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.
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SSO SS O S S O3
3 3
NH2 G I V E Q C C T S I C S L Y Q L E N Y C N COOH
Insulin Rece ptor Bindin g
Abstract SSO
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Insulin has virtually universal ability to lower blood glucose and is currently used in multiple forms by 1 40

millions of humans in the treatment of diabetes. DNA technology has facilitated the biosynthesis of insulin and SH SH 1 20

various related analogs in virtually unlimited quantity. Nonetheless, the relatively low potency of insulin renders it NH2 F V N Q H L C G S H L V E A L Y L V C G E R G F F X

% Specific Binding
1 00
a unique commercial challenge where yearly production is measured in tonnage. Furthermore, low potency is a amino acid amide,
significant obstacle in the development of non-invasive methods for insulin administration. or 26-30 acid 80

A recent report1 outlined replacements for TyrB26 in a C-terminally shortened insulin analog that yielded 60

unprecedented increases in potency. Bioactivity constituted measurement of glucose transport and insulin 40
receptor binding in isolated primary rat adipocytes and plasma membranes respectively. Insulin
N N MeH isB26D TI
N-methylHisB26 DTI, prepared via semi-synthesis, was reported to possess increased potency relative to native G
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Y
C
20
H is B26 Insulin
S
insulin in excess of 50-fold in receptor binding and 10-fold in stimulating glucose uptake. We have explored this V
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observation through synthesis of the same analog by chain combination of totally synthetic A and B chains. C
C T S I C S L Y
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Bioactivity and potency was measured relative to insulin in engineered cells that over-express human insulin X 1 E -3 0. 0 1 0 .1 1 10 1 00 10 00
S
receptor. Additionally, we have prepared a number of more acidic B26 amino acid insulin analogs to further F F
F [Peptide], nM

elaborate the molecular basis for the superagonism. Our results would suggest that the increased potency likely V
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resides in the nature of the bioassays. Further work is necessary to reconcile differences in observations H
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reported from primary cells with those we observe in engineered cells. C G S H L A L Y L V
V E

Insulin Receptor Binding

180
Insulin
Experimental Design & Results 160 AadB26D TI
NMeGluB26DT I
N-methyl HisB26 DTI (DLS-002-092H) 140
Phe(4carboxy)B26DT I

Synthesis of B-Chain Insulin Analogs: Boc His (Bom) was coupled to an mbha-amide resin and the N-MeHisB26 DTI
120 IGF-1

Boc group removed with TFA treatment. The alpha-amino group was N-methylated after conversion of the

% Specific Binding
100
20ul 0.4mg/ml Biorad AS-100 Theoretical MW= 5367.19
imidazole to the oNPS derivative. After oNPS removal, Boc Phe was coupled manually using 20ul .3mg/ml Biorad AS-100
0.46x5cm Zorbax C8
80

PyBop/DiEA/DCM, and followed with a second coupling using a symmetric anhydride. The remainder of the B- 0.46x15cm Vydac C18
1ml/min,45c,214nm,0.5A 60

chain was assembled using traditional single coupling methodology using an ABI 430A peptide synthesizer. A=0.1%TFA,
1ml/min,45C,214nm 40
The B-chain analog was cleaved from the resin using HF in the presence of m-cresol. The peptide was B=0.1%TFA/90%ACN

extracted into aq HOAc and purified over a preparative Kromasil C18 column using a linear gradient of A=0.1%TFA
10%B to 80%B over 10min
20

acetonitrile in aq. 0.1% TFA, while monitoring the UV at 214nm. All other analogs (AadB26; B=0.1%TFA/90%ACN 0

N-MeGluB26; and Phe, 4-carboxyB26) were prepared using an Fmoc-based synthesis employing a Rink amide -20

resin via HOBt active esters, on an ABI 433A instrument. The HisB26 (1-30) B-chain analog was also prepared 1 E -3 0. 01 0. 1 1 10 1 00 1000

using Fmoc chemistry starting with Fmoc Thr(OtBu)-Wang resin. Each peptide derived from an FMOC-based [Peptide], nM
synthesis was chromatographically purified as previously identified. Somatostatin 28 (M+H)+

Chain Combination: A modifed chain combination procedure was used to generate the respective insulin Ubiquitin (M+H)+

analog. An amount of B-chain was added to a molar equivalent of native A-chain S-sulfonate Ubiquitin (M+2H)+

(Eli Lilly) and a stiochiometric amount of DTT was added to reduce the remaining sulfhydryls. The reaction
Lyzozyme (M=2H)2+
Conclusions
was stirred in 0.1M glycine buffer (pH 10.5) at 4C for 22hrs. The reaction mixture was purified over a Zorbax
C8 column in a slightly alkaline NH4HCO3 through a linear gradient of acetonitrile, while observing the UV 214 1. N-MeHisB26 DTI appears to have insulin receptor binding activity similar to
absorption. The step yield for the isolated insulin analogs typically ranged between 10-20%. The theoretical native insulin under the specific assay conditions we utilized.
masses were confirmed by MALDI analysis.
2. The basis for the difference in our observation and the superagonism
Receptor Binding: The affinity of each peptide for the insulin receptor was measured in a competition B26 residue Insulin receptor (nM) n of N-MeHisB26 DTI reported previously is not immediately certain. We
binding assay utilizing scintillation proximity assay technology. Serial 3-fold dilutions of the peptides were presume it to reside in the nature of the over-expressing receptor engineered cells we
Tyr (1-30) 0.68+0.28 6 employed relative to the isolated rat hepatocytes of the published report.
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 TyrA14 His (1-30) 1.33 1
3. Other types of acidic moieties in the B26 position served as near-functional
insulin (Amersham Biosciences, Piscataway, NJ). An aliquot of 1-6 micrograms of plasma membrane equivalents for the native TyrB26 residue.
1.24+0.49 4
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 (1-26)amide 4. The aromatic nature of the native TyrB26 residue is not required for high affinity
(Amersham Biosciences, Piscataway, NJ) were added. After five minute of shaking at 800 rpm the plate was binding at the IR.
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 0.71+0.81 2 References
fourfold concentration excess of cold native ligand than the highest concentration in test samples. Total (1-
bound radioactivity was detected in the wells with no competitor. Percent specific binding was calculated as 26)amide (1)Zakova,L. et al, Shortened Insulin Analogs: Marked Changes in
following: % Specific Binding = (Bound-NSB / Total bound-NSB) x 100. IC50 values were determined by using Biological Activity Resulting from Replacement of TyrB26 and N-Methylation
Origin software (OriginLab, Northampton, MA). 0.46 1 of Peptide Bonds in the C-Terminus of the B-Chain, Biochemistry, 2004,
(1-26)amide 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.
0.6 1
(1-26)amide Acknowledgements
We would like to thank Beili Quan who assisted with the binding studies.