Structures of neuropeptide gamma from goldfish and mammalian neuropeptide gamma, as determined by 1H NMR spectroscopy.

Neuropeptide gamma belongs to tachykinin families which have a common C-terminal amino acid sequence (Phe-X-Leu-Met-NH2) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide gamma (NPgamma) from gold fish (G-NPgamma) and mammalian NPgamma (M-NPgamma), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G-NPgamma has a alpha-helical conformation in the region of His12-Met21 and a short helix in the N-terminal region, and has a beta-turn from Arg9 to Arg11 in between. In aqueous TFE solution, M-NPgamma also has alpha-helical conformations both in the C-terminal region and the N-terminal region and a beta-turn from His9 to Arg11 in between. In SDS micelle, the structure of G-NPgamma contains a stable alpha-helix from His12 to Met21 and a beta-turn from Arg9 to Arg11, while M-NPgamma has a short helix from Ser16 to Met21. The region from His12 to Met21 corresponds to the amino acid sequence of neurokinin A. Neuropeptide gamma may act as a precursor of neurokinin A and the post-translational processing of this peptide involves the enzymatic attack of the basic beta-turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G-NPgamma induces the biological actions corresponding to those of substance P in mammalian system. The structures of G-NPgamma and M-NPgamma contain alpha-helical structures at the C-terminus and this helix seems to promote the affinity for NK1 and/or NK2 receptor.     

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.     

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.     

Regional distribution of neuropeptide gamma and other tachykinin peptides derived from the substance P gene in the rat.

Substance P (SP) and multiple neurokinin A (NKA)-related peptides can be derived from alpha-, beta- and/or gamma-preprotachykinin (PPT) mRNAs. In this study, the relative concentrations of the tachykinin peptides derived from the SP gene in rat brain, duodenum, jejunum, submandibular gland, parotid gland, urinary bladder and vas deferens was determined using high-performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). In all tissues, SP levels were the highest. The relative abundance of NKA-related peptides was NKA greater than neuropeptide gamma (NP gamma) = neuropeptide K (NPK) greater than NKA(3-10). These results demonstrate that multiple tachykinin peptides are present in tissues where the SP gene is expressed, and that the NKA portion of the beta- and gamma-PPT precursors can be differentially processed posttranslationally in rat tissues into NKA, NPK, NP gamma and/or NKA(3-10).     

Structure-activity relationship study of anoplin.

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.     

Structure-activity relationship of human calcitonin-gene-related peptide.

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each others' receptors. The present study was undertaken to examine the structural prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxyamidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, alpha and beta, were equipotent, and were both at least 1000-fold were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxyamidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotryptic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala1-Lys35): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results suggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the alpha and beta forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.     

Structure-activity relationship of a series of diaminoalkyl substituted benzimidazole as neuropeptide Y Y1 receptor antagonists

A series of benzimidazoles (4) was synthesized and evaluated in vitro as potent and selective NPY Y1 receptor antagonists. Substitution of the piperidine nitrogen of 4 with appropriate R groups resulted in compounds with more than 80-fold higher affinity at the Y receptor compared to the parent compound 5 (R = H). The most potent benzimidazole in this series was 21 (Ki = 0.052 nM).     

Structure-activity relationship studies on a novel class of antiproliferative agents derived from Lavendustin A. Part I: Ring A modifications

The potent antiproliferative agent SDZ LAP 977, which has shown efficacy in a clinical proof of concept study in actinic keratosis patients, has been previously demonstrated to block the cell cycle in mitosis. In the present study, we further explored the mode of action: SDZ LAP 977 binds to the "colchicine binding site" on tubulin and, thus, inhibits tubulin polymerization in vitro. Moreover, we established structure-activity relationships for the effect of modifications in the 2,5-dimethoxyphenyl moiety ("ring A") of the molecule on in vitro antiproliferative activity.     

Structure-activity relationship of brassinosteroids

The plant growth-promoting activities of brassinolide and brassinosteroids with different side chains were investigated by means of the Raphanus an     

Structure-activity relationship of a hemagglutinin from Moringa oleifera seeds

The hemagglutinin from the seeds of Moringa oleifera (MoL) agglutinates human as well as rabbit erythrocytes; the affinity for the latter is almost 250 times more than that for the former. MoL was inhibited by glycoproteins namely thyroglobulin, fetuin and holotransferin indicating the complex sugar specificity of the lectin. The protein is a homodimer with molecular mass of 14kDa, subunits (7.1kDa) linked by the disulfide bond(s). The secondary structure elements of MoL are alpha-helix, 28%; beta-sheet, 23%; turn 20% and unordered 28%. While the activity and secondary structure were not affected at extreme pH and high temperature, they were drastically affected in presence of dithiothreitol at and above pH 7.0, indicating that disulfide linkages hold the active conformation of the protein.     

