Bivalent ACTH antagonists: influence of peptide and spacer components on potency enhancement

The antagonist potency of a series of bivalent adrenocorticotropic hormone (ACTH) peptides was examined using suspensions of inner zone rat adrenocortical cells. Bivalent antagonists were prepared by bis(maleimide) covalent cross-linking of carboxyl terminal cysteine sulfhydryl groups of synthetic ACTH peptides, Cys25 ACTH(7-25) and Cys39 ACTH(7-39). Antagonist potency enhancement was defined by shifts in ACTH(1-39) concentration-steroidogenic response curves relative to monovalent antagonist analogs. The EC50 values measured in the presence of 0.5 microM monovalent antagonist were 110 +/- 28 pM for Cys25 ACTH(7-25)-S-N-ethylsuccinimide and 44 +/- 9 pM for Cys39 ACTH(7-39)-S-N-ethylsuccinimide. Some bivalent ACTH antagonists displayed much greater antagonist potency than their monovalent analogs, which supports the findings of Stolz and Fauchere (Helv Chim Acta 71: 1421-1428, 1988). The level of potency enhancement, however, was found to be dependent upon the spacer used to link receptor binding domains and the length of the ACTH peptide used in bivalent antagonist synthesis. The most potent inhibitor, bis(Cys25 ACTH(7-25)-S-succinimidopropionyl)2-hydroxy-1,3-propanediamine, was 28 times more potent than its monovalent analog, Cys25 ACTH(7-25)-S-N-ethylsuccinimide. However, a bivalent Cys25 ACTH(7-25) peptide containing two bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacers that had been linked end-to-end via dithioerythritol showed no potency enhancement. Cys25 ACTH(7-25) based peptides containing one receptor binding domain and having the structure peptide-spacer-cysteine displayed no enhancement in antagonist potency. Bivalent Cys39 ACTH(7-39) linked by bis(succinimidopropionyl)2-hydroxy-1,3-propanediamine spacer exhibited only 4-fold enhancement in antagonist potency relative to Cys39 ACTH(7-39)-S-N-ethylsuccinimide. We therefore conclude that the potency enhancement observed with bivalent ACTH peptides: (1) is optimal with spacers less than approximately 40 A in length, (2) is not due to direct interactions between the spacer and cell surface, and (3) is dependent on the length of the ACTH peptide component. In addition, these results indicate that electrostatic interaction between bivalent ACTH peptides and plasma membrane lipids does not adequately account for the potency enhancements observed.