Kazushi Okada

Synthesis of a complete set of L-difluorophenylalanines, L-(F2)Phe, as molecular explorers for the CH/π interaction between peptide ligand and receptor

Abstract A complete set of difluorophenylalanines in the l -configuration [ l -(F 2 )Phe] (namely, l -(2,3-F 2 )Phe, l -(2,4-F 2 )Phe, l -(2,5-F 2 )Phe, l -(2,6-F 2 )Phe, l -(3,4-F 2 )Phe, l -(3,5-F 2 )Phe) was prepared and incorporated into the thrombin receptor-tethered ligand peptide SFLLRNP to identify the phenyl hydrogens of the Phe-2 residue involved in the CH/π receptor interaction.

Synergistic effect of basic residues at positions 14–15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.

Discriminatory synergistic effect of Trp-substitutions in superagonist [(Arg/Lys)14, (Arg/Lys)15]nociceptin on ORL1 receptor binding and activation

ORL1 is an endogenous G protein-coupled receptor for neuropeptide nociceptin. [(R/K)(14), (R/K)(15)]nociceptin is a superagonist that strongly activates the ORL1 receptor. We have previously found that substituting with Trp can reproduce the potentiation induced by Arg or Lys at position 14. In the present study, in order to ensure the effect of Trp-substitution on the activities of [(R/K)(14), (R/K)(15)]nociceptin, we synthesized [W(14), (R/K)(15)]nociceptin and [(R/K)(14), W(15)]nociceptin. [W(14), (R/K)(15)]nociceptin was found to exhibit threefold higher binding activity and 10-fold greater potency in a functional [(35)S]GTPgammaS functional assay as compared to wild-type nociceptin. However, when only Trp was placed in position 15, the resulting analogues, [(R/K)(14), W(15)]nociceptin, showed only a moderate enhancement of binding and biological activity (2-3 fold in both). These results indicate that the placement of Trp at position 14, unlike at position 15, enhances in a synergistic fashion the interaction of nociceptin with the ORL1 receptor. The results indicate that specific interactions feasible for Arg/Lys and Trp in common must be there for aromatic residues in ORL1, thus forming a cation/pi interaction or pi/pi hydrophobic interaction. The necessity for a favorable electrostatic interaction appears strict in position 15.

Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues

TheS-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [d-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [d-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [d-Ala2,Cys(Npys)12]dynorphin A-(1–13) amide and [d-Ala2,Cys(Npys)8]dynorphin A-(1–9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [d-Ala2, Cys(Npys)12]dynorphin A-(1–13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [d-Ala2,Cys(Npys)12]dynorphin A-(1–13) amide.

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg(14),Lys(15)]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg(14),Lys(15)]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg(14),Lys(15)]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg(14),Lys(15)]. Trp208Ala was found to bind strongly both nociceptin and [Arg(14),Lys(15)]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide and [-Ala2,Cys(Npys)8]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide.

Receptor Binding Site of Arg-Lys Triple Repeat in Nociceptin Superagonist

Nociceptin (FGGFTGARKSARKLANQ), an endogenous heptadecapeptide isolated from the mammalian brain [1,2], was reported to be a ligand for opioid receptor-like ORL1 receptor to produce a hyperalgesia. The major efforts in the structure-activity studies have been focused on the design of antagonists for anti-neuropathy drugs. Indeed, several attempts to obtain nociceptin antagonists have recently been reported. On the contrary, a highly potent agonist, or so-called superagonist, often elicits the receptor responses such as desensitization and internalization, providing antagonistic cellular responses. Nociceptin possesses two Arg-Lys (RK) dipeptide units at the positions 8–9 and 12–13. This basic region of nociceptin has been suggested to interact with a cluster of acidic amino acid residues in the second extracellular loop (EL2) of ORL1 receptor [3]. We previously reported that when additional RK dipeptide unit was placed at positions 6–7, 10–11, 14–15, or 16–17, respectively, [Arg-Lys14–15]noci-ceptin showed increased activities both in the receptor binding assay (about 3-fold) and in the functional assay using [35S]GTPyS (about 17-fold) [4]. In the present study, in order to explore the reason why such an activity enhancement was induced by Arg-Lys replacement, we carried out the structure-activity studies by preparing a series of nociceptin analogs and ORL1 recombinant receptor.