C-terminal Amide Group Research Articles

In vitro and in vivo characterization of opioid activities of C-terminal esterified endomorphin-2 analogs

Previously, we have synthesized a series of endomorphin-2 (EM-2) analogs by the substitution of C-terminal amide group. In the present study, to further our knowledge of the influence of C-terminal esterified modification on the pharmacological activities, we investigated the in vitro and in vivo opioid activities of C-terminal esterified EM-2 analogs 1– 3. Our results showed that the ED 50 values on contractions of the longitudinal muscle of distal colon induced by analogs 1– 3 were about 1.5-fold higher, 2- and 8-fold lower than EM-2, respectively. In addition, intravenous (i.v.) injections of analogs 1 and 2 dose-dependently decreased the system arterial pressure (SAP) and heart rate (HR) in anesthetized rats, but the degree of the hypotension and bradycardia was significantly smaller relative to the parent. Moreover, analog 3 was almost ineffective. Nevertheless, all these analogs produced potent antinociception in the tail-flick test after intracerebroventricular (i.c.v.) injection, and this antinociception was inhibited by naloxone, indicating an opioid mechanism. In summary, these results gave the evidence that the conversion of C-terminal amide to esterified modification may play an important role in the regulation of opioid affinities and pharmacological activities.

Involvement of Metals in Enzymatic and Nonenzymatic Decomposition of C-Terminal α-Hydroxyglycine to Amide: An Implication for the Catalytic Role of Enzyme-Bound Zinc in the Peptidylamidoglycolate Lyase Reaction

The peptide C-terminal amide group essential for the full biological activity of many peptide hormones is produced by consecutive actions of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-alpha-hydroxyglycine to an amidated peptide and glyoxylate. PAL contains 1 mol of zinc, but its role, catalytic or structural, has not yet been clarified. In this study, we found that a series of transition metals, Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), catalyze the nonenzymatic decomposition of the hydroxyglycine intermediate in a concentration-dependent manner. The second-order rate constant of the metal catalysis increased with elevation of pH, indicating that the hydrated metal acts as a general base. Extensive removal of the enzyme-bound metals remarkably diminished the PAL activity; k(cat) of the metal-depleted enzyme retaining 0.1 mol of zinc decreased to 3.2 s(-1) from 25.7 s(-1) of the wild-type enzyme. Among a series of divalent metals tested, Zn(2+), Co(2+), and Cd(2+) could fully restore the PAL activity of the metal-depleted enzyme. Especially, Zn substitution reproduced the steady-state parameters of the wild-type enzyme. On the other hand, Co and Cd substitution largely altered the kinetic parameters; the k(cat) increased 3- and 5-fold and the K(m) for the substrate increased 2.5- and 4-fold, respectively. These observations support that the enzyme-bound zinc plays a catalytic role, rather than a structural role, in the PAL reaction through the action of zinc-bound water as a general base.

Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

Structure- and Substrate-based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.

Structure–activity study of endomorphin-2 analogs with C-terminal modifications by NMR spectroscopy and molecular modeling

Endomorphin-2 (EM-2) is a putative endogenous μ-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substitution of the C-terminal amide group with –NHNH 2, –NHCH 3, –N(CH 3) 2, –OCH 3, –OCH 2CH 3, –OC(CH 3) 3, and –CH 2–OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4, and 7) or decreased (analogs 2, 3, 5, and 6) μ affinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K i (μ) values was 2 > 3; for the C-terminal esterified analogs 4– 6, the potency order with the K i (μ) values was 4 > 5 > 6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D 1H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analog conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.

