Tsugumi Fujita

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.

Pharmacological characterization of a novel AVP4-9 binding site in rat hippocampus

pGlu-Asn-Cys (Cys)-Pro-Arg-Gly-NH(2) (AVP(4-9)), a major metabolite C-terminal fragment of Arginine(8)-vasopressin (AVP), improves the disruption of the learning and memory, and is a far more potent in the mnemonic function than AVP. In this study, we pharmacologically characterized its putative binding site and mechanism of intracellular signaling. Radioligand binding assay showed that [35S]AVP(4-9) could detect specific binding sites in the rat hippocampus membrane preparations, and the binding site was specifically displaced by AVP(4-9) but not by either V(1) or V(2) antagonists. Furthermore, [35S]AVP(4-9) could not detect the cloned rat V(1a), V(1b) and V(2) vasopressin receptors. Even at a low doses (10-100 pM), AVP(4-9) caused an increase in both inositol(1,4, 5)-trisphosphate (Ins(1,4,5)P(3)) and intracellular calcium concentrations ([Ca(2+)](i)) in rat hippocampal cells. The AVP(4-9)-induced [Ca(2+)](i) increase was partially inhibited by the absence of Ca(2+) or by Ca(2+)-channel blocker, suggesting that AVP(4-9) caused the [Ca(2+)](i) increase via release from intracellular calcium store as well as influx from extracellular calcium. For the first time, this study provides evidence to show that AVP(4-9) activates Ins(1,4,5)P(3)/[Ca(2+)](i) pathway through a novel type of receptor in rat hippocampus, which might be potentially important in improving the mnemonic function.

A novel molecular design of thrombin receptor antagonist.

In a computer modeling of transmembrane domains of human thrombin receptor, Lys-158 was found near the ligand binding site. To capture this basic residue, analogs of peptide ligand containing a series of acidic amino acids were synthesized and assayed for human platelet aggregation, and Ser-(p-F)Phe-Aad(= alphaaminoadipic acid)-Leu-Arg-Asn-Pro-NH2 was found to be a potent antagonist.

Thrombin Receptor Aromatic Residues for Edge-to-Face CH/π Interaction with Ligand Phe-2-phenyl Group

Serine protease thrombin plays an important role in blood coagulation and possesses a specific receptor in the platelets. When thrombin cleaves the peptide bond between Arg-41 and Ser-42 in the N-terminal segment of receptor, the newly exposed N-terrninal peptide segment binds to the receptor itself as a tethered ligand which activates the receptor [1]. Synthetic heptapeptide Ser-Phe-Leu-Leu-Arg-Asn-Pro (SFLLRNP, one letter amino acid codes) corresponding to this tethered-ligand is able to activate the receptor without thrombin, and the Phe-2 residue of this SFLLRNP peptide was found to be essential for receptor recognition and activation. In the present structure-activity studies to elucidate the role of Phe-2 in receptor activation, the benzene hydrogens in the Phe-2-phenyl group were suggested to be in the edge-to-face CH/π interaction with the receptor aromatic groups. Computer modeling of thrombin receptor indicated that the aromatic amino acid cluster in the fifth transmembrane domain (TM5: YYAYYFSAFSAVFFF) is a binding site of this Phe-2-phenyl. In this study, in order to determine the genuine binding site of Phe-2-phenyl, we prepared mutant receptors in which Tyr in TM5 were replaced by Ala.

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.

Agonist-antagonist structure-activity relationships of thrombin receptor tethered ligand peptide

T. FUJITA1, T. NOSE1, M. NAKAJIMA2, Y. INOUE2, N. NAKAMURA2, T. INOUE3, T. COSTA4 and Y. SHIMOHIGASHI1 1 Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan 2 Research Division, The Green Cross Corp., Hirakata 573-1153, Japan 3 Institute of Genetic information, Kyushu University, Fukuoka 812-8582, Japan 4Laboratory di Farmacologia, Istituto Superiore di Sanita, 00161 Roma 299, Italy