Journal of Protein Chemistry, Vol. 9, No. 2, 1990

An Insulin-Like Compound Consisting of the B-Chain of Bovine Insulin and an A-Chain Corresponding to a Modified A- and the D-Domains of Human Insulin-Like Growth Factor 11

Satish Joshi, 2'3 G. Thompson Burke, z and Panayotis G. Katsoyannis 2'4

Received January 31, 1990

We report the synthesis and biological evaluation of a two-chain, disulfide-linked, insulin-like compound consisting of the B-chain of bovine insulin and an A-chain corresponding to the A- and D- domains of human insulin-like growth factor-I (IGF-I) in which the A-domain amino-acid residues -Phe49-ArgS°-Ser sl- found in IGF-I have been replaced by -Ala-Gly-Val-, the homologous region of sheep insulin. The compound is indistinguishable from a previously reported compound whose A-chain corresponds to the A- and D-domains of IGF-I without the substitution, in assays for insulin-like activity as well as in assays for growth-promoting activity. We conclude that these A-domain residues do not contribute significantly to the interaction of IGF-I with either insulin or IGF-I receptors.

KEY WORDS: Insulin analogue; insulin-like-growth factor; peptide synthesis; receptor binding assay; lipogenesis.

1. I N T R O D U C T I O N

Insulin-like growth factor-I ( IGF-I ) is a polypeptide in human plasma that is chemically related to proin- sulin (Rinderknecht and Humbel, 1978). IGF- I con- tains B- and A-domains with - 4 0 % homology to the B- and A-domains of proinsulin, which are separated from each other by a 12 amino-acid peptide, the C-domain. Unlike proinsulin, IGF- I contains an eight amino-acid extension peptide at its C-terminus, the D-domain. The primary structure of human IGF-I is shown in Fig. 1. The B-domain involves residues 1-30, the C-domain residues 31-41, the A-domain residues 42-62, and the D-domain residues 63-70. By model

1 A preliminary discussion of this work was presented (P.G.K.) to the 19th European Peptide Symposium, Porto Carras, Greece, 1986.

2 Department of Biochemistry, Box 1020, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 1OO29-6574.

3 Present address: Star Biochemicals, Inc., Torrance, California. 4 To whom all correspondence should be addressed.

building and computer-graphics studies (Blundell et al., 1978, 1983), it was shown that IGF-I can assume an insulin-like structure as far as the A- and B-chain domains and the hydrophobic core are concerned. The considerable homology and conformational simi- larity of insulin and IGF- I results in similar functional behavior of these compounds; their relative biological potencies, however, differ. Insulin is more potent than IGF-I in insulin-like effects (e.g., lipogenesis), whereas IGF- I is more potent than insulin in growth- promoting effects (Zapf et al., 1978, King and Kahn, 1981). A program is under way in our laboratory designed to identify the regions of IGF-I that con- tribute to its particular biological activity (Katsoyan- nis et al., 1987). This goal is being pursued through the synthesis and biological evaluation of disulfide- linked, two-chain, insulin-like molecules containing structural features of IGF- I and insulin. From the synthesis and biological evaluation of a number of such hybrid molecules (Ogawa et aL, 1984; De Vroede et al., 1985, 1986; Joshi et al., 1985a, b; Tseng et al., 1987; Chen et al., 1988; Schwartz et aL, 1988), we

235 0277-8033/90/0400-0235506.00/0 © 1990 Plenum Publishing Corporation

236 Joshi, Burke, and Katsoyannis

43

42

41

40

39

38

S S

44 45 46 471 48 49 50 51 521 53 54 55 56 57 58 59 60 61 62

Vii I .Asp. G I u • Cys ,Cys-Phe-Arg • Ser • CysoAspoLeu oArg oArg • Leu-GI u • Met.Ty r • Cys-AI a

lie I / Pro G!y H-O!y I S S Leu 64

I / Li Thr Pro 2 S S • Glu 3 I s/ Pro 66

Gin ThroLeu.CysoGlyoAleoGluoLeuoVoloAspoAlaoLeuoGInopheoValoCy 18 Ale 67 Pro. 4 5 6 7 8 9 I0 II 12 13 14 15 16 17 O!y 19 Lys 68 Ale Asp 20 S~r 69

A~g-Arg-SeroSer oSer • GlyoTyr-Gly,Thro ProoLysoAsnoPheoTyroPheoGlyoArg AI'o-OH -to

57 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21

Fig. 1,. Structure of human IGF-I.

have concluded that the determinants of growth- promoting activity reside in the A-domain of IGF-I. In view of this finding, we are now investigating the contribution of specific structural features of the A- domain of IGF-I to the growth-promoting activity of this molecule. In this paper, we describe the synthesis and biological evaluation of an insulin-like com- pound consisting of the B-chain of bovine insulin and a 29 amino-acid residue A-chain corresponding to the A- and D-domains of human IGF-I (sequence 42-70 in Fig. 1) in which the A-domain sequence -Phe49-ArgS°-Ser51- has been substituted by the homologous sequence -Ala-Gly-Val- found in sheep insulin (modified IGF-I [A-D] domain).

2. EXPERIMENTAL PROCEDURES AND RESULTS s

Details of the analytical procedures used are given in a previous publication (Kitagawa et al., 1984). [125I]insulin for receptor binding assays and [3-3H]glucose for lipogenesis were products of Dupont-NEN, and cellulose acetate filters were obtained from Sartorius. The scintillation fluids Soluscint-O and Filtron-X were purchased from National Diagnostics (Somerville, New Jersey). RIA-

s Abbreviations: Ac, acetyl; Bzl, benzyl; Boc, tert-butoxycarbonyl; BSA, bovine serum albumin; BrZ, 2-bromobenzyloxy-carbonyl; DCC, N,N'-dicyclohexylcarbodiimide; DMF, dimethylfor- mamide; CIzBzl , 2,6-dichlorobenzyl; Chex, cyclohexyl; Et, ethyl; HPLC, high-performance liquid chromatography; HOBT, 1- hydroxybenzotriazole; MBzl, p-methylbenzyl; NMM, N-methyl- morpholine; RIA, radioimmunoassay; Tos, p-toluenesulfonyl. TEA, triethylamine; TFA, trifluoracetic acid; Tris, tris(hydroxy- methyl)aminomethane; Z, benzyloxycarbonyl.

grade BSA was obtained from Arnel Products, New York, and fatty acid-free BSA from Boehringer- Mannheim.

2.1. Insulin-Receptor Binding

A rat liver plasma membrane fraction enriched in insulin receptors was prepared by differential cen- trifugation, essentially as previously described (Hor- vat et al., 1975). 0.2 ml binding assays, performed in triplicate, contained [125I]insulin, 3 × 10 -1° M, vary- ing concentrations of natural insulin or test com- pound, and membrane protein, 40-60/zg, in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.6% RIA-grade BSA. After 45 min at 24°C, the incubation mixtures were filtered on cellulose acetate membrane filters, which were subsequently rinsed with the same buffer, ice cold, containing 0.1% BSA, dried and dissolved in Filtron-X for liquid scintillation count- ing. Nonspecific binding of [125I]insulin, defined as radioactivity remaining on the filters when the incuba- tion mixture contained unlabeled insulin, 1 x 10 -5 M, was subtracted from all values. Relative potency was calculated from the ratio of the concentration of natural insulin to test compound required to inhibit 50% of the binding of [125I]insulin observed in the absence of competitor.

