Complement Activation by Animal Venoms

Abstract

During activation of complement (C) system, C4b and C3b attach covalently to targets such as cell membranes. Deposited C4b serve both as the focus for the assembly of the C3 convertases and as ligands for C3b receptors or inactivators. C4a, C3a and C5a, mediators of the early events of inflammation, are released into the fluid phase while C5b serves to organize the cytolytical complex C5b-C9. Among the Arthropoda the venom from some spiders (Loxosceles) contain activators of the mammalian C system, although the exact mechanism and point of the activation cascade in where they act were not yet elucidated. In the insect venoms as honeybee, yellow jacket, yellow hornet, white-facet hornet, caterpillars and wasp there are some components capable of reducing total haemolytic and serum levels. Interestingly, mellitin, a soluble haemolytic peptide present in bee venom shares structural homology with human C9. Scorpion venom apparently are not active on the C system. Snakes, belonging to ELAPIDAE, CROTALIDAE and VIPERIDAE families produce venoms containing components with a vast range of action on mammalian C system, some acting by cleaving directly a particular component, while others interact with C components, the resulting complex being able as an amplificator, to activate part of the C cascade. Soluble mediators of inflammation, cell bound fragments of C components recognizable by specific receptors disposed on immune or inflammatory cells, or the formation of multimolecular complex potentially capable of damage host cells are the presumable consequences of C activation by animal venoms.     

Activation of carboplatin by carbonate

Carboplatin, [Pt(NH3)2(CBDCA-O,O')], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, is in wide clinical use for the treatment of ovarian, lung, and other types of cancer. Because carboplatin is relatively unreactive toward nucleophiles, an important question concerning the drug is the mechanism by which it is activated in vivo. Using [1H,15N] heteronuclear single quantum coherance spectroscopy (HSQC) NMR and 15N-labeled carboplatin, we show that carboplatin reacts with carbonate ion in carbonate buffer to produce ring-opened products, the nature of which depends on the pH of the medium. The assignment of HSQC NMR resonances was facilitated by studying the reaction of carboplatin in strong acid, which also produces a ring-opened product. The HSQC NMR spectra and UV-visible difference spectra show that reaction of carboplatin with carbonate at pH > 8.6 produces mainly cis-[Pt(NH3)2(CO3(-2))(CBDCA-O)]-2, 5, which contains the mono-dentate CBDCA ligand and mono-dentate carbonate. At pH 6.7, the primary product is the corresponding bicarbonato complex, which may be in equilibrium with its decarboxylated hydroxo analogue. The UV-visible absorption data indicate that the pKb for the protonation of 5 is approximately 8.6. Thus, the reaction of carboplatin with carbonate produces a mixture of ring-opened species that are anions at physiological pH. HSQC NMR studies on 15N-labeled carboplatin in RPMI culture media containing 10% fetal bovine serum with and without added carbonate suggest that carbonate is the attacking nucleophile in culture media. However, because the rate of reaction of carbonate with carboplatin at physiological pH is small, NMR peaks for ring-opened carboplatin were not detected with HSQC NMR. The rate of disappearance of carboplatin in culture medium containing 9 x 10(8) Jurkat cells is essentially the same as that in carbonate buffer, indicating that the ring-opening reaction is not affected by the presence of cells. This work shows that carbonate at concentrations found in culture media, blood, and the cytosol readily displaces one arm of the CBDCA ligand of carboplatin to give a ring-opened product, which at physiological pH is a mixture of anions. These ring-opened species may be important in the uptake, antitumor properties, and toxicity of carboplatin.     

Activation of acetylcholinesterase by vanadate

Vanadate activated acetylcholinesterase in rat ventricular strips. This effect of probably due to the action of vanadate (V) form rather than to vanadyl (IV). Vanadate also activated purified acetylcholinesterase from the electric eel and also erythrocytes, suggesting a direct effect upon this enzyme system. It is possible that the increase in the activity of acetylcholinesterase produced by vanadate may contribute, at least in part, to the positive inotropic effect of vanadate.     

