STRUCTURAL MODIFICATIONS OF NONMAMMALIAN GONADOTROPIN-RELEASING-HORMONE (GNRH) ISOFORMS - DESIGN OF NOVEL GNRH ANALOGS

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH ) provided the structural basis upon which to design new GnRH agonists : [His(5),Trp(7),Leu(8)]-GnRH, dogfish (df) GnRH; [His(5),Asn(8)]-GnRH , catfish (cf) GnRH; and [His(5),Trp(7),Tyr(8)]-GnRH, chicken (c) GnRH -II. The synthetic peptides incorporated the position 6 dextro (D)-iso mers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination wi th an ethylamide substitution of position 10. The in vitro potencies f or LH and FSH release of these analogues were assessed using static cu ltures of rat anterior pituitary cells. Efficacious peptides were exam ined for their gonadotroplin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release w as measured using homologous radioimmunoassays, whereas goldfish growt h hormone and gonadotropin-II: release were determined using heterolog ous carp hormone radioimmunoassays, The receptor binding of the most p otent analogues was determined in bovine pituitary membrane preparatio ns. Substitution of D-Nal(6) into [His(5),Asn(8)]-GnRH increased the p otency over 2200-fold compared with the native ligand (cfGnRH) in cult ured rat pituitary cells, This was equivalent to a 55-fold greater pot ency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal(6) or D-Arg(6) into dfGnRH or cGnRH-II resulted in potencies tha t were related to the overall hydrophobicity of the analogues. The [D- Nal(6),Pro(9)NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal(6),Pro(9)NEt]-mGn RH (k(d) = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultur ed rat pituitary cells. All analogues tested released the same ratio o f FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower an alogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.