Small peptides mimicking substance P (1–7) and encompassing a C-terminal amide functionality

Some of the biological effects demonstrated after administration of substance P (SP) in vivo can indirectly be attributed to the fragmentation of the undecapeptide to its N-terminal bioactive fragment SP(1-7). This heptapeptide (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is a major bioactive metabolite from SP that frequently exerts similar biological effects as the parent peptide but also, in several cases, completely opposite actions. Specific binding sites for the heptapeptide SP(1-7) that are separate from the SP preferred NK receptors have been identified. In this study we demonstrate that (a) the C-terminal part of the SP metabolite SP(1-7) is most important for binding as deduced from an Ala scan and that a replacement of Phe(7) for Ala is deleterious, (b) truncation of the N-terminal amino acid residues of SP(1-7) delivers peptides with retained binding activity, although with somewhat lower binding affinities than SP(1-7) and (c) a C-terminal amide group as a replacement for the terminal carboxy group of SP(1-7) and for all of the truncated ligands synthesized affords approximately 5-10-fold improvements of the binding affinities.

Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L–H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm 2/h) measured after a 60-min incubation with 100 μmol/l of peptide in the lumen bathing fluid. The rates of flux, L–H and H–L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G 7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.

N-Methyldehydroamino acids promote a configuration cis of N-methylamide bond

Dehydroamino acids with a methylated N-terminal tertiary amide bond occur in natural small cyclic peptide toxins. To investigate their conformational preferences a systematic theoretical analysis was performed on N′-methylamides of N-acetyl- N-methyldehydroamino acids (Ac-Δ(Me)Xaa-NHMe, where Xaa = ( Z)-Abu, ( E)-Abu, Val, ( Z)-Phe, and ( E)-Phe) considering the configuration trans and cis of the tertiary amide bond. The ϕ, ψ potential energy surfaces were calculated at the B3LYP/6-31+G ∗∗//HF/3-21G level with inclusion of the solvent (water) effect (SCRF method). The conformers localised were fully optimised at the B3LYP/6-31+G ∗∗ in vacuo. The accessible areas of the potential energy surfaces; the number of conformers and the stabilising internal forces were compared for all the studied molecules. The main feature of the studied N-methyldehydroamino acids is their considerable tendency to adopt the configuration cis for the N-terminal tertiary amide bond. It results from the specific ability of these dehydroamino acids to be able to gain stability from the π-electron conjugation between the C α C β double bond and the neighbouring C-terminal amide group. This stabilising force concomitant with the short N H⋯N hydrogen bond makes the conformer cis C7 eq ( φ, ψ ∼ −105°, 8°) the lowest in energy. The data presented hereinto indicates that N-methyldehydroamino acids can potentially be new promoters of trans–cis isomerisation of the amide bond.

The conformation cis of N-acetyl-N-methyl-α,β-dehydroalanine N′-methylamide and saturated analogues

A series of three homologous amino acids derivatives: N-acetyl-N-methyl-α,β–dehydroalanine N′-methylamide (1), N-acetyl-N-methyl-L-alanine N′-methylamide (2), and N-acetyl-N-methyl-DL-alanine N′-methylamide have been synthesised. The racemic species undergoes spontaneous separation into L and D-enantiomers. From these two chiral forms, the structure of L-enantiomer (3) was analysed. The molecules of 1 – 3 adopt the cis arrangement of the N-terminal amide bond. The molecular conformations are similar for 1 (φ, ψ = 94.6(1)°, −1.7(1)°) and 3 (φ, ψ = 111.5(1)°, −23.8(1)°), and also 2 (φ, ψ = −114.8(2)°, 29.5(2)°), if inversion through the chiral C2 carbon is considered. They are stabilised by intramolecular N—H…N hydrogen bond. In the case of 1, the π—electron conjugation resulting from the planar arrangement of the C2—C21 double bond and C-terminal amide group is another stabilising force of this conformation. Thus, the N-methyldehydroalanine residue should promotes trans-cis isomerisation to a greater extend than the saturated analogue. The compounds 1 – 3 reveal similar associative pattern and form centrosymmetric dimers linked by the intermolecular N—H…O hydrogen bonds.

Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae

The degradation of synthetic cydiastatin 4 (ARPYSFGL-amide) and cydiastatin 4 analogues cydiastatin 4α (PPPPPARPYSFGL-amide) and cydiastatin 4β (PPPPPARPYSF[Acpc]L-amide) by enzymes associated with the midgut and/or haemolymph of the tobacco hawkmoth moth, Manduca sexta was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) combined with matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Cydiastatin 4 had an estimated half-life of c. 16.5 min when incubated with midgut tissue in vitro and c. 2.5 min with midgut lumen contents. Two degradation products were identified; cydiastatin 1–6, due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 2–8, due to cleavage of the N-terminal A residue. Both cydiastatin 4α and cydiastatin 4β had increased stability to gut and haemolymph enzymes, and full biological activity, but reduced potency compared to cydiastatin 4 when assayed on foregut peristalsis. The P-extended N-terminus of both analogues prevented hydrolysis by aminopeptidases and the replacement of the susceptible G residue with cyclopropylalanine ([Acpc]) counteracted carboxypeptidase activity. However, both analogues were susceptible to amidase-like activity giving an increase in one mass unit presumably due to the conversion of the C-terminal amide group to the free carboxylic acid. No metabolism of cydiastatin 4β occurred when incubated with larval M. sexta haemolymph over a 90 min period.

Novel 1D Hydrophobic Pore Formation by C-Terminal Amide-controlled Self-assembly of Leu–Leu–Leu–NH2 in Crystal

Abstract A unique hydrophobic pore is generated by the self-assembly of Leu–Leu–Leu–NH2 (1) in the crystal structure, in which the C-terminal amide groups induce the cylindrical arrangement of Leu side chains by intermolecular NH···O hydrogen bonds. This body-centered cubic-type pore provides a structural basis for devising the material to selectively extract low-molecular hydrophobic molecule such as methane.

Methyl Esterification Assisted MALDI FTMS Characterization of the Orcokinin Neuropeptide Family

Methyl esterification of a peptide converts carboxylic acids, such as those present on the side chains of aspartic (D) and glutamic acid (E) as well as the free carboxyl terminus, to their corresponding methyl esters. This method has been applied to peptide and protein quantitation, de novo sequencing, and reduction of nonspecific binding in immobilized metal affinity chromatography for enrichment of phosphorylated peptides. In this study, we investigate the application of this derivatization reaction to the identification and characterization of the orcokinin neuropeptide family by screening and localizing the acidic side chains in peptides. The methyl esterification reaction drastically improves the fragmentation efficiency of modified orcokinins due to blockage of the aspartate selective cleavage pathway of the native orcokinin peptides. With the improved sustained off-resonance irradiation-collisional-induced dissociation spectra, the number and the locations of D and E residues are easily deduced. In addition, a side reaction that occurs at the carboxamide group of asparagine (N) is studied. The deamidation followed by subsequent methyl esterification reaction mechanism is proposed based on the study of an isotope-labeled standard N*FDEIDR. Reaction kinetics is studied by elevating the temperature from room temperature to 37 degrees C. The deamidation-methyl esterification products are greatly enhanced with elevated reaction temperature. Furthermore, we also explore the utility of this side reaction for rapid screening and characterization of C-terminally amidated neuropeptides. This derivatization reaction is applied to both in situ direct tissue neuropeptide analysis and the analysis of HPLC fractions from the separation of complex neuronal tissue extracts. Overall, this study reports a simple and effective method for profiling and localizing acidic amino acid residues (D/E), amide-containing residues (N/Q), and the C-terminal amide group in a peptide.