2.2. Lipogenesis

Adipocytes were prepared from rat fat pads by incubation with collagenase, 1 mg/ml, in Krebs- Ringer bicarbonate buffer containing one half the recommended calcium, 0.5 mM D-glucose, and 3% fatty acid-free BSA. Triplicate incubation mixtures

Insulin-Like Compound 237

in plastic scintillation vials contained cell suspension (1.0 ml, corresponding to 20-40 mg dry-weight cells), and varying concentrations of insulin or text com- pound. The gas phase was 95% 02/5% CO2. After 45 rain at 37°C, [3-3H]glucose, 0.5/zCi, was added; the incubation was continued for 60 rain, and stopped by the addition of 5 N H2SO4, 0.2ml. Corn oil, 0.2 ml, was added to aid in the extraction of lipids. Soluscint-O scintillation fluid, 10 ml, was added to each vial, and the mixtures were shaken for 30 min before liquid scintillation counting. Under these con- ditions, unmetabolized labeled glucose is not extrac- ted into the organic phase, and is not counted. The stimulation of the conversion of [3-3H]glucose into organic-extractable material by insulin reached a maximum of 9.5- to 12.6-fold over the basal level of conversion in the absence of agonist. Relative potency was calculated from the ratio of the concentration of insulin to test compound required to achieve 50% of the maximum conversion.

2.3, General Aspects of Synthesis of Insulin-Like Compound Consisting of B-Chain of Insulin and A-Chain Corresponding to Modified [A-D] Domain of Human IGF-I

The synthesis of this compound was carried out by the interaction of the sulfonated forms of bovine B-chain and modified human IGF-I [A-D]-chain domain (Fig. 1, sequence 42-70 in which the -Phe 49- ArgS°-Ser 51- segment is substituted with -Ala-Gly- Val-) at pH 10.5 in the presence of dithiothreitol (Chance et al., 1981). The S-sulfonated bovine B- chain was prepared from bovine insulin as previously described (Katsoyannis et al., 1967a). The synthesis of the S-sulfonated modified [A-D]-chain of human IGF-I XXVI, patterned after the synthesis of the IGF-I [A-D]-chain domain (Schwartz et al., 1988) involved as the key intermediate in the construction of the protected nonacosapeptide XXV, containing the entire amino acid sequence of the modified IGF [A-D] domain. The synthesis of the protected non- acosapeptide XXV was done by a combination of the stepwise and fragment condensation approaches. N % Boc protection was used throughout the synthesis with the exception of the final step, where N%Z protection was employed. Side-chain protecting groups were as follows: -Bzl for serine, -ClzBzl for tyrosine, NC-Tos for arginine, N~-BrZ for lysine, -Chex or -Bzl for glutamic acid, -Chex for aspartic acid, and -MBzl for cysteine. For the synthesis of the protected nonacosapeptide XXV, the C-terminal

tridecapeptide XI, synthesized stepwise, was coupled with the adjacent tetrapeptide, and the resulting hep- tadecapeptide XII was elongated stepwise until the protected eicosapeptide XV was obtained. The latter compound was coupled with the adjacent tetrapep- tide XIX to give the C-terminal tetracosapeptide XX which, in turn, was elongated stepwise until the desired protected nonacosapeptide XXV was obtained. Deprotection of this compound on exposure to liquid hydrogen fluoride in the presence of p-cresol and 2-mercaptopyridine and sulfitolysis of the resulting reduced product led to the synthesis of the S-sulfonated modified IGF-I [A-D]-chain XXVI.

2.3.1. N%Boc-Lys(BrZ)-Ser(Bzl)-Ala.OBzl) (I)

A solution of N-Boc-Ser(Bzl)-Ala-OBzl (Schwartz et al., 1988) (7.2 g) in TFA (30 ml) was stored at room temperature for 30 rain and then con- centrated under reduced pressure to dryness. The residue was dried by the addition of toluene, followed by evaporation under reduced pressure. To a solution of this residue in DMF (65 ml), neutralized by TEA (-2.8ml), N%Boc-Lys(BrZ)-p-nitrophenyl ester [prepared in the usual way from N~-Boc-Lys.OH (Yamashiro and Li, 1973)] (10 g) was added. After 24 hr the mixture was diluted with AcOEt (500 ml) and saturated NaC1 (100ml). The organic layer was washed (1 N NH4OH, 5% citric acid and water), dried, and concentrated to a small volume. Addition of ether and petroleum ether to this solution caused the precipitation of the product as a low melting solid: weight 10.2g (81%); [a]~-18 ° (c 1, DMF). Anal. Calcd. for C39H49N409Br: C, 58.7; H, 6.19; N, 7.0. Found: C, 58.5; H, 6.19; N, 7.0.

2.3.2. Boc-Ala-Lys(BrZ)-Ser(Bzl)-Ala-OBzl (II)

A solution of compound I (9.7 g) in TFA-AcOH (7:3, v/v) (40 ml) was stored at room temperature for 45 rain and then concentrated under reduced pressure to dryness. The residue was dissolved in AcOEt (400 ml) and this solution was treated with cold 1 M Na2CO3 until the pH of the aqueous phase was -9.0. The organic layer was separated, washed with saturated NaC1, dried, and concentrated under reduced pressure to dryness. To a solution of this residue in tetrahydrofuran (40 ml) Boc-Ala. O H (4.1 g) and 2-ethyl- 1 (ethoxycarbonyl)- 1,2-dihydroquinoline (5.34g) were added. After 24hr the solvent was removed by evaporation and the residue, dissolved in AcEOt, was washed (1 M KHCO3, 0.5 N HCI and

238 Joshi, Burke, and Katsoyannis

water), dried, and concentrated to a small volume. Addition of ether and petroleum ether to this solution caused the precipitation of the product: weight 7.3 g (70%); mp 140-141°C; [a]~-19.4 ° (c 1, DMF). Anal. Calcd. for C42H54NsOloBr; C, 58.0; H, 6.27; N, 8.1. Found: C, 57.9; H, 6.37; N, 8.0.

