Vol. 58, Issue 5, 1035-1041, November 2000

Creation of a Selective Antagonist and Agonist of the Rat VPAC₁ Receptor Using a Combinatorial Approach with Vasoactive Intestinal Peptide 6-23 as Template

Jeppe Wegener Tams, Rikke Malene Jørgensen, Arne Holm, and Jan Fahrenkrug

Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark (J.W.T., J.F.); and Chemistry Department, The Royal Veterinary and Agricultural University, Copenhagen, Denmark (R.M.J., A.H.)

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 a K_i value of 90 nM. This analog was unable to stimulate cAMP production at 10⁶ M but was able to inhibit VIP-induced cAMP stimulation (K_b = 79 nM). The K_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 and K_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, the K_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.