 24 analogs did not exhibit a sizable reduction in binding affinity. The highly homologous B 24 Phe(4 -F) analog is a rare example of an l-amino acid with comparable potency to the native hormone, and represents an opportunity for structural study in the receptor recognition site with isotopically enriched amino acid. Further study at B 24 (Figure 4) illustrates that even a single methyl group added to the phenyl ring is deactivating and most notably when ortho to the ring. Phenylalanine heterocycle-based mimetics were studied. An appreciable difference was observed among two commonly used derivatives. The 5 -membered thiophene ring provided nearly native insulin affinity but the more alkaline 6 -membered pyridine ring was sizably reduced in potency. Additional study at position B 25 (Figure 5) demonstrated that movement of the phenyl ring closer or further from the peptide backbone had a significant deactivating impact upon binding affinity (2). Additional modifications that selectively introduced negative charge (4 -COOH) and size (4 -Br) relative to the native Phe were largely without effect. Although, the alkaline amino-methyl substitution (4 -CH 2 NH 2) reduced binding affinity to a significant degree. This observation is consistent with the more subtle reduction noted for the less alkaline 4 -NH 2 modification (Figure 3). 3. 15 20 CH 2 NH 2 0 0. 01 0. 1 1 10 1000 0. 55 Br Fig 5. Binding Affinity of Insulin Analogs Substituted at Position B 25 References CH 2 [Peptide], n. M Fig 2. Purified Insulin Analog Chromatographic & Mass Spectral Analysis 0. 83 COOH 40 1 E-3 Receptor Binding : The binding affinity of each insulin analog for the insulin receptor was measured in a competition binding assay utilizing scintillation proximity assay technology. Serial 3 -fold dilutions of the peptides were made in scintillation proximity assay buffer (0. 05 M Tris-HCl, p. H 7. 5, 0. 15 M Na. Cl, 0. 1% w/v bovine serum albumin) in a 96 well plate (Corning Inc. , Acton, MA) with 0. 05 n. M (3 -[125 I]-iodotyrosyl) Tyr. A 14 Insulin. An aliquot of 1 -6 micrograms of plasma membrane fragments prepared from cells over-expressing the human insulin receptor and 1 mg/well polyethyleneiminetreated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, NJ) were added to each well. The plate was incubated for twelve hours at room temperature and measurements made with Micro. Beta 1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). Non-specifically bound (NSB) radioactivity was measured in the wells with fourfold greater concentration of “cold” native ligand than the highest concentration in test samples and total bound radioactivity was detected in the wells with no competitor. Percent specific binding was calculated as following: % Specific Binding = Bound-NSB / Total bound- NSB x 100. IC 50 values were determined by using Origin software (Origin. Lab, Northampton, MA). 100 0 CH 3 1000 160 20 0 32 B 24(4 -pyridyl) B 24(4 -thienyl) Fig 1. Primary Structure of Human Proinsulin 0. 46 Χ 15 cm Vydac C 18 1 ml/min, 45 C, 214 nm, Linear gradient of CH 3 CN in 0. 1% TFA(aq) 1000 Fig 3. Binding Affinity of Insulin Analogs with Comparable Amino Acid Substitution at B 24 & B 25 30 30 LYS 6 0 16. 58 100 [Peptide], n. M 31 THR HIS IGF-1 40 CH 2 ARG S 60 Insulin Synthesis of B-chain analogs : B chain analogs with unnatural amino acids at B 24 and B 25 were successfully synthesized by solid phase methodology using Fmoc chemistry. Peptides were cleaved from the support in strong anhydrous acid and purified under acidic conditions by preparative high performance reverse-phase chromatography in the free sulfhydryl form. Each peptide was characterized by MALDI-MS and HPLC analysis to be at least 90% pure prior to chain combination. Chain Combination Reaction : Each B-chain analog was mixed with a molar equivalent of native A-chain S-sulfonate (gift from Eli Lilly) in a 0. 05 M glycine buffer, p. H 10. 5. A molar equivalent of DTT relative to each S-sulfonate was used to facilitate disulfide interchange. The reaction was conducted at 4ºC, overnight. The product was purified by preparative high performance reverse phase chromatography in a slightly alkaline NH 4 HCO 3 buffer with CH 3 CN elution. The insulin analogs were obtained in step yield that varied from 5 -30%, and characterized by MALDI-MS and HPLC analysis to be greater than 95% pure. 20 0. 91 33 21 80 F GLU ARG S GLN GLU ALA SER 37 NH 2 CH 2 %Specific Binding 2 1. 03 IGF-1 20 48 43 100 0. 72 6. 79 100 GLN 64 1 56 51 47 LEU SER GLY 59 PRO GLN 46 LEU ALA GLY ALA 60 PRO 45 LEU GLY 61 GLU 44 GLY 62 GLY SER LEU 63 65 57 52 0. 67 OH %Specific Binding 58 53 0. 67 80 120 54 CH 2 40 B 25(4 -CF 3) B 25(4 -F) B 25(4 -NH 2) B 25 (4 -OH) 120 %Specific Binding 100 B 25 IC 50 n. M %Specific Binding Our work has focused on two amino acids in the C-terminus of the Bchain that are central to insulin activity, specifically positions B 24 and 25. The selective introduction of a series of Phe-related derivatives at each position has yielded an insightful perspective on insulin receptor recognition. Position 24 has proven highly restrictive to changes that can be introduced and the results emphasize the importance of backbone conformation to full potency. In comparison, position B 25 is quite accommodating to sizable changes in side-chain structure but surprisingly highly demanding in that the phenyl ring must be positioned at the beta-carbon. These collective observations establish a foundation for application of unnatural amino acids as a route to insulin pharmacology that may not be obtainable with natural amino acids alone. B 24 IC 50 n. M B 24(4 -F) B 24(4 -NH 2) B 24 (4 -OH) B 24(4 -CF 3) 120 %Specific Binding Insulin constitutes a hormone of central importance in physiology and a vital element in glucose management. Its use in diabetes care has been of seminal significance for nearly a century. The advent of r. DNA biosynthesis provided human insulin in virtually unlimited quantity. More importantly, it provided a mechanism by which improved biosynthetic insulin analogs could be synthesized, evaluated and registered as new medicines. The advent of chemical biotechnology (biosynthesis with unnatural amino acids) provides a new venue for optimizing insulin pharmacology through the use of synthetic chemistries that otherwise would be prohibitively expense for commercialization. 1. Mirmira, R. G. , Nakagawa S. H. , and Tager H. S. (1991) J. Biol. Chem. 266, 1428 -1436 2. Nakagawa, S. H. And Tager, H. S. (1986) J. Biol. Chem. 261, 7332 -7341 Acknowledgements Beili Quan is financially supported by an Indiana University Gill Center Fellowship.