The β-turn scaffold of tripeptide containing an azaphenylalanine residue

The conformational preferences of azaphenylalanine-containing peptide were investigated using a model compound, Ac-azaPhe-NHMe with ab initio method at the HF/3-21G and HF/6-31G ⁎ levels, and the seven minimum energy conformations with trans orientation of acetyl group and the 4 minimum energy conformations with cis orientation of acetyl group were found at the HF/6-31G ⁎ level if their mirror images were not considered. An average backbone dihedral angle of the 11 minimum energy conformations is ϕ = ± 91° ± 24°, ψ = ± 18° ± 10° (or ± 169° ± 8°), corresponding to the i + 2 position of β-turn ( δ R) or polyproline II ( β P) structure, respectively. The χ 1 angle in the aromatic side chain of azaPhe residue adopts preferentially between ± 60° and ± 130°, which reflect a steric hindrance between the N-terminal carbonyl group or the C-terminal amide group and the aromatic side chain with respect to the configuration of the acetyl group. These conformational preferences of Ac-azaPhe-NHMe predicted theoretically were compared with those of For-Phe-NHMe to characterize the structural role of azaPhe residue. Four tripeptides containing azaPhe residue, Boc-Xaa-azaPhe-Ala-OMe [Xaa = Gly( 1), Ala( 2), Phe( 3), Asn( 4)] were designed and synthesized to verify whether the backbone torsion angles of azaPhe reside are still the same as compared with theoretical conformations and how the preceding amino acids of azaPhe residue perturb the β-turn skeleton in solution. The solution conformations of these tripeptide models containing azaPhe residue were determined in CDCl 3 and DMSO solvents using NMR and molecular modeling techniques. The characteristic NOE patterns, the temperature coefficients of amide protons and small solvent accessibility for the azapeptides 1–4 reveal to adopt the β-turn structure. The structures of azapeptides containing azaPhe residue from a restrained molecular dynamics simulation indicated that average dihedral angles [( ϕ 1, ψ 1), ( ϕ 2, ψ 2)] of Xaa-azaPhe fragment in azapeptide, Boc-Xaa-azaPhe-Ala-OMe were [(− 68°, 135°), (116°, − 1°)], and this implies that the intercalation of an azaPhe residue in tripeptide induces the βII-turn conformation, and the volume change of a preceding amino acid of azaPhe residue in tripeptides would not perturb seriously the backbone dihedral angle of β-turn conformation. We believe such information could be critical in designing useful molecules containing azaPhe residue for drug discovery and peptide engineering.

Conformational studies into N-methylation of alanine diamide models: A quantitative approach

A systematic theoretical analysis was performed on N-acetyl- l-alanine N′-methylamide (Ac- l-Ala-NHMe) and the analogues methylated on the N-terminus (Ac- l-(Me)Ala-NHMe), C-terminus (Ac- l-Ala-NMe 2), and both N/C-termini (Ac- l-(Me)Ala-NMe 2), to evaluate the influence of methylation of the amide group on the conformational properties of the affected residues. The ϕ, ψ potential energy surfaces were calculated at the B3LYP/6-31+G**//HF/3-21G level of theory with inclusion of the solvent (water) effect (SCRF method). The conformers localised were fully optimised at the B3LYP/6-31+G** in vacuo. The accessible areas of the potential energy surfaces; the number of conformers and the stabilising internal forces were compared for all the studied molecules. For comparison, data drawn from the Cambridge Crystallographic Data Centre has been presented. The conformational freedom of residue methylated either on the N-terminal ( trans and cis) or C-terminal amide group is reduced, in contrast to residue methylated on both the N/C-terminal amide groups. For all the studied molecules the lowest energy conformers have the methylated amide bond in the trans configuration. N H⋯O hydrogen bonds stabilise the lowest conformers of Ac- l-Ala-NMe 2 and all the conformers of trans-Ac- l-(Me)Ala-NHMe, but not those of cis-Ac- l-(Me)Ala-NHMe where N H⋯N operates. For all the methylated residues studied, the weaker C H⋯O hydrogen bonds and the carbonyl C O▶⋯◀C O attractions seem to play an important role.

