1. References (1)Zakova,L. et al, “Shortened Insulin Analogs: Marked Changes in Biological Activity Resulting from Replacement of TyrB26 and N-Methylation of Peptide Bonds in the C-Terminus of the B-Chain”, Biochemistry, 2004, 43, 2323-2331. (2)Miller,S and Scanlan T., “Site-Selective N-methylation of Peptides on Solid Support”, J.A.C.S., 1997, 119, 2301-2302. Acknowledgements We would like to thank Beili Quan who assisted with the binding studies. Exploration and Elaboration of Reported Insulin Superagonism through Site-Selective Replacement at TyrB26 D. Smiley, Ma Boaquan, V. Gelfanov, and R. DiMarchi, Department of Chemistry Indiana University, Bloomington Indiana 47405 U.S.A. Experimental Design & Results Synthesis of B-Chain Insulin Analogs :Boc His (Bom) was coupled to an mbha-amide resin and the Boc group removed with TFA treatment. The alpha-amino group was N-methylated after conversion of the imidazole to the oNPS derivative. After oNPS removal, Boc Phe was coupled manually using PyBop/DiEA/DCM, and followed with a second coupling using a symmetric anhydride. The remainder of the B- chain was assembled using traditional single coupling methodology using an ABI 430A peptide synthesizer. The B-chain analog was cleaved from the resin using HF in the presence of m-cresol. The peptide was extracted into aq HOAc and purified over a preparative Kromasil C18 column using a linear gradient of acetonitrile in aq. 0.1% TFA, while monitoring the UV at 214nm. All other analogs (Aad B26 ; N-MeGlu B26 ; and Phe, 4-carboxy B26 ) were prepared using an Fmoc-based synthesis employing a Rink amide resin via HOBt active esters, on an ABI 433A instrument. The His B26 (1-30) B-chain analog was also prepared using Fmoc chemistry starting with Fmoc Thr(OtBu)-Wang resin. Each peptide derived from an FMOC-based synthesis was chromatographically purified as previously identified. Chain Combination: A modifed chain combination procedure was used to generate the respective insulin analog. An amount of B-chain was added to a molar equivalent of native A-chain S-sulfonate (Eli Lilly) and a stiochiometric amount of DTT was added to reduce the remaining sulfhydryls. The reaction was stirred in 0.1M glycine buffer (pH 10.5) at 4ºC for 22hrs. The reaction mixture was purified over a Zorbax C8 column in a slightly alkaline NH 4 HCO 3 through a linear gradient of acetonitrile, while observing the UV 214 absorption. The step yield for the isolated insulin analogs typically ranged between 10-20%. The theoretical masses were confirmed by MALDI analysis. Receptor Binding: The affinity of each peptide 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, pH 7.5, 0.15 M NaCl, 0.1% w/v bovine serum albumin) and mixed in 96 well plates (Corning Inc., Acton, MA) with 0.05 nM (3-[125I]-iodotyrosyl) A Tyr A14 insulin (Amersham Biosciences, Piscataway, NJ). An aliquot of 1-6 micrograms of plasma membrane fragments prepared from cells over-expressing the human insulin receptors were present in each well and 1 mg/well polyethyleneimine-treated wheat germ agglutinin type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, NJ) were added. After five minute of shaking at 800 rpm the plate was incubated for 12h at room temperature and then analyzed with a MicroBeta1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). Non-specifically bound (NSB) radioactivity was measured in the wells with a fourfold concentration excess of “cold” native ligand than the highest concentration in test samples. 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 (OriginLab, Northampton, MA). Abstract Insulin has virtually universal ability to lower blood glucose and is currently used in multiple forms by millions of humans in the treatment of diabetes. DNA technology has facilitated the biosynthesis of insulin and various related analogs in virtually unlimited quantity. Nonetheless, the relatively low potency of insulin renders it a unique commercial challenge where yearly production is measured in tonnage. Furthermore, low potency is a significant obstacle in the development of non-invasive methods for insulin administration. A recent report 1 outlined replacements for TyrB26 in a C-terminally shortened insulin analog that yielded unprecedented increases in potency. Bioactivity constituted measurement of glucose transport and insulin receptor binding in isolated primary rat adipocytes and plasma membranes respectively. N-methylHisB26 DTI, prepared via semi-synthesis, was reported to possess increased potency relative to native insulin in excess of 50-fold in receptor binding and 10-fold in stimulating glucose uptake. We have explored this observation through synthesis of the same analog by chain combination of totally synthetic A and B chains. Bioactivity and potency was measured relative to insulin in engineered cells that over-express human insulin receptor. Additionally, we have prepared a number of more acidic B26 amino acid insulin analogs to further elaborate the molecular basis for the superagonism. Our results would suggest that the increased potency likely resides in the nature of the bioassays. Further work is necessary to reconcile differences in observations reported from primary cells with those we observe in engineered cells. Conclusions 1.N-MeHis B26 DTI appears to have insulin receptor binding activity similar to native insulin under the specific assay conditions we utilized. 2. The basis for the difference in our observation and the superagonism of N-MeHis B26 DTI reported previously is not immediately certain. We presume it to reside in the nature of the over-expressing receptor engineered cells we employed relative to the isolated rat hepatocytes of the published report. 3. Other types of acidic moieties in the B26 position served as near-functional equivalents for the native Tyr B26 residue. 4. The aromatic nature of the native Tyr B26 residue is not required for high affinity binding at the IR. N-MeHisB26 DTI 20ul 0.4mg/ml Biorad AS-100 0.46x5cm Zorbax C8 1ml/min,45c,214nm,0.5A A=0.1%TFA, B=0.1%TFA/90%ACN 10%B to 80%B over 10min Ubiquitin (M+H) + Lyzozyme (M=2H)2 + Ubiquitin (M+2H) + Somatostatin 28 (M+H) + Theoretical MW= 5367.19 N-methyl HisB26 DTI (DLS-002-092H) 20ul.3mg/ml Biorad AS-100 0.46x15cm Vydac C18 1ml/min,45C,214nm A=0.1%TFA B=0.1%TFA/90%ACN B26 residueInsulin receptor (nM) n Tyr (1-30)0.68+0.286 His (1-30)1.331 1.24+0.494 0.71+0.812 0.461 0.61 (1-26)amide FVNQHLC SH GSHLVEALYLVCGERGFF G NH 2 IVEQCCTSICSLYQLENYCNCOOH SSO 3 - SSO 3 - SSO 3 - SSO 3 - S S S S S S X + G I V E Q C C T S IC S L Y Q L E N Y C N F V N Q H L C G S H L VE A L Y L V C G E R G F F X amino acid amide, or 26-30 acid SH NH 2