Mark J. Dufton

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summaryα-neurotoxins from elapid snake venoms andα-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (anα-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smallerα-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snakeα-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to theα-conotoxins to show that the extent of the erabutoxin b/α-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namelyα-cobratoxin fromNaja naja siamensis and cytotoxin VII4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with theα-conotoxins decreased in the series erabutoxin b>α-cobratoxin>cytotoxin VII4, and this also relates the order of similarity to the pharmacological properties of theα-conotoxins.Theα-conotoxin-like area of the snakeα-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snakeα-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.

The structural evolution of cobra venom cytotoxins

SummaryIn order to analyze the evolutionary behavior of the cobra venom cytotoxins, their probable tertiary structure was predicted using computer graphics. The 41 amino acid sequences known show that the major evolutionary changes have taken place in two particularly exposed areas of the molecular surface. In each area, neighboring residue positions seem to have evolved interdependently, but there is no obvious interdependence between the two areas. Indeed, the relative evolution of these two areas prompts a subdivision of the sequence set into four groups. According to the known cytotoxin circular dichroism spectra, one of these four groups could be characterized by a difference in molecular secondary structure. Sine the two variable areas have functional associations, it is suggested that their evolution may be governed by a target with several similar binding sites.

Enkephalin-processing oligopeptidases in cobra venom : Inhibition by thiorphan and bestatin reveals co-operative actions

The peptidase inhibitors thiorphan and bestatin were tested for their ability to inhibit the actions of the oligopeptidases contained in the venom of the Taiwan cobra (Naja naja atra). With methionine enkephalin (TyrGlyGlyPheMet) as substrate, thiorphan was an effective inhibitor of cleavage of the GlyPhe peptide bond while bestatin inhibited cleavage of the TyrGly peptide bond. Thiorphan and bestatin also inhibited subsequent cleavage of the fragments GlyGlyPheMet and TyrGlyGly respectively. These inhibitors reveal an interplay between the venom oligopeptidases in which the enzymes provide additional substrates for each other following their initial competitive attack on the neuropeptide. A possible explanation is that the system is intended to ensure a steady release of Tyr, GlyGly and PheMet over time. Significantly, Tyr is the favoured substrate of the L-amino acid oxidase present in the venom, which rapidly transforms this aromatic amino acid into phenolic derivatives. The efficacies of these inhibitors also suggest that there are similarities between the venom oligopeptidases and the peptidases associated with the processing of enkephalin in its normal contexts.

Genetic code redundancy and the evolutionary stability of protein secondary structure.

The genetic code has an inherent bias towards some amino acids because of the variable number of synonymous codons per amino acid. The extent to which these biases are expressed in protein secondary structure is described through the analysis of the overall amino acid compositions of the alpha-helix, beta-sheet, beta-turn and random coil segments elucidated by X-ray crystallography. Given the concept of neutral mutation in proteins, the allocation of synonyms in the genetic code appears to protect secondary structures from amino acid changes and discourages the appearance of chemically complex residues. The level of protection is similar for each structural form, despite their clear preferences for certain amino acids. The organization of the code is therefore relevant to the preservation of conformation seen in the evolution of many protein families.

Metabolomic profiling of the synergistic effects of melittin in combination with cisplatin on ovarian cancer cells

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 μg/mL melittin + 2 μg/mL cisplatin) and A2780CR (combination treatment 2 μg/mL melittin + 10 μg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R2) and goodness of prediction (Q2), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 μg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Characteristics of the peptidase activity contained in Taiwan cobra (Naja naja atra) venom

Cobra venoms (Naja species) contain a little-understood peptidase activity which shows specificity towards small peptides containing glycine and non-polar aromatic/aliphatic residues. We have examined the ability of whole cobra venom to degrade several types of peptide with emphasis on the action of Taiwan cobra (Naja naja atra) venom on L-alanylglycylglycine and glycylglycyl-L-phenylalanine. These are competing substrates, and it proved possible to generate inhibitors of the degradation of glycylglycyl-L-phenylalanine by synthesizing L-alanylglycylglycine analogues in which the peptide bond between the second and third residues had been replaced by different linkages. These analogues were themselves resistant to hydrolysis. The peptidase activity can also be inhibited by bestatin, captopril and chloromethyl ketones. Kinetic analyses suggested that even the best substrate discovered was of poor efficacy, so the natural peptide substrate remains to be identified. In unsuccessful attempts to devise a reliable chromogenic assay, it was found that the venom had activity against N-blocked amino acid p-nitrophenol esters, but not against leucine p-nitroanilide or ester substrates for trypsin-like and chymotrypsin-like enzymes.