2.3.3. Boc-Pro-Ala-Lys(BrZ)-Ser(Bzl)-Ala.OBzl (III)

A solution of compound II (6.8 g) in TFA-AcOH was stored at room temperature for 45 min and then concentrated under reduced pressure to dryness. The solid product obtained on trituration of the residue with ether was collected, dissolved in DMF (25 ml), and to this solution, cooled to 0°C, NMM (1 ml) was added followed by ether. The precipitated free base of the tetrapeptide derivative was filtered off, washed with ether, dried, and mixed with the carboxyl com- ponent which was preactivated as follows. Boc Pro-OH (1.75 g) was dissolved in DMF (15 ml) and to this solution cooled to 0°C were added HOBT (1.09 g) and DCC (1.66 g). After stirring at 0°C for 1 hr, and at room temperature for 1 hr,'the urea by- product was filtered off and the filtrate mixed with the free base of the deblocked tetrapeptide prepared as just described. After 24 hr at room temperature, the mixture was diluted with AcOEt (500 ml), and this solution was washed (1 M KHCO3, 0.5 N HC1 and water), dried, and concentrated to a small volume. Addition of petroleum ether caused the pre- cipitation of the product, which was collected and reprecipitated from a mixture of ethanol-AcOEt (20:80, v/v) by the addition of petroleum ether: weight 7.15 g (95%); mp 175-177°C; [c~]~C34.9 ° (c 1, DMF). Anal. Calcd. for C47H61N6OI1Br; C, 58.4; H, 6.36; N, 8.7. FoundzC, 58.2; H, 6.47; N, 8.4. An acid hydrolysate gave the following molar ratios: ProLo Alazo Lyslo SerLo.

2.3.4. N % Boc-L ys(BrZ)-Pro-Ala-L ys(BrZ)-Ser(Bzl)- Ala.OBzl (IV)

Compound III (6.75 g) was deblocked with TFA- AcOH (30 ml) as described above. The resulting tri- fluoroacetate salt was disolved in DMF (35 ml) and to this solution, cooled to 0°C, was added TEA (1.8 ml) followed by N~-Boc-Lys(BrZ)-p-nitrophenyl ester (4.41 g). The reaction mixture was processed as in the synthesis of compound III and the product purified by precipitating from AcOEt-ether: weight 6.5 g (71%); mp 115-117°C; [a]~-29.3°C (c 1, DMF). Anal. Calcd. for C61HTsNaO14Br2: C, 56.0, H, 6.01; N, 8.6. Found C, 55.9; H, 5.95; N, 8.4. An acid

hydrolysate gave the following molar ratios: PrOLo Alazo Lyszo SerLo.

2.3.5. Boc-Leu-Lys(BrZ)-Pro-Ala-Lys(BrZ)-Ser(Bzl). Ala.OBzl (V)

Compound IV (6.0 g) was deblocked with TFA- AcOH (25 ml) as described above. The resulting tri- fiuoroacetate salt was disolved in DMF (30 ml), and to this solution was added TEA (2 ml) followed by Boc-Leu-p-nitrophenyl ester (Vogler et al., 1965) (1.94g). The reaction mixture was processed as described in the synthesis of compound IV: weight 5.7 g. (88%); mp 98-100°C; [a]~-37.5°C (c 1, DMF). Anal. Calcd. for C67Hs9N9Oa5Br2: C, 56.6; H, 6.31; N, 8.9. Found: C, 56.4; H, 6.13; N, 8.8. An acid hydrolysate gave the following molar ratios: ProL2 Alazo Seho LeuL0 Lys2o.

2.3.6. Boc-Pro-Leu-Lys(BrZ)-Pro-Ala-Lys(BrZ)- Ser(Bzl)-Ala.OBzl (VI)

Compound V (5.5 g) was deblocked with TFA- AcOH (25 ml) and the free base of the resulting product was prepared as described in the synthesis of compound II. The free base was then added to Boc-Pro.OH (1.67 g), which was preactivated with HOBT (1.04 g) and DCC (1.6 g) in DMF (20 ml) as described in the synthesis of compound III. The reac- tion mixture was processed in the usual way and the final product was obtained by precipitation from AcOEt-ether: weight 5.3g (90%); mp 113-115°C; [c~]~-47.1 ° (c 1, DMF). Anal. Calcd. for C72H96NloO16Br2: C, 57.0; H, 6.37; N, 9.2; Found: C, 56.9; H, 6.28; N, 9.2. An acid hydrolysate gave the following molar ratios: Prozo Alazo SerE0 LeuLo Lys2.2.

2.3.7. Boc-Ala-Pro-Leu-L ys(BrZ)-Pro-Ala-L ys(BrZ)- Ser(Bzl)-Ala.OBzl (VII)

This compound was synthesized in exactly the same way as described in the synthesis of compound VI. The materials used in this synthesis were as fol- lows. Compound VI (5.0 g), TFA-AcOH (25 ml), Boc- Ala-OH (1.25 g), DMF (20 ml), HOBT (0.88 g), and DCC (1.35 g): weight 4.3 g (84%); mp 121-122°C; [a]~-49.9 ° (c 1, DMF). Anal. Calcd. for C75HlolN11017Br2: C, 56.6; H, 6.40; N, 9.7. Found C, 56.1; H, 6.45, N, 9.5. An acid hydrolysate gave the following molar ratios: Prozo Ala3o Serlo Leujo Lys2.2.

Insulin-Like Compound 239

2.3.8. N-Boe-Cys(MBzl)-Ala-Pro-Leu-Lys(BrZ)-Pro- Ala-Lys(BrZ)-Ser(Bzl)-Ala.OBzl (VIII)

Compound VII (4.0 g) was deblocked with TFA- AcOH (20ml) and the free base of the resulting product was prepared as described in the synthesis of compound II. To a solution of this product in DMF (25 ml) N-Boc-Cys(MBzl)-p-nitrophenyl ester [prepared in the usual way from N-Boc- Cys(MBzl).OH] (1.4 g) was added. After 24 hr the mixture was diluted with AcOEt (400 ml) and this solution was washed (in NH4OH, 0.5 N HC1 and water), dried, and concentrated to a small volume. Addition of ether caused the precipitation of the product: weight 3.8g (88%); mp 134-136°C; [a]~-40.1 ° (c 1, DMF). Anal. Calcd. for C86Hl14N12OlsSBr2: C, 57.5; H, 6.39; N, 9.3. Found: C, 56.9; H, 6.04; N, 8.9. An acid hydrolysate gave the following molar ratios: Prozo Ala3o Ser~.o Leuo8 Lyszz. S-p-methylbenzylcysteine was not determined.

2.3.9. Boc- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-Ala-L ys(BrZ)-Ser(Bzl)- Ala.OBzI (IX)

A solution of compound VIII (3.0 g) in TFA- AcOH (20 ml) and anisole (3 ml) was stored at room temperature for 45 rain and then concentrated under reduced pressure to dryness. The solid residue was washed with ether, dried, and dissolved in cold DMF (15 ml) containing NMM (0.5 ml). To this solution was added the carboxyl component preactivated in the usual way. Materials used for the preactivation reation are as follows: Boc-Tyr-(C12Bzl).OH (1.3 g), DMF (15 ml), HOBT (0,4 g), and DCC (0.6 g). After 24 hr the reaction mixture was filtered and the filtrate diluted with 2-propanol (50 ml) and ether (500 ml). The precipitated product was collected and purified from 95% ethanol: weight 3.2g (90.4%); mp 173- 175°C; [ a ] ~ - 4 8 . 9 ° (c 1, DMF). Anal. Calcd. for C1o2H127N13OzoSBr2C12: C, 57.8; H, 6.04; N, 8.6. Found: C, 57.6; H, 5.84; N, 8.4. An acid hydrolysate gave the following molar ratios: Pro2.1 Ala3.o Ser~o LeuL~ Tyrlo Lys2.2. S-p-methylbenzylcysteine was not determined.

