Probing the GnRH receptor agonist binding site identifies methylated triptorelin as a new anti-proliferative agent.

D-amino acid substitutions at glycine postion 6 in GnRH-I decapeptide can possess super-agonist activity and enhanced in vivo pharmacokinetics. Agonists elicit growth-inhibition in tumorigenic cells expressing the GnRH receptor above threshold levels. However, new agonists with modified properties are required to improve the anti-proliferative range. Effects of residue substitutions and methylations on tumourigenic HEK293_[SCL60] and WPE-1-NB26-3 prostate cells expressing the rat GnRH receptor were compared. Peptides were ranked according to receptor binding affinity, induction of inositol phosphate production and cell growth-inhibition. Analogues possessing D-Trp⁶ (including triptorelin), D-Leu⁶ (including leuprolide), D-Ala⁶, D-Lys⁶, or D-Arg⁶ exhibited agonist and anti-proliferative activity. Residues His⁵ or His⁵,Trp⁷,Tyr⁸, corresponding to residues found in GnRH-II, were tolerated, with retention of sub-nanomolar/low nanomolar binding affinities and EC₅₀s for receptor activation and IC₅₀s for cell growth-inhibition. His⁵D-Arg⁶-GnRH-I exhibited reduced binding affinity and potency, effective in the mid-nanomolar range. However, all GnRH-II-like analogues were less potent than triptorelin. By comparison, three methylated-Trp⁶ triptorelin variants showed differential binding, receptor activation and anti-proliferation potency. Significantly, 5-Methyl-DL-Trp⁶-Triptorelin was equipotent to triptorelin. Subsequent studies should determine whether pharmacologically enhanced derivatives of triptorelin can be developed by further alkylations, without substitutions or cleavable cytotoxic adducts, to improve the extent of growth-inhibition of tumour cells expressing the GnRH receptor.