N-ALKYLATED DERIVATIVES OF [D-PRO(10)]DYNORPHIN A-(1-11) ARE HIGH-AFFINITY PARTIAL AGONISTS AT THE CLONED RAT KAPPA-OPIOID RECEPTOR

As part of an effort to develop peptides with selective kappa-opioid a ntagonist activity, a series of N-alkylated [D-Pro(10)]dynorphin A-(1- 11) derivatives were made through solid-phase peptide synthesis: R-Tyr -Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cy clopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These deriva tives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid recept or binding affinity and potency as inhibitors of adenylyl cyclase. Equ ilibrium competition binding experiments using [H-3]diprenorphine (app roximate to 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opi oid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 mu M) adenylyl cyclase activity. Displacement of [H-3]diprenorphine specific binding by these peptides was observed with a rank order of affinity (K-i, nM) = [D-Pro(10)]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N- cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl-(12.6) approxi mate to N-benzyl- (18.3) approximate to N,N-diallyl-[D-Pro(10)]dynorph in A-(1-11) (26.0). A similar rank order was observed for potency of a denylyl cyclase inhibition (IC50, nM): [D-Pro(10)]dynorphin A-(1-11) ( 0.12) approximate to dynorphin A-(1-13)NH2 (0.19)> N-cyclopropylmethyl (2.7) > N,N-dicyclopropylmethyl (13.2) approximate to N,N-diallyl (18 .0) approximate to N-benzyl-[D-Pro(10)]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cycl ase activity: dynorphin A-(1-13)NH2 (100%) approximate to N-cyclopropy lmethyl- (94.3%) approximate to [D-Pro(10)]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) >> N,N-dicyclopropylmethyl- (23.6%) approximate to N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) (18.9%). As the N,N-dicyclo propylmethyl- and N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) derivative s were found to have only weak partial agonist activity with respect t o adenylyl cyclase inhibition, they were evaluated for their ability t o reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro(10)]dynorph in A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N, N-diallyl-[D-Pro(10)]dynorphin A-(1-11) alone. This weak partial agoni sm combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) compounds promising lea ds for further attempts to synthesize peptide kappa-opioid receptor an tagonists. (C) 1997 Elsevier Science B.V.