Mechanical Activation of Pharmaceutical Systems

Mechanical activation has become a phenomenon of general significance in pharmaceutics. This report describes the extent of activation induced by relevant processes. With the use of the Eyring equation the transformation of structurally stored energy into chemical energy and the implied free enthalpy as well as the excess free enthalpy (activity) were evaluated from the rate of an indicator reaction. A comparison was made between different kinds of milling and tabletting with respect to pharmaceutical conditions. An optimization of these processes was derived.     

Activation mechanisms to chemical toxicity

The pathobiology of chemical toxicity may involve acute lethal injury (necrosis), autoxidative injury (oxygen toxicity), immunological injury (neoantigen formation), and malignancy. Toxic chemicals may be activated by reduction, conjugation, radical formation, or oxidation. Oxidative activation may be effected by cytochromes P-450/P-448, flavoprotein monooxygenases, or hydroxyl radicals. The alternative pathways of oxidative metabolism of toxic chemicals, namely, detoxication and activation, are catalysed by the phenobarbital-induced cytochromes P-450 and by the 3-methylcholanthrene-induced cytochromes P-448 respectively. Oxidative metabolism by cytochromes P-450 is followed by conjugation and detoxication, whereas oxidative metabolism by cytochromes P-448 yields reactive intermediates which are not readily conjugated, and thus react with vital intracellular macromolecules, resulting in necrosis, redox cycling and oxygen radical formation, neoantigen production, and mutations. The molecular dimensions of specific substrates, inhibitors and inducers of the PB-cytochromes P-450 indicate that they are globular and are different from those of the cytochromes P-448 which are planar, suggesting that the active sites of the two families of enzymes are different. Oxidative metabolism of planar substrates of cytochromes P-448 results in conformationally-hindered oxygenations, which inhibits subsequent conjugations. Cytochrome P-448 activity may be quantified by the oxidative deethylation of 7-ethoxyresorufin which, unlike benzo(a)pyrene hydroxylation (AHH) is a specific reaction for this family of enzymes. Oxidative metabolism of chemicals varies inversely with the body weight of the animal species, so that chemical toxicity involving oxidative activation, redox cycling, and reactive oxygen is greater the smaller the animal species.Dedicated to Professor Dr. Herbert Remmer on the occasion of his 65th birthday     

Activation mechanisms of nucleoside phosphoramidate prodrugs

A series of thymidine and tetrahydrofurfuryl phosphoramidates bearing haloethyl or piperidyl substituents was synthesized and used to investigate the activation mechanisms of nucleoside phosphoramidate prodrugs. Structure assignments for the tetrahydrofurfuryl reaction products were confirmed by comparison to authentic samples. Structural assignments for thymidine phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl products. Generation of the phosphoramidate anion leads to cyclization and subsequent nucleophilic attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the observed products. Nucleophilic attack by water at carbon and phosphorus occurs without selectivity, supporting a mechanism of action of haloethylamine nucleoside prodrugs involving intracellular release of the nucleotide. Activation of the benzotriazolyl piperidyl phosphoramidates is followed by P-N bond hydrolysis; this reaction is subject to specific acid catalysis and to nucleophilic catalysis by 1-hydroxybenzotriazole. These results suggest that the mechanism of action of the piperidyl nucleoside phosphoramidates involves the intracellular release of the active nucleotide following P-N bond cleavage, presumably by the action of an endogenous phosphoramidase.     

