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The 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label mu and delta opioid receptors. When rat brain membranes were incubated with [D-Ala2,Leu(CH2SNpys)5]enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpys-containing enkephalin. It was found that this enkephalin analog occupies mu receptors highly specifically (EC50 = 51 nM) and almost 100 times more selectively than delta receptors. In contrast, [D-Ala2,Leu5]enkephalyl-Cys(Npys)6 attached covalently to delta receptors (EC50 = 34 nM) about 150 times more selectively than to mu receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin-bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1,000 times) for delta receptors than for mu receptors. The present results indicated that both mu and delta receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala2,Leu5]enkephalyl-Cys6 and delta receptors appears to be exposed, while that between [D-Ala2,Leu(CH2-SNpys)5] enkephalin and mu receptors is shielded.

Dipeptide amides H-D-Leu-Phe-NH-R have been found to assume a conformation induced by the CH/π interaction and to inhibit chymotrypsin strongly. A series of benzyl amide derivatives H-D-Leu-Phe-NH-[CH2]n-C6H5(n= 0–4) have been assayed for chymotrypsin. They inhibit the enzyme in a competitive manner and the highest inhibition is achieved by the amide of n= 1 (Ki= 3.6 × 10–6M). The activity enhancement is dependent upon the length of methylene chain, not upon the increase in molecular hydrophobicity, indicating the presence of an optimal distance between dipeptide backbone and C-terminal phenyl group for chymotrypsin inhibition. The C-terminal phenyl group has been found to interact with chymotrypsin stereospecifically. The R-isomer of H-D-Leu-Phe-NH-CH(CH3)-C6H5 is as active as the benzyl amide, while the S-isomer is about twenty-fold less active. When the fluorine atom is introduced at a para-position of the C-terminal phenyl group, the resulting dipeptide H-D-Leu-Phe-NH-CH2-C6H4F-p exhibits about six-times increased inhibitory activity (Ki= 6.1 × 10–7M; this dipeptide is one of the most potent chymotrypsin inhibitors to date). 1H NMR conformational analyses of these dipeptide amide derivatives show the CH/π interaction between D-Leu-isobutyl and Phe-phenyl as a key structural element for chymotrypsin inhibition. These structural examinations strongly suggest that in the inhibitory conformation the C-terminal phenyl group fits the chymotrypsin S1 site, while the hydrophobic core constructed by D-Leu-Phe CH/π interaction fits the chymotrypsin S2 or S1′ site.

A synthetic peptide, H–Ser–Phe–Leu–Leu–Arg–Asn–Pro–NH2, which corresponds to a ligand peptide tethered to a human thrombin receptor, was able to activate the thrombin receptor with no thrombin. In order to inspect the structural requisites of two consecutive leucines (Leu-3 and Leu-4) in receptor activation, two sets of analogs with substitutions at either position 3 or 4 were synthesized and evaluated for their ability to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells. The replacement of Leu-4 by Ala drastically decreased the activity of the parent peptide (only about 4% activity), while that of Leu-3 retained about 50% activity. A similar result was obtained when replaced by Gly. Substitution by Phe for Leu-3 sustained full activity. Although the Leu-4/Phe substitution exhibited a slight reduction in activity, Leu-4/Ile substitution unexpectedly diminished the activity (20%). These results suggested that two consecutive leucines have different roles in receptor activation; i.e., Leu-3 beh...

A series of Ile-containing elastin-derived peptide-analogs, (Ile-Pro-Gly-Val-Gly)n (n = 7-10) possessing remarkable and reversible coacervation property were newly synthesized. In comparison with the known elastin-derived peptide-analogs, which were so-called polypeptides, the obtained 35 to 50 mer peptides, (IPGVG)n (n = 7-10) were significantly low molecular sized-polypeptides. However, they clearly exhibited coacervation property as same as the polypeptides did. Because of their low molecular size, spectrographic analyses of (IPGVG)n (n = 7-10) became feasible to carry out. As results of secondary structural analyses by CD and FT-IR, it was found that the coacervation property of the peptides is clearly attributed to the ordered secondary-structures, mainly, type II β-turn.

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavensis (himehabu snake) venom, releases specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for α-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N -glycanase. Okinaxobin II was much more basic (pI=8.1) than okinaxobin I (pI=5.4). The N-terminal sequence was highly similar to those of okinazobin I and some other snake venom coagulant enzymes such as flavoxobin ( Trimeresurus flavoviridis ), batroxobin ( Bothrops atrox and Bothrops moojeni ), and catroxobin ( Crotalus atrox ). Okinaxobin II hydrolyzed tosyl-L-arginine methly ester and benzoyl-L-arginine p -nitroanilide. The esterase activity was strongly inhibited by di iso propylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethly ketone, indicating that the enzyme is a serine protease like α-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to α-thrombin.