2.3.10. Boc-Met(O)- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro- Leu-Lys(BrZ)-Pro-Ala-Lys(BrZ)-Ser(Bzl)- Ala.OBzl (X)

Compound IX (2.75 g) was debtocked with TFA- AcOH (15 ml) containing anisole (2.5 ml), and the free base of the resulting product was isolated as

described in the synthesis of compound II. To a solution of this product in DMF (20 ml) were added Boc-Met(O).OH (1.03 g) and 2-ethyt-l-(ethoxycar- bonyl)-l,2-dihydroquinoline (0.96 g). After 24 hr the mixture was diluted with AcOEt (300 ml) and this solution was washed (1 M KHCO3, 0.5 N HC1, and water), dried, and concentrated to a small volume. Addition of petroleum ether completed the precipita- tion of the product, which was reprecipitated from 95% ethanol: weight 2.2g (74.8%); mp 183-185°C; [c~]~-47.1 ° (c 1, DMF). Anat. Calcd. for ClovHls6N14022S2BrzC12: C, 56.7; H, 6.0; N, 8.6. Found: C, 56.6; H, 5.76; N, 8.4. An acid hydrolysate gave the following molar ratios: Pro:l Sero.9 Ala3.o Meto.6 Leul.o Tyr~.o Lys2.2. S-p-methylbenzylcysteine was not determined.

2.3.11. Boc-Glu(OBzl)-Met(O)- Tyr(Cl2Bzl)- Cys( MBzl)-Ala-Pro-Leu-L ys(BrZ)-Pro-Ala- Lys(BrZ)-Ser(BzO-Ala.OBzl (XI)

Compound X (1.8 g) was deblocked as described above and the resulting trifluoroacetate salt was dis- solved in DMF (8 ml). To this solution, neutralized with NMM (-0.8 ml), was added the carboxyt com- ponent, which was preactivated in the usual way. Materials used in this step are as follows. Boc- Glu(OBzl)-OH (1.07 g), DMF (8 ml), HOBT (0.43 g), and DCC (0.65 g). After 24 hr the mixture as filtered and the filtrate mixed with 1 M KHCO3 (300 ml). The precipitated product was collected, washed with water, dried, and reprecipitated from 70% aqueous ethanol: weight 1.6g (80%); mp 195-197°C; [~]~-47.7 ° (c 1, DMF). Anal. Calcd. for C119H149N15025S2Br2C12: C, 57.5; H, 6.04; N, 8.4. Found: C, 57.3; H, 6.29; N, 8.2. An acid hydrolysate gave the following molar ratios: Pro22 Sero.9 Ala3o Meto.6 GlUl.o Leul.o Tyrl.o Lys21. S-p-methylbenzyl- cysteine was not determined.

2.3.12. Boc-Leu-Arg(Tos)-Arg(Tos)-Leu-Glu(OBzl)- Met(O)- Tyr(CleBzl)- Cys(MBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-Ala-Lys(BrZ)-Ser(Bzl)- Ala.OBzl (XII)

Compound XI (1.5g) was deblocked as described in the synthesis of compound IX. To a cold (0°C) solution of the resulting tridecapeptide tri- fluoroacetate in 1-methylpyrrolidinone (15 ml), TEA (0.8 ml) was added, followed by ether (400 ml). The precipitated free base was collected and dissolved in DMF (10 ml). To this solution was then added Boc- Leu-Arg(Tos)-Arg(Tos)-Leu.OH (Schwartz et al.,

240 Joshi, Burke, and Katsoyannis

1988) (1.65g) followed by DCC (0.34g) and N- hydroxy-5-norbornene-2,3-dicarboxylic acid imide (0.34 g). After 72 hr at 4°C, the reaction mixture was poured into cold water (100ml) containing 1 M KHCO3 (20 ml). The precipitated heptadecacapep- tide derivitive was collected, washed with water, dried, and reprecipitated from 95% ethanol: weight 1.4g (70%); mp> 250°C; [a]~-39.1 ° (c 1, DMF). An acid hydrolysate gave the following molar ratios: Pro2.0 Sero.9 GlUl.o Ala2.7 Meto.7 Leu3.o Tyro.9 Lys2.o Argzo. S-p-methylbenzylcysteine was not determined.

2.3.13. Boc-Asp(OChex)-Leu-Arg(Tos)-Arg(Tos)- Leu-Otu(OBzO-Met(O): Tyr(Cl2Bzl)~ Cys(MBzl)-Ala-Pro-Leu:Lys(BrZ)-Pro-Ala- Lys(BrZ)-Ser(Bzl)-Ala.OBzl (XIII)

Compound XII (6.0 g) was deblocked with TFA- AcOH (35 ml) and anisole (5 ml) and converted to the free base as described above. To a solution of this product in DMF (20 ml) was added the carboxyl component preactivated in the usual way. Materials used in this step are as follows. Boc-Asp(OChex).OH (2.27g), DMF (15 ml), HOBT (0.8g), and DCC (1.17 g). After 24 hr at room temperature the mixture was filtered and the filtrate poured into ether (800 ml). The precipitated product was collected and reprecipi- tated from DMF-ether: weight, 4.6g (72%); mp> 250°C; [a]~-30.3 ° (c 1, DMF). Amino acid analysis: Pr02.a Serl.0 Asp1.0 GlUl,o Ala3.0 Meto.4 Leu3,3 Tyrl.o Lys2.2 Argl.9. S-p-methylbenzylcysteine was not determined.

2.3.14. Boc-Cys(MBzl)-Asp(OChex)-Leu-Arg(Tos)- Arg( Tos)-Leu-Glu(O BzO-Met(O)- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-ala-Lys(BrZ)-Ser(Bzl)- Ala.OBzl (XIV)

Compound XIII (4.2g) was deblocked with TFA-AcOH (30 ml) and anisole (4 ml), and the result- ing trifluoroacetate salt was dissolved in DMF (20 ml) and neutralized with TEA (0.9 ml). This solution was mixed with a solution of Boc-Cys(MBzl).OH (2.3 g) in DMF (15ml) preactivated in the presence of HOBT (1.0 g) and DCC (1.46 g) as described pre- viously. After 24 hr the mixture was filtered and the filtrate poured into cold 1 M KHCO3 (500 ml). The precipitated product was filtered off and reprecipi- tated from DMF-ether: weight 3.9g (88%); rap> 230°C; [a]~-30.8 ° (c 1, DMF) Amino acid analysis: AspH Sero.9 Proz5 GlUl.o Ala3.o Leu3.o Tyro.9 Lys2.o

Arg2.1. Methionine and S-p-methylbenzylcystiene were extensively destroyed and not quantitated.

