HUMAN GROWTH HORMONE-RELEASING HORMONE HGHRH(1-29)-NH2 - SYSTEMATIC STRUCTURE-ACTIVITY RELATIONSHIP STUDIES

Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle(27 )]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-p oint replacement of each amino acid by alanine led to the identificati on of [Ala(8)]-, [Ala(9)]-, [Ala(15)]- (Felix et al. Peptides 1986 198 6, 481), [Ala(22)]-, and [Ala(28),Nle(27)]-hGHRH(1-29)-NH2 as being 2- 6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly co mplete loss of potency was seen for [Ala(1)], [Ala(3)], [Ala(5)], [Ala (6)], [Ala(10)], [Ala(11)], [Ala(13)], [Ala(14)], and [Ala(23)], where as [Ala(16)], [Ala(18)], [Ala(24)], [Ala(25)], [Ala(26)], and [Ala(29) ] yielded equipotent analogues and [Ala(7)], [Ala(12)], [Ala(17)], [Al a(20)], [Ala(21)], and [Ala(27)] gave weak agonists with potencies 15- 40% that of the standard. The multiple-alanine-substituted peptides [M eTyr(1),Ala(15,22),Nle(27)]-hGHRH(1-29)-NH2 (29) and eTyr(1),Ala(8,9,1 5,22,28),Nle(27)]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. I ndividual substitution of the nine most potent peptides identified fro m the Ala series with the helix promoter a-aminoisobutyric acid (Aib) produced similar results, except for [Aib(8)] (doubling vs [Ala(8)]), [Aib(9)] (halving vs [Ala(9)]), and [Aib(15)] (10-fold decrease vs [Al a(15)]). A series of cyclic analogues was synthesized having the gener al formula ),Ala(15),Xaa(25),Nle(27),Yaa(29)]-GHRH(1-29)-NH2, where Xa a and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i+4)] lactam ring. The ring size, bridgehead amino acid chirality , and side-chain amide bond location were varied in this partial serie s in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified ,Ala(15),DAsp(25),Nle( 27),Orn(29)]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging e lement (19-membered ring) in this region of the molecule. This analogu e (46) was 17 times more potent than the standard. Equally effective w as an [i-(i+3)] constraint yielding the 18-membered ring ),Ala(15),Glu (25),Nle,(27)Lys(28)]-hGHRH(1-29)-NH2 (51) which was 14 times more pot ent than the standard. A complete [i-(i+3)] scan of Ala(15),Glu(i),Lys ((i+3)),Nle(27)]-hGHRH(1-29)-NH2 was then produced in order to test th e effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% r elative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i+3)] cycles between r esidues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined r esults from these systematic studies allowed for an analysis of struct ural features in the native peptide that are important for receptor ac tivation. Reinforcement of the characteristics of amphiphilicity, heli city, and peptide dipolar effects, using recognized medicinal chemistr y approaches including introduction of conformational constraints, has resulted in several potent GHRH analogues.