Identification of a 4-mer Peptide Inhibitor that Effectively Blocks the Polymerization of Pathogenic Z α1-Antitrypsin

alpha(1)-Antitrypsin (AT) is a major proteinase inhibitor within the lung. The Z variant of AT (E342K) polymerizes within the liver and lung, resulting in hepatic aggregation of AT and tissue deficiency, predisposing to early onset of cirrhosis and emphysema, respectively. Polymerization of the aberrant protein can be prevented in vitro by specific peptides such as FLEAIG. This peptide serves as a lead molecule to design a shorter peptide that may be effective as a therapeutic agent. In this study we employed a systematic chemical approach using alanine scanning of Ac-FLEAIG-OH and subsequent peptide shortening to study the binding of shorter peptides to Z-AT. While two additional 6-mer peptides Ac-FLAAIG-OH and Ac-FLEAAG-OH were found to bind to Z-AT, their daughter peptides Ac-FLEAA-NH(2) and Ac-FLAA-NH(2) also bound avidly to Z-AT and prevented polymerization of the protein. Further comparative studies revealed that the binding of Ac-FLAA-NH(2) was more specific for Z-AT. The peptide-AT complex formation was enhanced by the presence of C-terminal amide group on the peptide, and circular dichroism analysis demonstrated that a random coil rather than a beta-helical conformation favored binding of the peptide to AT. In summary, this study has identified novel small peptides that inhibit Z-AT polymerization, and are a significant advance towards the treatment of Z-AT-related cirrhosis and emphysema.

Structural function of C‐terminal amidation of endomorphin

To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)), an endogenous micro-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D (1)H-NMR measurement and molecular modelling calculation. Both peptides were in equilibrium between the cis and trans rotamers around the Tyr--Pro w bond with population ratios of 1 : 1 to 1 : 2 in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans/cis rotamer ratio of 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure. Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans/cis rotamer form. On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH. Most of these folded conformers took an F1-type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH(3) (+)...COO(-)(Phe4), observed in its crystal structure. These results show that the substitution of a carboxyl group for the C-terminal amide group makes the peptide structure more flexible and leads to the ensemble of folded and open conformers. The conformational requirement of EM2 for binding to the micro-opioid receptor and the structural function of the C-terminal amide group are discussed on the basis of the present conformational features of EM2 and EM2OH and a possible model for binding to the micro-opioid receptor, constructed from the template structure of rhodopsin.

Phenethyl amides as novel noncovalent inhibitors of hepatitis C virus NS3/4A protease: discovery, initial SAR, and molecular modeling.

The discovery of novel, reversible and competitive tripeptide inhibitors of the Hepatitis C virus NS3/4A serine protease is described. These inhibitors are characterized by the presence of a C-terminal phenethyl amide group, which extends into the prime side of the enzyme. Initial SAR together with molecular modeling and data from site-directed mutagenesis suggest an interaction of the phenethyl amide group with Lys-136.

Neuropeptides in cnidarians

Cnidarians are the lowest animal group having a nervous system. In the primitive nervous systems of cnidarians, peptides play important roles as neurotransmitters or neurohormones. So far, we have isolated and sequenced about 35 neuropeptides from different cnidarian classes (Hydrozoa, Scyphozoa, Anthozoa). All these neuropeptides have a C-terminal amide group, which protects against C-terminal degradation, but which also is important for receptor recognition. Also the N-termini of the cnidarian neuropeptides often contain different kinds of protecting groups (such as <Glu residues, L-3-phenyllactyl groups, and X-Pro or X-Pro-Pro sequences). Cnidarian neuropeptides are located in neuronal dense-core vesicles and are synthesized as preprohormones, which can contain up to 41 copies of a neuro peptide sequence. From Hydra, six different neuropeptide genes have been cloned so far. Each gene is expressed by a specific population of neurons, but in two instances coexpression of neuropeptide genes has been found. We have also cloned some of the cnidarian prohormone processing enzymes, among them the enzymes necessary for C-terminal amidation. These enzymes are closely related to their mammalian counterparts. All these data show that the primitive nervous systems of cnidarians have already acquired some of the sophisticated principles that we know from higher animals.

Structural requirements of nociceptin antagonist Ac‐RYYRIK‐NH2 for receptor binding

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 mu 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.

Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein.

Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.