2.3.15. Boc- Val-Cys(MBzl)-Asp(OChex)-Leu- Arg(Tos)-Arg(Tos)-Leu-Glu(OBzO-Met(O)- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-Ala-Lys(BrZ)- Ser(Bzl)Ala.OBzl (XV)

Compound XIV (3.9g) was deblocked with TFA-AcOH (30 ml) in the presence of anisole (4 ml), and the resulting trifluoroacetate salt was dissolved in DMF (20 ml) and neutralized with TEA (0.9 ml). This solution was mixed with a solution of Boc- Val.OH (0.93 g) in DMF(12 ml), preactivated in the presence of HOBT (0.6 g) and DCC (0.9 g). The mix- ture wasprocessed as described in the synthesis of compound XIV: weight 3.6g (87%); mp>230°C; [a]~-33.5 ° (c 1, DMF). Amino acid analysis: Aspl.o Serl.o Pro2.2 Glul.0 Ala3.o Meto.5 Val0.9 Leu3.o Tyrl.o LySl.8 ArgL9. S-p-methyl-benzylcysteine was not determined.

2.3.16. Boc-Ala-Gly.OMe (XVI)

To a solution of Boc-Ala.OH (18.9 g) in tetrahy- drofuran (200ml) containing NMM (11.2ml) and cooled to -10°C, isobutyl chloroformate (12.8 ml) was added followed, 10 min later, by a solution of H.Gly.OMe, HC1 (12.5 g) in DMF (20 ml) and NMM (11.2 ml). After 24 hr at room temperature, the mix- ture was filtered and the filtrate concentrated under reduced pressure to dryness. A solution of the residue in AcOEt (600 ml) was washed (1 M KHCO3, 5% citric acid and water), dried, and concentrated to dryness. The product was obtained as an oil (18 g, 70%). This product was homogeneous on thin-layer chromatography and was used in the synthesis of the following compound without any further characteriz- ation.

2.3.17. Boc-Cys(MBzl)-Ala-Gly.OMe (XVII)

Compound XVI (8.3 g) was deblocked with TFA (25 ml) as described in the synthesis of compound I. To a solution of the resulting trifluoroacetate salt in DMF (50 ml) neutralized with TEA (-4.5 ml), Boc- Cys(MBzl)-p-nitrophenyl ester (14.2g) was added. After 24hr, the mixture was diluted with AcOEt (600ml) and saturated NaC1 (100 ml). The organic layer was washed (1 N NH4OH, 5% citric acid and water), dried, and concentrated to a small volume. Upon addition of petroleum ether, the product pre-

Insulin-Like Compound 241

cipitated: weight 13.2g (88%); mp 58-60°C; [c~]~-14.8 ° (c 1, DMF). Anal. Calcd. for C22H33N3068: C, 56.5; H, 7.10; N, 9.0. Found: C, 56.7; H, 7.34; N, 8.8.

2.3.18. Boc-Cys(MBzl)-Cys(MBzl)-Ala-Gly-OMe (XVIII)

Compound XVII (13.1 g) was deblocked with TFA (50ml) in the presence of anisole (10ml) as described above. The resultant trifluoroacetate salt was dissolved in DMF (50 ml) and to this solution, neutralized with TEA ( - 4 m l ) Boc-Cys(MBzl)-p- nitrophenyl ester (13.7 g) was added. The mixture was processed as described in the synthesis of com- pound XVII, and the final product was purified by precipitation from 95% ethanol containing DMF (5%, v/v): weight 13.5 g (72%); mp 148-150°C; [a]~-27.0 ° (c 1, DMF). Anal. Calcd. for C33H46N40782: C, 58.4; H, 6.83; N, 8.2. Found: C, 58.7; H, 7.22; N, 8.2.

2.3.19. Boc-Cys(MBzl)-Cys(MBzl)-Ala-Gly.OH (xix)

To a solution of compound XVIII (4.9 g) in a mixture of dioxane-methanol-water (5:5: 1, v/v) (50ml) 1 N NaOH (9ml) was added over a 2hr period, maintaining the pH of the reaction at 12.5 with the use of a pH meter. Subsequently, the mixture was adjusted to pH 2.5 with 1 N HC1 and then diluted with water (100 ml) and AcOEt (400 ml). The organic layer was separated, washed with water, dried, and concentrated to a small volume. Upon addition of petroleum ether, the product precipitated: weight 4.3 g (89%); mp 135-138°C; [a]~-16.8 ° (c 1, DMF). Anal. Calcd. for C32H44N407S2: C, 57.8; H, 6.68; N, 8.4. Found: C, 58.0; H, 6.99; N, 8.2.

2.3.20. Boc-Cys(MBzl)-Cys(MBzI)-Ala-Gly- Val- Cys(MBzl)-Asp(OChex)-Leu-Arg(Tos)- Arg(Tos)-Leu-Glu(OBzO-Met(O)- Tyr(Cl2Bzl)- Cys(MBzl)-Ala-Pro-Leu- L ys(BrZ)-Pro-Ala-L ys(BrZ)-Ser(Bzl)- Ala.OBzl (XX)

The deblocking of compound XV (3.2 g) with TFA-AcOH (30 ml) and anisole (3 ml) was done in the usual way. The resulting trifluoroacetate salt was dissolved in DMF (20ml), neutralized with TEA (~0.5 ml), and mixed with a solution of compound

XIX (2.22 g) in DMF (15 ml), which was preactivated with HOBT (0.45 g) and DCC (0.69 g) as described previously. After 24 hr, the mixture was filtered and the filtrate poured into ether (500 ml). The precipi- tated tetracosapeptide was collected and reprecipi- tated from DMF-ether: weight 3.2g (87%); mp >230°C; [~]~-19.5 ° (c 1, DMF). Amino acid analy- sis: Asplo SerH Pro1.9 Glu~o Ala4.o Glyl.o Valo.9 Leu3.3 Tyrl.o LyszA Argo8. Methionine and S-p-methylben- zylcysteine were extensively destroyed and were not quantitated.

2.3.21. Boc-Glu(OChex)-Cys(MBzl)-Cys(MBzl)-Ala- Gl~- Val-Cys(MBzl)-Asp(OChex)-Leu- arg(Tos)-Arg(Tos)-Leu-Glu(OBzO-met(O)- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro-Leu- L ys(BrZ)-Pro-Ala-L ys(BrZ)-Ser(Bzl)- Ala.OBzl (XXI)

Compound XX (3.2g) was deblocked as described above. To a solution of the resulting prod- uct in DMF (12 ml), containing TEA (0.6 ml), a sol- ution of Boc-Glu(OChex).OH (1.0g) in DMF (10 ml), preactivated with HOBT (0.4g) and DCC (0.6 g) was added. After 24 hr, the mixture was filtered and the filtrate poured into cold 1 M KHCO3 (200 ml). The precipitated product was collected, washed, and dried: weight 2.8 g (84%); mp >230°C; [a]~-27.3 ° (c 1, DMF). Amino acid analysis: Aspl.o SerH Pro1.9 Glu2.3 Glyo9 Alas.8 Valo.9 Leu3.3 Tyrl.o Lys2.o Arga.9. Methionine and S-p-methylbenzyl-cysteine were not determined.

