Design of Potent Dicyclic (4−10/5−8) Gonadotropin Releasing Hormone (GnRH) Antagonists

Abstract

With the ultimate goal of identifying a consensus bioactive conformation of GnRH antagonists, the compatibility of a number of side chain to side chain bridges in bioactive analogues was systematically explored. In an earlier publication, cyclo[Asp(4)-Dpr(10)]GnRH antagonists with high potencies in vitro and in vivo had been identified.(1) Independently from Dutta et al. (2) and based on structural considerations, the cyclic [Glu(5)-Lys(8)] constraint was also found to be tolerated in GnRH antagonists. We describe here a large number of cyclic (4-10) and (5-8) and dicyclic (4-10/5-8) GnRH antagonists optimized for affinity to the rat GnRH receptor and in vivo antiovulatory potency. The most potent monocyclic analogues were cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Asp(4),DArg(6),Xaa(10)]GnRH with Xaa = D/LAgl (1, K(i) = 1.3 nM) or Dpr (2, K(i) = 0.36 nM), which completely blocked ovulation in cycling rats after sc administration of 2.5 microgram at noon of proestrus. Much less potent were the closely related analogues with Xaa = Dbu (3, K(i) = 10 nM) or cyclo(4-10)[Ac-DNal(1), DFpa(2),DTrp(3),Glu(4),DArg(6),D/LAgl(10)]GnRH (4, K(i) = 1.3 nM). Cyclo(5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Glu(5),DArg++ +(6),Lys(8), DAla(10)]GnRH (13), although at least 20 times less potent in the AOA than 1 or 2 with similar GnRHR affinity (K(i) = 0.84 nM), was found to be one of the most potent in a series of closely related cyclo(5-8) analogues with different bridge lengths and bridgehead chirality. The very high affinity of cyclo(5,5'-8)[Ac-DNal(1), DCpa(2),DPal(3),Glu(5)(betaAla),DArg(6),(D or L)Agl,(8)DAla(10)]GnRH 14 (K(i) = 0.15 nM) correlates well with its high potency in vivo (full inhibition of ovulation at 25 microgram/rat). Dicyclo(4-10/5-8)[Ac-DNal(1),DCpa(2),DTrp(3),Asp (4),Glu(5),DArg(6), Lys(8),Dpr(10)]GnRH (24, K(i) = 0.32 nM) is one-fourth as potent as 1 or 2, in the AOA; this suggests that the introduction of the (4-10) bridge in 13, while having little effect on affinity, restores functional/conformational features favorable for stability and distribution. To further increase potency of dicyclic antagonists, the size and composition of the (5-8) bridge was varied. For example, the substitution of Xbb(5') by Gly (30, K(i) = 0.16 nM), Sar (31, K(i) = 0.20 nM), Phe (32, K(i) = 0.23 nM), DPhe (33, K(i) = 120 nM), Arg (36, K(i) = 0.20 nM), Nal (37, K(i) = 4.2 nM), His (38, K(i) = 0.10 nM), and Cpa (39, K(i) = 0.23 nM) in cyclo(4-10/5,5'-8)[Ac-DNal(1),DCpa(2),DPal(3),Asp(4),G lu(5)(Xbb(5')), DArg(6),Dbu,(8)Dpr(10)]GnRH yielded several very high affinity analogues that were 10, ca. 10, 4, >200, 1, ca. 4, >2, and 2 times less potent than 1 or 2, respectively. Other scaffolds constrained by disulfide (7, K(i) = 2.4 nM; and 8, K(i) = 450 nM), cyclo[Glu(5)-Aph(8)] (16, K(i) = 20 nM; and 17, K(i) = 0.28 nM), or cyclo[Asp(5)-/Glu(5)-/Asp(5)(Gly(5'))-Amp(8)] (19, K(i) = 1.3 nM; 22, K(i) = 3.3 nM; and 23, K(i) = 3.6 nM) bridges yielded analogues that were less potent in vivo and had a wide range of affinities. The effects on biological...

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

“…Our data also suggest the proximity between the pepfide termini, which although surprising for a nonapepfide sequence, is also in agreement with Reddy and co-workers' results [13], and a tendency of the N-terminus to form a stable conformation proposed also by Rivier and co-workers [15]. NMR studies of the native hormone indicated that is essentially a random coil, in three different solvent conditions (aqueous solution, DMSO solution and lipid-bound form in model membranes) [7].…”

Structure–Function Studies of Linear and Cyclized Peptide Antagonists of the GnRH Receptor

“…There is a threshold in in vitro activity, but a binding affinity of K D Յ 1 nM is no guarantee for prolonged testosterone suppression in vivo. Peptides with similar high binding affinity displayed very different efficacy in the animal, a phenomenon observed by others as well (27,28). The pharmacokinetics of Cetrorelix and D-63153 (Fig.…”

“…For the cyclo [1][2][3][4][5][6][7][8][9][10] peptides D-23620 (Cyclorelix) and D-24236 (Cycloantarelix), this strategy was not successful since binding affinity was reduced up to 80-fold. Nevertheless, highly potent cyclo [1][2][3][4] and cyclo [4 -10] peptides were described in the literature and structural analysis of a cyclo [4 -10] analog revealed the presence of a ␤-hairpin conformation within residues 5-8, stabilized by two hydrogen bonds, with a type IIЈ ␤-turn around positions 6 and 7 (27,28,30,31). Consistent with these data, cyclo [3][4][5][6][7][8] decapeptides were found to be highly potent (27,28,32).…”

“…Antagonists have reached the market late with Cetrorelix (Cetrotide, ASTA Medica AG) or Ganirelix (Orgalutran; NV Organon) now used clinically in assisted fertilization protocols and Abarelix developed by Amgen Inc. for treatment of prostate cancer (12,25,26). Although many GnRH analogs were synthesized, systematic studies evaluating receptor binding and antagonistic potency toward different GnRH receptor proteins and the relevance of these parameters for efficacy in animal models are rare (27,28). Therefore we evaluated a comprehensive set of structurally different antagonists using defined recombinant cell systems, expressing the human and rat GnRH receptor or the W 101 A and N 102 A receptor mutants.…”

“…Nevertheless, highly potent cyclo [1][2][3][4] and cyclo [4 -10] peptides were described in the literature and structural analysis of a cyclo [4 -10] analog revealed the presence of a ␤-hairpin conformation within residues 5-8, stabilized by two hydrogen bonds, with a type IIЈ ␤-turn around positions 6 and 7 (27,28,30,31). Consistent with these data, cyclo [3][4][5][6][7][8] decapeptides were found to be highly potent (27,28,32). In contrast to the cyclo [6 -10] analog D-24418, the cyclo [3][4][5][6][7][8][9][10] analog D-52391 still fits the structural requirements needed for high binding affinity, which is in accordance to the published data.…”