• Design, synthesis and pharmacological evaluation of orphanin FQ(1-13)amidogen analogs

      Charoenchai, Laksana; Aldrich, Jane V. (2001)
      The novel orphanin (ORL1) receptor and its endogenous ligand orphanin FQ or nociceptin (OFQ/N) are similar to the opioid receptors and ligands, particularly the Kopioid receptor and dynorphin A (Dyn A). OFQ exhibits various physiological and pharmacological effects but the role of OFQ in nociception is not well understood. Thus OFQ analogs are useful as pharmacological tools to study the ORL1 receptor. One goal of this research was to prepare constrained analogs of OFQ(1-13)NH2 to explore the structural and conformational requirements of this peptide for receptor binding and activation. Initially linear D/L-Asn and D/L-Met OFQ(1-13)NH 2 analogs were synthesized to determine possible positions and linkage for the design of constrained OFQ(1-13)NH2 analogs. Other linear OFQ(1-13)NH2 analogs with substitutions of various amino acids at positions 2 to 7 (excluding position 4) were also prepared to examine the effects of different side chain functional groups on ligand-receptor interaction. Two series of cyclic peptides with a lactam linkage were prepared with the constraint incorporated into different regions of the parent peptide. Hybrid OFQ peptides were also designed by incorporating novel N-terminal sequences from Dyn A analogs into OFQ(1-13)NH2 to examine the effects on ORL1 receptor affinity versus the Dyn A analogs. The peptides were prepared by solid phase synthesis using the Fmoc (9-fluoronylmethoxycarbonyl)/tert-butyl synthetic protocol. The compounds were evaluated for their affinity and efficacy for cloned human orphanin receptor (hORL1) expressed in Chinese hamster ovary (CHO) cells using a radioligand binding and the [35S]GTPgammaS assays, respectively. Modification with a variety of L/D-amino acids in the middle of the peptide sequence resulted in high affinity analogs. [D-Ala7]OFQ(1-13)NH2 was a full agonist in the GTPgammaS assay, in contrast to the literature report' that [D-Ala7]OFQ is a partial agonist. Cyclization via a lactam linkage in the middle of the OFQ sequence resulted in analogs with high affinity (Ki = 0.27-7 nM) and high to moderate potency (EC50 = 1.6-157 nM) in the GTPgammaS assay. Two cyclic analogs exhibited higher affinity and potency than the parent peptide. Hybrid OFQ analogs which had novel N-terminal sequences from Dyn A analogs generally exhibited low or no binding affinity at the ORL1 receptor. Thus the structural requirements in the N-terminal sequence of OFQ and Dyn A are different. These structure-activity relationship (SAR) studies provide additional information on the structural features for consideration in the further design of novel OFQ ligands. The high affinity analogs described here, particularly the cyclic peptides, will be useful pharmacological tools to study the roles of the orphanin system.
    • Synthesis and pharmacological evaluation of dynorphin A analogs constrained in the "message" sequence

      Vig, Balvinder Singh; Aldrich, Jane V. (2001)
      kappa-Opioid receptors and their endogenous ligand dynorphin A (Dyn A) are of tremendous interest because of their diversity of functions and potential for analgesics with fewer side effects. Compared to other opioid peptides such as the enkephalins, the study of the structure-activity relationships (SAR) of Dyn A have been limited, and the conformational preferences of the three key pharmacophoric groups, the N-terminal basic amine, Tyr1, and Phe4, in the "message" sequence of Dyn A are not yet clear. The main objectives of this research were to develop conformationally restricted analogs of Dyn A with high affinity and/or selectivity that can be used to explore the structural and conformational requirements for interaction of Dyn A with kappa-opioid receptors, and to explore the structure-activity relationships (SAR) for agonist vs. antagonist activity at kappa receptors. The central hypotheses of this research were that the spatial orientations of the three key pharmacophoric groups in the "message" sequence are important for the high affinity and activity of Dyn A for kappa-opioid receptors and that the spatial requirements of these groups for agonist activity are different from those for antagonist activity. Incorporation of conformational constraints in the "message" sequence will restrict and/or alter the relative position of these pharmacophoric groups thus effecting the affinity, selectivity and/or efficacy of the resulting peptides. Synthetic strategies were explored for the synthesis of side chain-to-side chain and novel head-to-side chain cyclic analogs of Dyn A. Modifications in the "message" sequence of cyclo[D-Asp2, Dap5]Dyn A-(1-11)NH2 resulted in peptides with high affinity for kappa receptors and high selectivity for kappa over delta receptors; however the selectivity of these peptides for kappa over mu receptors was low to modest. Substitution of Pro at position 3 of cyclo[D-Asp 2,Dap5]Dyn A-(1-11)NH2 resulted in a peptide with reduced efficacy for kappa receptors. The synthesis of novel head-to-side chain cyclic analogs of Dyn A with non-basic N-terminus resulted in peptides with nanomolar affinity and reduced efficacy for kappa-opioid receptors. The N-terminal cyclic analogs of Dyn A with basic N-terminus resulted in reduced affinity for opioid receptors. Results obtained during this research suggested that the spatial orientation of the key pharmacophoric groups is critical for opioid receptor affinity and activity of Dyn A analogs.