Structure-activity relationships of neuropeptide FF and related peptidic and non-peptidic derivatives

Neuropeptide FF, a member of the RFamide family of peptides, has demonstrated an interesting array of pharmacological effects. To date however, little information has been obtained as to the exact pharmacological roles of the individual NPFF1 and NPFF2 receptors. Through peptide analogs of NPFF and related peptides, the essential pharmacophore has emerged somewhat. Yet, the field is lacking small molecule ligands selective for each receptor. This review of the structure-activity relationships of the reported NPFF peptide analogs and some non-selective small molecule ligands highlights the current understanding of the pharmacophoric elements required for affinity and activity at the NPFF receptors. The lack of mutagenesis data on the receptor as well as a crystal structure has also hindered the understanding of ligand recognition at the receptor level. If the targets can be further investigated as to their requirements for ligand recognition, the successful development of highly selective ligands should follow.     

Structure-activity relationship study on human urotensin II.

The vasoactive cyclic undecapeptide urotensin-II (U-II) has been identified as an endogenous ligand for the G-protein coupled receptor now referred to as the UT receptor. The U-II/UT receptor system might be relevant for cardiovascular functions. A structure-activity study of human U-II investigating 31 peptides in the rat aorta bioassay is reported. Ala- and D-scan investigations indicated that the sequence Phe6-Trp7-Lys8-Tyr9 is essential for biological activity and that Lys8 and Tyr9 are particularly important. These two residues were substituted with a series of coded and non-coded amino acids. These studies demonstrated that the positive charge of the primary aliphatic amine at position 8 and its relative spatial orientation is crucial for both receptor occupation and activation, while the only chemical requirement at position 9 is the presence of an aromatic moiety. Moreover, this study led to the identification of UT receptor partial agonists (compounds 23 and 24) which can be used as chemical templates for further investigations aimed at the identification of selective antagonists.     

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.     

Structure-activity relationship in buffalo spleen cathepsin B.

Effect of pH, urea, and guanidine hydrochloride on the activity and structure of buffalo spleen cathepsin B was investigated. At alkaline pH, there was an irreversible loss of the structure as well as the activity of the buffalo enzyme. At acidic pH, however, the inactivation of the enzyme was reversible. The enzyme reversibly lost most of its activity at denaturant concentrations which did not cause a significant change in its secondary structure. The inactivation could be attributed to minor perturbations in the environment of the amino acid residue(s) at and/or around the active site of the enzyme. High urea/guanidine hydrochloride concentrations leading to the structural changes in cathepsin B made the inactivation process irreversible.     

Structure-activity analysis of an antimicrobial peptide derived from bovine apolipoprotein A-II

We previously showed that bovine apolipoprotein A-II (apoA-II) has antimicrobial activity against Escherichia coli in PBS, and its C-terminal residues 49-76 are responsible for the activity using synthetic peptides. In order to understand the structural requirements of peptide 49-76 for the antimicrobial activity, the N- or C-terminus was truncated and then the charged (Lys or Asp) or Ser residues were replaced by Ala. Deletion of the first or last three amino acids and replacement of Lys-54/55 or 71/72 by Ala caused a substantial decreases in alpha-helical content in 50% TFE, showing the possible presence of helices in N- and C-terminal regions, respectively. The anti-Escherichia coli activity of the peptide correlated with its liposome-binding activity. Replacement of Lys-54/55 or 71/72 by Ala resulted in an almost complete loss of anti-E. coli activity with a substantial decrease in liposome-binding activity. Moreover, deletion of the last three amino acids caused a reduction to 1/17 of the original anti-E. coli activity with a moderate decrease in liposome-binding activity. In contrast, replacement of Ser-65/66, Asp-59, or Asp-69 by Ala hardly affected the anti-E. coli activity. These findings suggest that Lys-54/55 and Lys-71/72 on the putative helices are critical for antimicrobial activity, and the C-terminal 3 amino acids are important for the structural integrity of the C-terminal region for effective antimicrobial activity.     

Structure-activity study at positions 3 and 4 of human neuropeptide S

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe(2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg(3) and Asn(4) that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO2) or Arg(Me)2 but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn(4) side chain for NPS bioactivity.     

Structure-activity relationship of antibacterial chalcones

The antibacterial activity of 31 chalcones was tested against bacterial strains, Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923. Some of the tested chalcones showed fair to significant activity against Gram-positive bacteria. By comparison of the results obtained, the antibacterial activity can be related to features such as the presence of a C-4 hydroxyl group, a C-4' oxygenated substituent or a C-3' isoprenoid side chain, while the C-2' hydroxyl group might have importance for the stability of the molecule. The inhibitory effect of chalcones on human pathogenic microorganisms can be correlated with the substitution patterns of the aromatics rings.