A synthetic heptapeptide H-Ser-Phe-Phe-Leu-Arg-Asn-Pro-NH 2 , which corresponds to the ligand peptide latent in rodent thrombin receptors, was able to activate the thrombin receptor with no thrombin. In order to evaluate the structural requisites of two consecutive phenylalanines, three sets of analogs with substitutions at position either 2 or 3 were synthesized and examined for their stimulatory activity in phosphoinositide turnover in SH-EP epithelial-like cells. The replacement of Phe-2 by Ala completely eliminated the activity, while that of Phe-3 retained about 50% activity with a full stimulation. The Phe/Leu substitution resulted in a large increase (37-fold) in EC 50 value for Phe-2, but in insignificant change for Phe-3. Substitution of para -fluorophenylalanine (( p -F)Phe) for Phe-2 enhanced strongly (4-fold) the activity, in contrast to a reduction by the Phe-5/( p -F)Phe substitution. Elimination of either Phe-2 or Phe-3 resulted in a complete loss of activity. These results indicated that Phe-2 and Phe-3 play different roles in the receptor activation. A highly specific aromatic π-π interaction was suggested between Phe-2-phenyl and thrombin receptor binding site, while Phe-3 appeared to be important for retaining a bioactive conformation.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.

In order to inspect the structural elements of Ser-1 in receptor activation by SFLLRNP (one-letter amino acid code), a ligand peptide tethered to the thrombin receptor, a series of analogs with such replacements as D-Ser, Ala, Thr, and Ac-Ser have been synthesized. These analogs were evaluated for their ability to hydrolyze the phosphoinositide in human neuroblastoma SH-EP cells. It was found that the α-amino group and L-configuration of Ser-1 are very important in the activation of receptors. N-Acetylation or deletion of Ser-1 completely eliminated the activity of SFLLRNP (a half-maximal effective concentration, EC50 = 0.89 μM (1 M = 1 mol dm−3)), and these modifications induced no antagonist activity. Incorporation of D-Ser also drastically diminished the activity, but retained about 50% activity of the maximal response by 100 μM SFLLRNP. The Ser/Ala substitution sustained 30% of the activity of SFLLRNP to elicit a full stimulation. The Ser/Thr substitution, however, enhanced the activity (20%) in spite...

Hepatocyte growth factor (HGF) has a strong affinity for heparin. About one fourth of HGF secreted from MRC-5 human embryonic lung fibroblast cells was found to be associated with heparin and heparan sulfate proteoglycan on the cell surface and extracellular matrix. To identify heparin-binding sites within the HGF molecule, we constructed variously deleted mutant HGFs and examined their binding ability to an immobilized heparin column. Native HGF and mutant HGFs, including d-K1 (deletion of the first kringle domain), d-K3 (deletion of the third kringle domain), d-K4 (deletion of the fourth kringle domain), d-beta (deletion of beta-chain), and HK1K2 (consisting of the N-terminal hairpin loop and the first two kringle domains), tightly bound to a heparin column, but d-H (deletion of the N-terminal hairpin loop) and d-K2 (deletion of the second kringle domain) markedly decreased binding ability to the column. These observations suggest that the N-terminal hairpin loop and the second kringle domain are essential for the heparin-binding of HGF. The finding that HK1K2 competed the binding of 125I-HGF to immobilized heparin provided additional evidence that the N-terminal half of HGF alpha-chain is the principal heparin-binding site. The hairpin loop in HGF possesses a cluster of basic amino acid residues and a highly positive net charge, when compared with hairpin loop structures in the other proteins, plasminogen and HGF-like protein. The second kringle domain in HGF has the basic amino acid cluster in the central region. Thus, it is likely that the basic clusters in these domains cooperatively contribute to the binding of HGF to the anionic heparin or heparan sulfate molecule.

T. Fukagawa , T. Nose , Y. Shimohigashi , T. Ogawa , N. Oda , K. Nakashima , C.-C. Chang and M. Ohno . Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon31, 957–967, 1993.—Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [ Oda et al. (1991) Toxicon29, 157; Fukagawa et al. (1992) Toxicon30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca2+-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3–5.2 × 10−4 M) for Ca2+ between these PLA2s and Tyr-28-containing PLA2s. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2s to Ca2+.

