Creation of a Selective Antagonist and Agonist of the Rat VPAC1Receptor Using a Combinatorial Approach with Vasoactive Intestinal Peptide 6–23 As Template

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 VPAC(1) receptor. The most efficient amino acid replacement was a substitution of alanine at position 18 to diphenylalanine (Dip), increasing the displacement efficiency of I-125-VIP by 370-fold. The [Dip(18)]VIP(6-23) was subsequently used to find a second replacement, employing the same approach. Tyrosine at position 9 was selected and the resulting [Tyr(9),Dip(18)]VIP(6-23) analog has a K-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). The K-i values of [Tyr(9),Dip(18)]VIP(6-23) using the rat VPAC(2) and PAC(1) receptors were 3,000 nM and >10,000 nM, respectively. Thus, [Tyr(9),Dip(18)]VIP(6-23) is a selective VPAC(1) receptor antagonist. The C-terminally extended form, [Tyr(9),Dip(18)]VIP(6-28), displays improved antagonistic properties having a K-i and K-b values of 18 nM and 16 nM, respectively. On the contrary, the fully extended form, [Tyr(9),Dip(18)]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 VPAC(1) receptor was high, the K-i values for the VPAC(2) and PAC(1) receptors were 53 nM and 3,100 nM, respectively. Seven other analogs with the [Tyr(9),Dip(18)] replacement combined with previously published VIP modifications have been synthesized and described in this work.