2.3.22. Boc-Asp(OChex)-Glu(OChex)-Cys(MBzl)- Cys(MBzl)-Ala-Gly- Val-Cys(MBzl)- Asp(OChex)-Leu-Arg(Tos)-Arg(Tos)-Leu- Glu(OBzO-Met(O)- Tyr(Cl2Bzl)-Cys(MBzl)- Ala-Pro-Leu-L ys(BrZ)-Pro-Ala-L ys(BrZ)- Ser(Bzl)-Ala.OBzO (XXII)

This compound was synthesized by the same procedure used in the synthesis of compound XXI. The materials used are as follows. Compound XXI (2.7g), DMF (22ml), TEA (0.5 ml), Boc- Asp(OChex).OH (0.74 g), HOBT (0.32 g), and DCC (0.48 g). The resulting product was triturated with a mixture of AcOEt-ethanol-ether (4: 4: 2, v/v) (25 ml): weight 2.4g (85%); mp>230°C; [a]~-26.3 ° (c 1, DMF). Amino analysis: Asps.9 Serlo Pr%.o Glu2.o Gly0.9 Ala37 Meto5 Val0.9 Leu3.4 Tyrl.0 Lysl.9 Arga.9. S-p-methylbenzyl-cysteine was not determined.

242 Joshi, Burke, and Katsoyannis

2.3.23. Boc- Val-Asp(OChex)-Glu(OChex)- Cys(mBzl)-Cys(mBzl)-Ala-Gly- Val- Cys(MBzl)-asp(OChex)-Leu-arg(Tos)- Arg(Tos)-Leu-Glu(OBzO-Met(O)- Tyr(Cl2Bzl)-Cys(mBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-ala-Lys(BrZ)-Ser(Bzl)- Ala.OBzl (XXIII)

This compound was prepared as described above. The materials used are as follows. Compound XXII (2.2g), DMF (20ml), TEA (0.3ml), Boc- Val.OH (0.4 g), HOBT (0.25 g), and DCC (0.4 g). The reaction mixture was processed as above: weight 2.0 g (89%); mp >230°C; [a]~-23.6 ° (c 1, DMF). Amino acid analysis: Asp1.9 Serl.o Pro2.o Glu2.o Glyo.9 Ala38 Met0.5 VaI2.o Leu3.3 Tyr~.o Lysl.9 Argl . 9. S-p-methyl- benzylcysteine was not determined.

2.3.24. Boc-Ile- Val-Asp(OChex)-Glu(OChex)- Cys(mBzO-Cys(MBzl) ala-Gly- Val- Cys(mBzl)-asp(OChex)-Leu-arg(Tos)- Arg(Tos)-Leu-Glu(OBzO-Met(O)- Tyr(Cl2Bzl)- Cys(MBzl)-Ala-Pro-Leu- Lys(BrZ)-Pro-Ala-Lys(BrZ)-Ser(Bzl)- Ala.OBzl (XXIV)

This compound was synthesized as described above. The materials used are as follows. Compound XXIII (2.1g), DMF (20ml), TEA (0.3ml), Boc- Ile.OH (0.4g), HOBT (0.23 g), and DCC (0.36 g). The product was reprecipitated from DMF-ether: weight 1.8g (84%); mp >230°C; [o~]~-28.3 ° (c 1, DMF). Amino acid analysis: Asp1.9 Sero.9 Pro1.8 Glu2.o Gly0.9 Ala3.s Meto.4 Ileo4 Vall.7 Leu28 Tyrl0 Lys2o Argz2. S-p-methylbenzylcysteine was not determined.

2.3.25. Z-Gly-Ile- Val-Asp(OChex)-Glu(OChex)- Cys(MBzO- Cys(MBzl)-Ala-Gly- Val- Cys(MBzl)-Asp(OChex)-Leu-arg( Tos)- Arg(Tos)-Leu-Glu(OBzO-Met(O)- Tyr(Cl2Bzl)-Cys(MBzl)-Ala-Pro-Leu- L ys(BrZ)-Pro-Ala-L ys(BrZ)-Ser(Bzl)- Ala.OBzl (XXV)

This nonacosapeptide was prepared as described above. The materials are as follows. Compound XXIV (1.6g), DMF (20ml), TEA (0.25ml), Z-Gly.OH (0.34 g), HOBT (0.22 g), and DCC (0.33 g). The prod- uct was purified by precipitation from DMF-ether: weight 1.3 g (80%); mp >230°C. Amino acid analysis: Asp1.8 Sero.9 Pro1.9 Glu2.o Glyl.8 Ala3.s Meto.s Ileo.s Vall.7 Leu2.9 Yyrl.o Lys2.1 Arg2.4. S-p-methylbenzylcys- teine was not determined.

2.3.26. H.Gly-Ile- Val-Asp-Glu- Cys(SSO j )-Cys(SSO ; )- ala-Gly- Val- Cys(SSO ~ )- Asp-Leu-Arg-Arg-Leu-Glu-Met- Tyr-Cys(SSO ~ )-Ala-Pro-Leu-L ys-Pro.Ala- Lys-Ser-Ala.OH (Modified IGF-I [A-D]- Chain S-Sulfonate) (XXVI)

The preparation of this compound from the fully protected derivative, XXV, was carried out essentially by the same procedure used previously for the syn- thesis of the corresponding unmodified [A-D]-chain (Schwartz et al., 1988). Briefly, compound XXV (200 mg) was treated with anhydrous liquid hydrogen fluoride (10 ml) in the presence of p-cresol (1 g) and 2-mercaptopyridine (200 mg) for 1 hr at 0°C. After removal of the liquid hydrogen fluoride, the residue was triturated with AcOEt (3 x 50 ml each) and with petroleum ether (2 x 50 ml each) and dried over KOH. To a solution of this product in 8 M guanidine hydro- chloride (25 ml), adjusted to pH 8.9 with dilute NH4OH, were added sodium sulfite (1.0g) and freshly prepared sodium tetrathionate (Gilman et aL, 1946) (0.8 g). After 4hr at room temperature, the mixture was placed in Spectrapor membrane tubing No. 3 and dialyzed against four changes of distilled water (4 L each) at 4°C for 24 hr. Upon lyophilization of the dialysate, the crude modified human IGF-I [A-D]-chain S-sulfonate was obtained as a white powder. For a preliminary purification this material was chromatographed on a Sephadex G-15 column (4.2x50cm) equilibrated and eluted with 0.015 M NH4HCO3. The effluent corresponding to the main peak, as monitored by an ISCO spectrophotometer, was lyophilized and the S-sulfonated modified human IGF-I [A-D]-chain was obtained as a white fluffy material: weight 130 rag.