A complete series of configurationally isomers (L-L, L-D, D-L AND D-D) of a dipeptide Leu-Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz 1H NMR, the L-D and D-L isomers, but not hte L-L and D-D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu-βCH2 and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe-βCH2, the NOE enhancement observed between Leu-δCH3 and Phe-phenyl groups revealed that these groups are in close proximity. These data indicated that L-D and D-L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac-Try-OEt as a substrate, the L-L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D-L isomer was strongest with Ki of 2.2 × 10−5M. It was found that the D-L isomer was only slowly hydrolysed but the L(or D)-D isomer was not. H-D-Phe-L-Leu-OBzl with the inverse sequence of H-D-Leu-L-Pre-OBzl inhibited chymotrypsin more strongly (Ki = 6.3 × 10−6M). Since the free acid analogue of the D-L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester-benzyl group fits the S1 site of chymotrypsin, while the side chain-side chain complexing hydrophobic core fits the S2 site.

[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.

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.

We established a novel screening method to survey endocrine-disrupting chemicals by means of in silico docking calculations. Endocrine disruptors target the human nuclear receptor, which bind a chemical in a pocket presenting in the ligand-binding domain (LBD). The LBD alters its conformation, depending upon the binding of either agonist or antagonist. We discovered that the chemicals can be differentiated into either agonist or antagonist by the docking calculations of the chemical for the LBD. We used the crystal structures of both agonist-bound LBDs and antagonist-bond LBDs as templates in the docking calculations, and estimated binding energies to discriminate between agonist and antagonist bindings. This agonist/antagonist differential-docking screening (AADS) method predicted, for example, 4-(1-adamantyl)phenol as an agonist of the human estrogen receptor alpha (hERalpha). Indeed, this compound, one of the essential raw materials for nanoporous organosilicate thin films, was confirmed to exhibit strong agonist activity in the reporter-gene assay for hERalpha with a high binding affinity. The AADS method is an approach that appears to foresee both the binding ability and the agonist/antagonist function of chemicals for the target nuclear receptors.

Analysis of the amino acid composition of prion protein using a newly developed program for radar-chart deviation analysis has identified an abnormality or irregularity of the N-terminal flexible domain. Aromatic amino acids Trp and His together with Gly are abnormally abounding in this N-terminal domain, in which octapeptide GQPHGGGW is connected four times in tandem. This tetrarepeat structure has been suggested to be essential for the prion protein not only to play an intrinsic functional role in the physiological condition, but also to bring on structural abnormalities in prion disease.

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, [RK14–15]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPγS-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 GTPγS 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.

A computer-aided docking study was carried out to quickly clarify the binding structure of the ligand-receptor complex between bisphenol A (BPA), a well-known endocrine disruptor, and estrogen-related receptor gamma (ERRgamma). The resulting complex indicated that BPA binds to the ligand-binding pocket of ERRgamma without any disruptions of the activation conformation.

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH 2 is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH 2 , where acyl (R-CO) possesses a series of alkyl groups, R = C n H 2 n +1 ( n = 0–5). The isovaleryl derivative with the C 4 H 9 (=(CH 3 ) 2 CHCH 2 –) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH 2 and producing pure antagonist activity.

Endogenous opioid peptides consist of a conserved amino acid residue of Phe(3) and Phe(4), although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the mu opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe(3) and Phe(4) in binding to the mu receptor, we synthesized a series of analogs in which Phe(3) and Phe(4) were replaced by various amino acids. It was found that the aromaticity of the Phe-beta-phenyl groups of Phe(3) and Phe(4) is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe(3) and Phe(4) of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp(3)]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses.

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

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean beta-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%-94%) and similarity (89%-100%) to insect PDFs and also to the crustacean beta-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure–activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(=Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8–13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the µ opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1–7) or -(14–17) binds. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.

