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 antagonist 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-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [H-3]diprenorphine (approximate to 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid 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) approximate to N-benzyl- (18.3) approximate to N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl 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 cyclase activity: dynorphin A-(1-13)NH2 (100%) approximate to N-cyclopropylmethyl- (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-dicyclopropylmethyl- and N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro(10)]dynorphin 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 agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro(10)]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists. (C) 1997 Elsevier Science B.V.