For purification, the material (30 mg) was dissol- ved in 2 ml of 0.02 M Tris-HC1 buffer (pH 7.4) and placed on a Whatman DE-52 cellulose column (1.2 x 24 cm) equilibrated with the same buffer. Elution of the column was carried out with a linear NaC1 gradient formed by adding to the above buffer (200 ml) 0.5 M NaC1 in the same buffer (200 ml). The chromatographic pattern, as monitored by an ISCO spectrophotometer and a conductivity meter (Radio- meter, Copenhagen) is shown in Fig. 2. The effluent corresponding to the main peak (240-280 ml) was dialized in Spectrapor membrane tubing No. 3, as described above, and lyophilized to give the purified S-sulfonated modified human IGF-I [A-D]-chain as a white powder: weight 16 m_g. An acid hydrolysate gave the following composition expressed in molar

Insu l in -L ike Compound 243

(--) 0..I0

E = 0.08

~ 0.06

0.04 0 0.02

%

(---} 20

j~ .-" 16g ~ s I~11s

i -'~-'" , , , , , i / o 50 Ioo mo 200 250 300 350 400-

EFFLUENT VOLUME (ml)

Fig. 2. Chromatography of crude S-sulfonated modified human IGF-I [A-D]-chain domain on a 1.2 × 24 cm Whatman preswollen microgranular diethylaminoethyl cellulose (DE 52) column equili- brated with 0.02 M Tris-HC1 buffer (pH 7.4) and eluted with a linear NaC1 gradient formed by adding to the above buffer (200 ml) buffer 0.4 M NaCI in the same buffer (200 ml). The column was monitored by an ISCO spectrophotometer and a conductivity meter (Radiometer, Copenhagen) . The S-sulfonated chain was recovered by dialysis and lyophilization of the effluent (240-280 ml).

ratios, in agreement with the theoretically expected values (shown in parentheses): Asp1.7(2) Sero.9(l) Pr02.2(2) Glu2.2(2) Glyl.9(2) Ala3.8(4) Va11.4(2) Meto.9(l) Ileo.4(1) Leu3.3(3) Tyro.9(1) Lys2.3(2) Arg2.3(2). Cys was not determined. This compound was completely digested by aminopeptidase M.

2.4. S - S u l f o n a t e d B-Cha in of Bovine Insul in

This compound was prepared by oxidative sulfitolysis of bovine insulin followed by CM- cellulose chromatography of the resulting S-sulfon- ated A- and B-chains by the procedure described previously (Katsoyannis et aL, 1967a), with the only difference being that the sulfitolysis was carried out for 3 hr instead of 24 hr.

2.5. Synthes i s and Iso lat ion o f the Insu l in -Like Compound Cons i s t ing o f the Modi f ied [ A - D ] Chain of H u m a n IGF-I and the B-Cha in of Bovine Insul in

The synthesis of this compound by the combina- tion of the S-sulfonated modified [A-D]-chain of human IGF-I and bovine B-chain was carried out as described previously for the synthesis of the corre- sponding unmodified hybrid molecule (Schwartz et al., 1988). Briefly, to a cold (4°C) solution of modified human IGF-I [A-D] chain S-sulfonate (22 mg) and bovine B-chain S-sulfonate (22 mg) in 0.l M glycine

buffer (pH 10.5; 8 ml), dithiothreitol (5.5 mg) was added. After 24 hr at 4°C, the mixture was diluted with glacial acetic acid (2 ml) and chromatographed on a Sephadex G-50 column (2.5x48 cm), equili- brated, and eluted with 1 M acetic acid. The effluent from the peak representing the monomer fraction (-114-141 ml; using insulin as a standard) was iso- lated and lyophilized ( - 3 mg). For purification, this product was chromatographed on a CM-Sepharose column (0.9 x 23 cm), equilibrated with a buffer con- sisting of 7 M urea in 0.1 M acetic acid. Elution of the column was carried out with a linear NaC1 gradient (from 0-0.2 M) in the same buffer. The elu- tion pattern, as monitored with an ISCO spec- trophotometer, is shown in Fig. 3. The effluent under the main peak (146-180 ml) was dialyzed as described above and concentrated to a small volume ( - 8 ml). From this solution the active material was isolated via picrate as the hydrochloride following the pro- cedure we have employed previously for the recovery of insulin and analogues (Katsoyannis et aL, 1967b). This product was subjected to reversed-phase HPLC using a C18 ~Bondapak column (0.39 x30 cm) con- nected to a Beckman liquid chromatography system. Solvents: A, 0.1% TFA in water; B, 0.1% TFA in 70% aqueous acetonitrile. A gradient was formed from 20-70% of B over 50 min at a flow rate 1 ml/min. The chromatographic profile is shown in Fig. 4A. The effluent under the peak emerging at 15.2 rain was collected, concentrated, and rechromatographed on the same column and under identical conditions as

0. I0

E_ 008 t

i.u ~ O061- m~ 0.04

~ 0"02 ~ / V ~ ~

o ; ' ' I00 150 EFFLUENT VOLUME (ml)

Fig. 3. Chromatography of a combinat ion mixture of the S-sulfon- ated modified h u m a n IGF-I [A-D]-chain domain and the S-sulfon- ated bovine insulin B-chain on an 0.9 × 23 CM-Sepharose (Phar- macia) column equilibrated with a 7 M urea in 0.1 M acetic acid buffer. Elution of the column was done with a linear NaCI gradient formed by adding to the above buffer (150 ml) 0.2 M NaCI in the same buffer (150 ml). The column effluent was monitored by an ISCO spectrophotometer and a conductivity meter. The active material was recovered (146-180 ml of the effluent) via picrate as the hydrochloride.

200

244 Joshi, Burke, and Katsoyannis

A. B. ~5.3

15.2

Fig. 4. (A) Reversed-phase HPLC of the active material recovered from Fig. 3 on a /zBondapack C18 column at 1 ml/min with a 20-70% linear gradient of 70% aqueous acetonitrile in 0.1% TFA over 50min. (B) Rechromatography of the material eluted at 15.2 rain in A, using the same column and identical conditions as in A.

described above. The elution pattern is shown in Fig. 4B. The effluent under the single peak was collected and used for biological studies. Amino acid analysis of this purified product after acid hydrolysis gave the following molar ratios in agreement with the theoreti- cally expected values (shown in parentheses): Asp3.00) Thro.9(l) Ser2.3(2) Pro2.6(3) Glus.o(5) GIy4.6(5) Alas.6(6) Va14.1(5) Meto.4(~) I1%.4(1) Leu7a(7) Tyr2.4(3) Phe2.6(3) Lys2.8(3) His2.o(2) Arg2.9(3). Cys was not deter- mined.

