A highly potent insulin: des-(B26-B30)-[AspB10,TyrB25-NH2]insulin(human).

Abstract

An insulin analogue that embodies two distinct structural modifications, each of which independently increases insulin activity, has been synthesized and evaluated for biological activity. The analogue, des-(B26-B30)-[AspB10, insulin is the most potent insulin analogue yet described; it displays an 11-to 13-fold higher activity than natural insulin. The rmdings are discussed with regard to the receptor-binding domains of insulin.In a recent publication, we described the synthesis of [AspBlolinsulin, an analogue of human insulin that displays a potency ca. 4-5 times greater than that of insulin (1). This finding in conjunction with previous studies carried out in our laboratory involving modifications of insulin at the B10 position (2)(3)(4) (20), was prepared by oxidative sulfitolysis of porcine insulin and separation of the resulting S-sulfonated A and B chains by column chromatography (21). The S-sulfonated doubly substituted des-(B26-B30) B chain was assembled by stepwise solid-phase synthesis (22, 23) by using 4-methylbenzhydrylamine resin as the solid support (0.5 mmol of amine per g; 1 g). The t-butoxycarbonyl group was used for N" protection except for the N-terminal phenylalanine residue, which was protected by the benzyloxycarbonyl group. Side-chain protecting groups were as follows: benzyl for serine, 2,6-dichlorobenzyl for tyrosine, NG-p-toluenesulfonyl for arginine, cyclohexyl for glutamic and aspartic acids, benzyloxymethyl for histidine, and 4-methylbenzyl for cysteine. A manual double-coupling protocol was followed (24) with activated protected amino acids (1-hydroxybenzotriazole/ dicyclohexylcarbodiimide in dimethylformamide) in 3-fold excess. The completion of the reaction was monitored by the qualitative ninhydrin test (25) and was negative after each double coupling.After the chain was assembled, the peptide-resin was washed extensively with methylene chloride and methanol and dried: weight, 3.0 g. A portion of this product (700 mg) was deprotected by the low/high hydrogen fluoride procedure (26).In the first step the peptide-resin was treated with a mixture consisting of p-cresol (1 ml), dimethyl sulfide (6.5 ml), and liquid hydrogen fluoride (2.5 ml). After 2 hr at 0°C, the mixture was concentrated under reduced pressure, and the residue was treated with liquid hydrogen fluoride (10 ml) for 1 hr at 0°C. The hydrogen fluoride was then removed, and the residue was triturated with ethyl acetate and petroleum ether. To a suspension ofthis product in 8 M guanidine hydrochloride (20 ml) buffered with 0.1 M Tris HCl (pH 8.8) were added sodium sulfite (700 mg) and sodium tetrathionate (500 mg). After 3 hr at room temperature, the reaction mixture was filtered to remove the resin, and the filtrate was placed in Spectra/por membrane tubing no. 3 and dialyzed against four changes of Abbreviation: 1251-insulin, 125I-labeled insulin.

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective deuterium and carbon-13 labeling with application to protein design

“…In DKP-insulin no significant interresidue NOEs are observed to AspB 10, suggesting that it projects from the surface of the protein as shown in Figure 1A. The apparent absence of structural "cross-talk" between the B10 site and residues B23-B26 (assigned in part III; below) is consistent with observations by Katsoyannis and colleagues that modifications at these two sites exhibit additive effects on the potency of insulin analogues (Schwartz et al, 1989).…”

Mini-proinsulin and mini-IGF-I: homologous protein sequences encoding non-homologous structures

“…There are three possible reasons why such residual activity was readily observed in the present study. First, the analogue contains the substitution His B10 3 Asp, which has been shown to enhance the binding and activity of insulin analogues (Schwartz et al, 1987(Schwartz et al, , 1989Shoelson et al, 1992). Second, at high protein concentrations (>1 mM) native insulin begins to form dimers and higher-order oligomers (Goldman & Carpenter, 1974).…”

“…The S-sulfonated single-chain polypeptide was prepared by solid-phase synthesis of the protected 50-residue polypeptide followed by deblocking and sul®tolysis. The crude S-sulfonated 50mer was puri®ed by ionexchange HPLC chromatography using a Synchropak AX300 column (10 mm  250 mm) as described (Schwartz et al, 1989). Conversion of the S-sulfonated derivative to a mini-proinsulin analogue was accomplished by: (i) reduction to the sulfhydryl form by 25% (v/v) b-mercaptoethanol in 6 M guanidine-HCl and 10 mM Tris-HCl (pH 8.4); (ii) precipitation of the reduced protein in 1 N HCl by acetone (40:1, v/v) on ice; and (iii) oxidative refolding in 0.09 M Tris-acetate (pH 8.2) in the presence of oxidized and reduced glutathione as described in studies of bovine pancreatic trypsin inhibitor (Ahmed et al, 1975).…”

Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and carbon-13-labeled-isotope-aided 2D-NMR studies of [PheB24.fwdarw.Gly]insulin with implications for receptor recognition

“…The mechanism by which insulin binds to its receptor is not understood despite extensive study of insulin's structure in multiple crystal forms (Adams et al, 1969;Peking Insulin Structure Group, 1971;Blundell et al, 1972;Bentley et al, 1978;Smith et al, 1984;Derewenda, et al, 1989;Badger et al, 1991). Structure-function relationships have been probed by comparative studies of species variants (Wood et al, 1975;Emdinetal., 1977;Pullen etal., 1976;Steiner, 1989),diabetesassociated mutant human insulins (Tager et al, 1979;Inouye etal., 1981aInouye etal., ,b, 1983Shoelson etal., 1983aShoelson etal., ,b, 1984, synthetic or engineered analogues (Inouye et al, 1979(Inouye et al, , 1982Assoian et al, 1982;Kobayashi et al, 1982;Fisher et al, 1985;Nakagawa & Tager, 1986Casaretto et al, 1987;Schwartz etal., 1987;Brange etal., 1988;Schwartz et al, 1989;Mirmira et al, 1991;Wang et al, 1991;Shoelson et al, 1992), and analogues containing chemical cross-links between the Aand B-chains (Brandenburg et al, 1972;Cutfield, et al, 1981Cutfield, et al, , 1986.…”