Abstract

We have used combinatorial chemistry with amino acid mixtures (X) at positions 6 to 23 in vasoactive intestinal peptide (VIP) to optimize binding affinity and selectivity to the rat VPAC₁ receptor. The most efficient amino acid replacement was a substitution of alanine at position 18 to diphenylalanine (Dip), increasing the displacement efficiency of ¹²⁵I-VIP by 370-fold. The [Dip¹⁸]VIP(6–23) was subsequently used to find a second replacement, employing the same approach. Tyrosine at position 9 was selected and the resulting [Tyr⁹,Dip¹⁸]VIP(6–23) analog has aK _i value of 90 nM. This analog was unable to stimulate cAMP production at 10⁻⁶ M but was able to inhibit VIP-induced cAMP stimulation (K _b = 79 nM). TheK _i values of [Tyr⁹,Dip¹⁸]VIP(6–23) using the rat VPAC₂ and PAC₁ receptors were 3,000 nM and >10,000 nM, respectively. Thus, [Tyr⁹,Dip¹⁸]VIP(6–23) is a selective VPAC₁ receptor antagonist. The C-terminally extended form, [Tyr⁹,Dip¹⁸]VIP(6–28), displays improved antagonistic properties having a K _i andK _b values of 18 nM and 16 nM, respectively. On the contrary, the fully extended form, [Tyr⁹,Dip¹⁸]VIP(1–28), was a potent agonist with improved binding affinity (K _i = 0.11 nM) and ability to stimulate cAMP (EC₅₀ = 0.23 nM) compared with VIP (K _i = 1.7 nM, EC₅₀ = 1.12 nM). Furthermore, the specificity of this agonist to the VPAC₁ receptor was high, theK _i values for the VPAC₂ and PAC₁ receptors were 53 nM and 3,100 nM, respectively. Seven other analogs with the [Tyr⁹,Dip¹⁸] replacement combined with previously published VIP modifications have been synthesized and described in this work.