An anticoagulant protein named AaACP was isolated from Agkistrodon actus (hundred-pace snake of Taiwan, Viperidae) venom. AaACP inhibited the factor Xa-induced plasma coagulation in a concentration-dependent manner. Thus, AaACP seems to bind to factor Xa in prothrombinase complex. AaACP was composed of A and B chains linked by disulphide bond(s). The amino acid sequences of A and B chains of AaACP were analysed with a few residues unidentified which were complemented from the nucleotide sequences of their cDNAs. The A chain consisted of 129 amino acid residues and the B chain 123 amino acid residues. Their amino acid sequences were highly similar to those of A and B chains of a series of anticoagulant proteins which had been purified from the venoms of some Viperidae snakes. The A and B chains structurally belong to C-type lectin-like protein family of snake venom origin. Construction of phylogenetic tree of C-type lectins and C-type lectin-like proteins based on their amino acid sequences indicated that their A and B chains diverged before speciation of snake species. The comparison of the nucleotide sequences of the cDNAs encoding A and B chains of AaACP and of Trimeresurus flavoviridis (Viperidae) venom-gland factors IX/X-binding protein and factor IX-binding protein showed that the mature protein-coding region is much more variable than the signal peptide-coding domain and the 5′- and 3′-untranslated regions, being in contrast to the case of the ordinary isoprotein genes. The ratios of the numbers of nucleotide substitutions per nonsynonymous site (KA) and per synonymous site (KS) in the mature protein-coding region in the cDNA pairs were about three times greater than those for the ordinary isoprotein genes, suggesting that these genes have been evolving in an accelerated manner. Taking account of the functional diversities of venom-gland C-type lectins and C-type lectin-like proteins including factors IX and/or X-binding proteins, it can be said that their functional diversities have been acquired by accelerated evolution.

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.

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.

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.

In the ligand/receptor interaction, the side chain phenyl group of phenylalanine (Phe) is involved in a so-called hydrophobic interaction, in which the Phe-phenyl group functions as a p element or merely as a hydrophobic element. The thrombin receptor-tethered ligand SFLLRNP consists of the Phe-2 residue essential for receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl group, a complete set of S/Phe/LLRNP peptides comprising six different difluorophenylalanine isomers [(F(2))Phe] was newly synthesized and assayed to evaluate their ability to induce the aggregation of human platelets. The assay results clarified several important structural elements to conclude that Phe-2-phenyl of S/Phe/LLRNP is in the edge-to-face CH/pi interaction with the receptor aromatic group, utilizing the Phe-phenyl edge along with adjacent benzene hydrogens at positions (2-3) or (5-6). It was also found that the fluorine atom at position 4 increases the acidity of the hydrogen mainly at its ortho position, resulting in a reinforcement of the CH/pi interaction and thus in an enhancement of biological activity. The H-->F replacement in the benzene ring was found to provide an effective structural examination to the Phe residue; i.e., to identify the hydrogens in the CH/pi interaction, and to strengthen the CH/pi interaction.

The dipeptide D-leucyl-L-phenylalanyl p-fluorobenzylamide (D-Leu-Phe-NH-BzlF) inhibits chymotrypsin strongly in a competitive manner with the Ki value of 0.61 microM [Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Ide, Y., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. & Ohno, M. (1996) J. Chem. Soc. Perkin Trans. 1, 2479-2485]. The structure/activity studies have suggested a unique inhibitory conformation, in which the C-terminal benzyl group fits the chymotrypsin S1 site and the hydrophobic core constructed by the side chains of D-Leu-Phe fits the S2 or S1' site. To verify this assumption, the molecular structure of the complex between the dipeptide and gamma-chymotrypsin has been determined crystallographically. Gamma-chymotrypsin itself was first crystallized and refined at 1.6-A resolution. The refined structure was virtually identical to the conformation reported and the electron density at the active site was interpreted as a pentapeptide Thr-Pro-Gly-Val-Tyr derived from autolysis of the enzyme (residues 224-228). The chymotrypsin-dipeptide complex was obtained by soaking the crystals of gamma-chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8-A resolution to a crystallographic R-factor of 18.1%. The structure of gamma-chymotrypsin in the complex agreed fairly well with that of gamma-chymotrypsin per se with a rmsd of 0.13 A for all the C alpha carbons. Two inhibitor molecules were assigned in an asymmetric unit, i.e. one in the active site and the other at the interface of two symmetry-related enzyme molecules. In both sites dipeptides adopted very similar folded conformations, in which side chains of D-Leu-Phe are spatially proximal. In the active site where the binding of dipeptide was judged to be a direct cause of inhibition, C-terminal p-fluorobenzylamide group of the dipeptide, NH-BzlF, was found in the S1 hydrophobic pocket. At the bottom of this pocket, the p-fluorine atom hydrogen bonded with a water molecule, probably to enhance the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C-terminal NH-C*H(CH3)-C6H5 was well explained by the space available for methyl replacement in the complex. The hydrophobic core constructed by side chains of D-Leu-Phe was found at the broad S2 site. Interestingly, a novel interaction was found between the inhibitor Phe residue and chymotrypsin His57, the phenyl of Phe and the imidazole of His being in a pi-pi stacking interaction at a distance 3.75 A.

