Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer methylene blue.

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via π-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of (1)O(2) by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp(84) and Trp(279), present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of (1)O(2) on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

A combined spin trapping/EPR/mass spectrometry approach to study the formation of a cyclic peroxide by dienolic precursor autoxidation.

The spontaneous addition of air oxygen to a dienolic compound, yielding a cyclic peroxide, was followed by spin trapping (ST) combined with EPR spectroscopy and mass spectrometry (MS). Using two different nitrones, the ST/EPR study allowed the detection of the spin adduct of a radical intermediate, and the radical centre in the addend was identified after similar experiments performed with two different (13)C-labelled analogues of the substrate. The media were also submitted to electrospray ionisation, in both positive and negative modes, for structural characterisation of the spin adducts by tandem mass spectrometry. This allowed the structure of the hydroxylamine derivatives of the nitroxides formed to be identified. Following these results, a mechanism pathway was proposed for this autoxidation.

Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms.

Ants use venom for predation, defense, and communication; however, the molecular diversity, function, and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modeling, stability studies, and in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t 1/2 = 13 h) than its homodimers or monomers (t 1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability, a concept that is highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides.

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease.

Exposure to (bi)sulfite (HSO3-) and sulfite (SO32-) has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bi)sulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3-), peroxymonosulfate (-O3SOO.), and especially the sulfate (SO4. -) anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO), which has been shown to form protein radicals. Although formation of (bi)sulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bi)sulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bi)sulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bi)sulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX) pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bi)sulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bi)sulfite-derived protein radical formation and show the involvement of protein radicals in a mouse model of human lung disease.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening.

BACKGROUND: Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. METHODS: Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. RESULTS: Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. CONCLUSIONS: This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Site-specific detection of radicals on α-lactalbumin after a riboflavin-sensitized reaction, detected by immuno-spin trapping, ESR, and MS.

Free radicals and other oxidation products were characterized on α-lactalbumin with electron spin resonance (ESR), immuno-spin trapping, and mass spectrometry (MS) after riboflavin-mediated oxidation. Radicals were detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in immuno-spin trapping with both enzyme-linked immunosorbent assay (ELISA) and Western blotting and further characterized with mass spectrometry. A DMPO-trapped radical was identified at His68 and another at one of the tyrosine residues, Tyr50 or Tyr36, respectively, generated by a type II or I mechanism. Not all tyrosyl radicals were trapped, as the secondary oxidation product, 3,4-dihydroxyphenylalanine (DOPA), was detected by mass spectrometry at Tyr18 and Tyr50. A further oxidation of DOPA resulted in the DOPA o-semiquinone radical, which was characterized by ESR. Both surface exposure and the neighboring residues in the local environment of the tertiary structure of α-lactalbumin seem to play a role in the generation of DMPO trapped radicals and secondary oxidation products.

Sulfite-mediated oxidation of myeloperoxidase to a free radical: immuno-spin trapping detection in human neutrophils.

Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide ((•)SO3(-)), peroxymonosulfate ((-)O3SOO(•)), and sulfate (SO4(•-)) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.

The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.(AU)

Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Background Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia. Methods Herein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion. Results Using this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4- C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom. Conclusions This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study.

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((•)SO(3)(-)) and sulfate (SO(4)(•-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(•-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.

5-Hydroxy-2,2,6,6-tetramethyl-4-(2-methylprop-1-en-yl)cyclohex-4-ene-1,3-dione, a novel cheletropic trap for nitric oxide EPR detection.

The title compound behaves as an efficient cheletropic trap for both NO and NO(2) radicals in the presence of oxygen, yielding EPR observable nitroxide and alkoxynitroxide, respectively.

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: a combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study.

Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species.