The drug discovery portal: a computational platform for identifying drug leads from academia.

The Drug Discovery Portal (DDP) is a research initiative based at the University of Strathclyde in Glasgow, Scotland. It was initiated in 2007 by a group of researchers with expertise in virtual screening. Academic research groups in the university working in drug discovery programmes estimated there was a historical collection of physical compounds going back 50 years that had never been adequately catalogued. This invaluable resource has been harnessed to form the basis of the DDP library, and has attracted a high-percentage uptake from the Universities and Research Groups internationally. Its unique attributes include the diversity of the academic database, sourced from synthetic, medicinal and phytochemists working an academic laboratories and the ability to link biologists with appropriate chemical expertise through a target-matching virtual screening approach, and has resulted in seven emerging hit development programmes between international contributors.

Conversion of tyrosine to phenolic derivatives by Taiwan cobra venom.

We have examined the ability of Taiwan cobra (Naja naja atra) venom to transform in vitro the amino acid tyrosine to phenolic oxidation products via 4-hydroxyphenylpyruvate. This amino acid can be released from neuropeptide substrates by oligopeptidases present in the venom. Using a variety of analytical techniques to probe a complicated series of reactions, we confirm that the L-amino acid oxidase present in the venom initially releases the keto form of 4-hydroxyphenylpyruvic acid and hydrogen peroxide after reacting with the tyrosine. Thereafter, there is evidence that a tautomerase in the venom promotes a partial conversion of the keto-form 4-hydroxyphenylpyruvic acid into an enol form. The enol is oxidised primarily to 4-hydroxybenzaldehyde and 4-hydroxyphenol (hydroquinone). The keto form is oxidised through to 4-hydroxyphenylacetic acid by the hydrogen peroxide co-released by the L-amino acid oxidase. The venom promotes both these spontaneous oxidation routes and also generates traces of other phenolics, some of which are as yet unidentified. We propose that reactions between the precursors of the major oxidation products may be responsible for generating unusual short-lived phenolics, possibly giving rise to special bioactivities that are relevant to venom action.

The action of Taiwan cobra venom on methionine enkephalin: A useful assay for oligopeptidase content

The pentapeptide methionine enkephalin is readily hydrolysed by the oligopeptidase activity contained in Taiwan cobra (Naja naja atra) venom. It is a significantly better substrate than the peptides previously used to identify the presence of this enzyme, but it retains many of the sequence characteristics shared by these other peptides. Analysis of the manner of hydrolysis by means of high-performance liquid chromatography and electrospray mass spectrometry revealed the simultaneous actions of at least two types of oligopeptidase on the neuropeptide, producing two routes of initial breakdown. By one route, an endopeptidase cleaved the Gly-Phe bond of enkephalin first to release Tyr-Gly-Gly and Phe-Met. By the other route, an aminopeptidase was able to release Tyr and Gly-Gly-Phe-Met by cleaving the Tyr-Gly bond first. From amongst the various peptide fragments produced, Tyr-Gly-Gly was subject to immediate aminopeptidase action to release Tyr and Gly-Gly. The free Tyr produced in these reactions was in turn quickly transformed by the L-amino acid oxidase in the venom. The kinetic qualities of the enkephalin hydrolysis, and the conversions of the fragments Tyr-Gly-Gly and Tyr, were measured. Methionine enkephalin has potential as a routine assay for venom oligopeptidases, either in testing the venoms from other species or in attempts to purify these enzymes. Moreover, the ease of hydrolysis of this bioactive peptide, coupled with the revelation of the other enzymic steps involving the fragments generated, may provide important clues as to the possible role of the oligopeptidases (and L-amino acid oxidase) in the venom.