2.6. Biological Evaluation of the Insulin-Like Compound Consisting of the Modified [A-D]-Chain Domain of IGF-I and the B-Chain of Insulin

Figure 5 depicts the stimulation of lipogenesis in rat adipocytes by natural insulin and the hybrid compound. The hybrid compound is a full agonist, reaching the same maximum stimulation as that seen with insulin, and its calculated potency is 13% relative to natural insulin. Figure 6 shows the effect of natural insulin and the hybrid compound on the binding of [lesI]-insulin to insulin receptors in rat liver plasma membranes. Inhibition of [125I]insulin is concentra- tion-dependent, and the calculated potency of the hybrid compound is 23% relative to natural insulin. Preliminary assays indicate that the hybrid compound stimulates the incorporation of [3H]thymidine in fibroblasts with a potency indistinguishable from that

1 0 0 09

LLI

& ' 2

LL o~

5 0 Z

I - o <~

I-- u)

, . , ,

10-,1 10-~o lO-g

[ A g o n i s t ] , M

Fig. 5. Effect of bovine insulin (O) and the hybrid compound (O) on the stimulation of lipogenesis in rat adipocytes (see under Experimental Procedures). The stimulation of lipogenesis, expressed as a percentage of maximum, is presented as a function of agonist concentration. The data points represent the means of triplicate determinations in each of three separate assays, in which the maximum stimulation of lipogenesis ranged from 9.5- to 12.6- fold over the basal rate of conversion of [3 -3 H]glucose into organic- extractable material.

of a previous compound (Schwartz et al., 1988), embodying the sequence of IGF-I in positions A-8, A-9, and A-10 (data not shown). We conclude that the substitution of the insulin sequence in these posi- tions in the present compound is without effect on its growth-factor activity.

3. DISCUSSION

Despite considerable homology and conforma- tional similarity between IGF-I and insulin (Rinder- knecht and Humbel, 1978; Blundell et al., 1978, 1983), these compounds display dramatic quantitative differences in the type of biological response each evokes. Insulin is a potent activator of short-term metabolic processes, such as glucose transport, but it is a weak mitogen; IGF-I, on the other hand, is a potent activator of long-term growth effects, such as the stimulation of DNA synthesis, but exhibits low activity in assays for insulin-like metabolic activity (Zapf et al., 1978; King and Kahn, 1978). We are pursuing studies aimed at the elucidation of the struc- tural features of IGF-I and insulin, which are respon-

Insulin-Like Compound 245

1 0 0

Cl

m

ii I

g3 5(?

m

, i , i . . . . . . . . i . . . . . . . . i . . . . . . . . , . . . . . . .

10 -5 1 Q-9 10 -e 10 -z 10-6

[Compet i to r ] , M

Fig. 6. Effect of bovine insulin (0) and the hybrid compound (©) on the binding of [125I]instflin to insulin receptors in rate liver plasma membranes (see under Experin~ental Procedures). The inhibition of specific binding, expressed as a percentage of maximum, is presented as a function of competitor concentration. The data points represent the means of triplicate determinations, in which [~25I]insulin bound in the absence of competitor amounted to 7-11% of input radioactivity.

sible for their particular biological activities through the synthesis and biological evaluation of two-chain, disulfide-linked molecules, which in turn embody various domains of these compounds (Katsoyannis et al., 1987). Based on the properties of several such molecules (Ogawa et al., 1984; De Vroede et al., 1985, 1986; Tseng et al., 1987; Joshi et al., 1985a, 1985b; Chen et al., 1988; Schwartz et al., 1988), we have drawn the following conclusions: (1) the A-domain of IGF-I, but not its B-domain or D-domain, contains determinants for its growth-promoting activity; (2) the B-domain is important for recognition of IGF carrier proteins, which transport IGF in the plasma and appear to modulate its delivery to target tissues (Nissley and Rechler, 1984); (3) the D-domain of IGF-I does not contribute directly to its growth- promoting activity or to carrier protein recognition, but it diminishes its insulin-like activity. It is noteworthy that molecular modeling studies indicate that the putative receptor-binding region of insulin, much of which is conserved in IGF-I, is partially covered by the D-domain (Blundell et al., 1978, 1983). This presumably accounts for the low insulin-like activity displayed by IGF-I.

Having established that important growth- promoting determinants reside in the A-domain of IGF-I, we undertook efforts to delineate the contribu- tion of specific structural features in that domain to growth-promoting activity. We have previously reported that a two-chain molecule consisting of the B-chain of bovine insulin and an A-chain correspond- ing to the A- and D-domains of human IGF-I, sequence 42-70 in Fig. 1, displayed growth-promoting activity four- to six-fold greater than insulin, but about 15% of the activity of the single chain IGF-I (Schwartz et al., 1988; Tseng et al., 1987). In assays for insulin-like activity, this compound displayed 21% potency relative to bovine insulin. In the present communication, we report the synthesis and biologi- cal evaluation of a compound consisting of the B- chain of bovine insulin and an A-chain corresponding to the A- and D-domains of human IGF-I, in which the sequence -Pheag-ArgS°-Ser 51- has been replaced by -Ala-Gly-Val-, the homologous region of sheep insulin. This compound was indistinguishable in assays for growth-factor activity from the synthetic material just described (data not shown). Figure 5 shows the behavior of the present compound in assays for the stimulation of lipogenesis in rat adipocytes. The compound is a full agonist, reaching the same maximum stimulation as that seen with bovine insulin, and it displays a calculated potency of 13% relative to bovine insulin. Figure 6 shows the effect of the compound on the inhibition of the binding of [125I]insulin to insulin receptors derived from rat liver. Inhibition is dose-dependent, and the synthetic com- pound displays a calculated potency of 23% relative to bovine insulin. The present compound is thus indistinguishable in all assay systems employed from the compound whose A-chain corresponds to the unsubstituted A- and D-domains of human IGF-I. This indicates that the A-chain sequence -Phe-Arg- Ser- in the two-chain molecules does not contribute either to their growth-promoting activity or to their insulin-like activity. It is noteworthy that the homologous residues, -AS-A9-AI°-, are among the most variable in the sequence of several mammalian insulins (Smith, 1966). Substitution of the -Phe 49- ArgS°-Ser 5L sequence in the A-domain of human IGF-I with the homologous sequence -Thr-Ser-Ile- from human insulin by site-directed mutagenesis led to observations similar to ours: the substituted com- pound was indistinguishable from natural IGF-I in binding to both the insulin receptor and to the IGF-I receptor, as well as in the stimulation of thymidine in- corporation in a muscle cell line (Cascieri et aL, 1989).

246 Joshi, Burke, and Katsoyannis

ACKNOWLEDGMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Dis- eases, U.S. Public Health Service (DK-12925 and DK-29988). The authors wish to express their appreciation to Dr. Uma Roy for the amino acid and enzymatic analyses.

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