The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K1 = 4.5 x 10(-6) M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)n-C6H5 (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K1 = 9.1 x 10(-7) M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-beta OH (shifts in ppm, 0.09-0.17), D-Thr-beta CH (0.23-0.32), and D-Thr-gamma CH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-gamma CH3 and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/pi interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/pi interaction binds at the S2 or S1' site.

Thrombin receptor-derived peptide SFLLRNP (one-letter amino acid code) which corresponds to the N-terminal heptapeptide of tethered ligand is able to activate thrombin receptor and to stimulate the phosphoinositide (PI) turnover. The replacement of Phe-2 by Ala eliminated this activity completely, showing the crucial role of the Phe-phenyl group in receptor activation. It was found that substitution of para-fluorophenylalanine ((p-F)Phe) for Phe-2 enhanced several times the PI-turnover activity of SFLLRNP. This is the first example to date of a substitution with one order of magnitude greater increase in receptor activation. The Phe-2/Tyr substitution diminished the activity drasticaliy (almost 2% of SFLLRNP), indicating the importance of hydrophobicity of Phe2-phenyl. The Phe-2/Leu substitution, however, diminished also the activity (less than 2% of SFLLRNP). These results suggested that highly specific hydrophobic interaction exists between Phe-2 of the tethered ligand and its binding site in thrombin receptor.

When papain was reacted with l -1-acetyl-2,3-dihydropyrrolo[2,3- b ]indole-2-carboxamide at pH 8.0, inactivation occurred accompanied by modification of Cys-25 in the active site. Plots of pseudo first-order rate constants against the reagent concentrations yielded an anomalous sigmoidal curve, suggesting that papain responded to this reagent in an allosteric manner. This is supported by the fact that the presence of a moderate concentration (a twenty-fold molar excess) of N α -acetyl- l -tryptophanamide over papain accelerated the inactivation.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.

Phenylalanine at position 2 of thrombin receptor-tethered ligand peptide (SFLLRNP) is crucially important for the activation of thrombin receptor. Its substitution by para-fluorophenylalanine [(p-F)Phe] enhanced several times the activity in human epithelial-like SH-EP cells [Nose et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. To clarify the interaction mode of Phe-2-phenyl in receptor activation, a series of analogs having chemical modifications on the benzene ring of Phe-2 were synthesized and examined for their ability to induce the aggregation of human platelets. When the fluorine atom was placed at the meta or ortho position, the resulting analogs exhibited considerably diminished activity (about 10-20% of para-derivative), indicating that the substitution is allowed only at the para position. The derivative with pentafluorophenylalanine was totally devoid of activity. These results suggested that Phe-2 requires hydrogen atom(s) on the benzene ring presumably for interaction with the receptor. No activity enhancement was observed for analogs with para-chloro-, bromo-, or iodophenylalanine, indicating the importance of the high electronegativity of fluorine to intensify the dipole of CH(s) remaining in the Phe-2-benzene ring. Inactivity of analogs having para-iodophenylalanine and homophenylalanine indicated the importance of the size of para substituents, and the placement of hydroxyl, nitro, and trifluoromethyl groups at the para position led to no activity. The interaction of Phe-2 of SFLLRNP appeared to be structurally restricted to a limited space in the receptor. The results suggested the presence of face-to-edge pi-pi interaction based upon the CH/pi interaction between the ligand Phe-2-phenyl group and the receptor aromatic group.

Synthetic heptapeptide of the human thrombin receptor tethered-ligand peptide, H–Ser–Phe–Leu–Leu–Arg–Asn–Pro–NH2 (SFLLRNP), activates fully the thrombin receptor without thrombin. The functional role of Arg-5 was examined using a series of analogs having amino acid substitutions at position 5 in this assays was to assess the abilities to hydrolyze phosphoinositide in human neuroblastoma SH-EP cells and to aggregate the human platelet. The replacement of Arg-5 by Ala reduced the activity (9% activity of the parent peptide) in the PI-turnover assay, and abolished completely the platelet aggregation activity. SFLL/Lys/NP was also active, but moderately: 36% in PI-turnover and 12% in platelet aggregation. These results indicated that the electrostatic interaction of the Arg-guanidino group is important for a peptide to interact with the receptor. When citrulline or glutamine was placed at position 5 instead of arginine, the resulting SFLL/citrulline/NP and SFLL/Gln/NP were found to be potent in both assays. S...