Activation of insulin receptors by lagerstroemin

Lagerstroemin, an ellagitannin isolated from the leaves of Lagerstroemia speciosa (L.) Pers. (Lythraceae), was examined for its biological activities. In rat adipocytes, the compound increased the rate of glucose uptake and decreased the isoproterenol-induced glycerol release. In Chinese hamster ovary cells expressing human insulin receptors, it increased the Erk activity. These insulin-like actions were accompanied by the increased tyrosine-phosphorylation of the beta-subunit of the insulin receptors. Tryptic digestion of the extracellular sites of the insulin receptors markedly increased the effective concentrations of insulin without changing those of lagerstroemin. Thus lagerstroemin was considered to cause its insulin-like actions by a mechanism different from that employed by insulin.     

Prothrombin Activation by Snake Venom Proteases

Abstract

Snake venom prothrombin activators can be classified into four groups on the basis of their structural and functional properties in prothrombin activation. Group I activators (the best known example of which is the activator present in Echis carinalus venom) are metalloproteases that efficiently convert prothrombin into meizothrombin. Prothrombin activation by these activators is not affected by the presence of the prothrombinase cofactors, negatively charged phospholipids, CaCl₂ or factor Va. Group II activators (e.g. the Notechis scutatus scutatus activator) are serine proteases that require the presence of negatively charged phospholipids, CaCl₂ and factor Va for efficient prothrombin activation. Activators belonging to group III (e.g. Oxyuranus scutellatus scutellatus) are serine proteases that are also stimulated by negatively charged phospholipids and CaCl₂ but not by factor Va. These activators have a high molecular weight (Mr > 250,000) and are composed of a catalytic unit that is tightly associated with a factor Va-like cofactor unit An additional group of activators can be defined that consists of snake venoms which do not activate prothrombin but which convert prothrombin into inactive precursor forms of thrombin. In this review we describe a representative example of each group using experimental techniques currently available for the analysis of prothrombin activation.     

Co-Crystals of Etravirine by Mechanochemical Activation

The co-crystals formation of etravirine with three carboxylic acids was investigated. New co-crystals of etravirine with adipic acid, benzoic acid, and 4-hydroxybenzoic acid have been synthesized by wet milling of ingredients for 120 min. The novelty of these solid forms was first evidenced by powder X-ray diffraction. Their different morphology was evidenced by SEM microscopy. Spectroscopic analyses (FT-IR, MAS-NMR, and XPS) highlighted the hydrogen bonds between etravirine and co-formers, as a result of the solid-state reaction of the ingredients by wet milling. Thermal analyses pointed out that the milling process caused in co-crystals a reduction in the fusion enthalpy and the melting temperature, compared to the values obtained for etravirine. These co-crystals are stable up to four months on storage under extreme conditions, excepting the co-crystal with benzoic acid which begins to transform into a polymorph of etravirine after 30 days. The UV absorption spectra of the samples tested in three simulated physiological media with pH values of 6, 6.3, and 7 have evidenced the conformation change of etravirine due to hydrogen bonds between etravirine and carboxylic acids.     

Activation of carboxylic acids by pyrocarbonates

Activation of carboxylic acids was achieved via dialkyl pyrocarbonates (ROCO₂O, R= C₂H₅, i-C₃H₇, sec-C₄H₉, tert.-C₄H₉) in aprotic solvents in the presence tertiary amines. A convenient procedure for the preparation of carboxylic acid anhydrides from carboxylic acids and di-tert.-butyl pyrocarbonate in the presence of pyridine is reported. Analogously, di-isopropyl- or diethyl pyrocarbonate may be used in the presence of N-methylmorpholine (triethylamine). With pyridine, di-isopropyl- or diethyl pyrocarbonate carboxylic acids form isopropyl- or ethyl esters, respectively. A wide variety of esters were prepared in good yields in a one-pot procedure from carboxylic acids, including N-protected amino acids, and alcohols or from phenols by means of di-tert.-butyl pyrocarbonate in the presence of pyridine (Boc₂O-pyridine system). t-Butyl esters of carboxylic acids were obtained by the same procedure with 4-dimethylarninopyridine. In the absence of carboxylic acid, with 4-dimethylaminopyridine Boc₂O and alcohols generate alkyl tert.-butyl carbonates.