Synthesis and evaluation of nitrostyrene derivative compounds, new snake venom phospholipase A2 inhibitors.

Several nitrostyrene derivatives were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Some compounds were very efficient as inhibition agents against edema-inducing, enzymatic and myotoxic activities. Data revealed that the size of the substitute and substitution position in the nitrostyrene moiety had important influence on the inhibition capacities. The enzymatic kinetic studies show that the nitrostyrene derivatives compounds inhibit PLA(2) in a non-competitive manner. The electronic, molecular and topologic parameters were calculated using ab initio quantum calculations (density functional theory-DFT) and analyzed by chemometric methods (principal component analysis (PCA) and hierarchical cluster analysis (HCA)) in order to build models able to establish relationships between the electronic features and the structure-activity presented by the target compound. Compounds with the nitro group in the ortho, meta and para position (compounds 2-4) on the aromatic ring were more efficient in the inhibition of PLA(2) activity in all tests. These results indicate that the influence of the nitro group in the aromatic ring is, in fact, important. In addition, quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) present lower values of highest occupied molecular orbital (HOMO) energy and polarizability, suggesting the formation of a charge-transferring complex between the nitrostyrene compounds and PLA(2).

[Allergens]. / Les allergenes.

The clinical signs linked with immediate hypersensitivity, correspond with an abnormal reactivity of the immune system, which appears following contact with external substances, the allergens. Over the last few years there has been considerable progress in research on these substances in the domain of structural characterisation, biochemistry and immunological properties. Besides the definition of the word allergen linked to its immunological characteristics, our usual language maintains a certain ambiguity in its use which may characterise different states, including successive steps of the manufacture of an allergen extract. In effect the word allergen may designate the agent that is responsible for the allergic disease, for example cat, but also the raw material used for the manufacture of the corresponding extract, whether it be hair or squames; it may also apply to the final allergenic extract, the extract of cat hair or squames, as well as a precise molecule such as the major allergen of cat Fel d1 in the same example. After having reviewed several definitions as well as the nomenclature we will study the general characteristics of pneumoallergens and trophallergens, those of recombinant allergens, then the parameters of manufacture of allergen extracts.

[Allergy to cypress pollen: preparation of a reference and standardization extract in vivo]. / Allergie au pollen de cypres: preparation d'un extrait de référence et standardisation in vivo.

Development of Cypress allergy frequency led to the standardization of commercial cypress extract used for diagnosis and immunotherapy. Previous in vitro studies on two cypress pollen species (Cupressus sempervirens and Cupressus arizonica) allowed us to produce an allergenic solution composed by a mixture of both extracts for in vivo standardization. Dilutions of this allergenic solution were tested by prick-test on 44 patients with clinical allergy to cypress pollen to define the dilution that corresponds to a 6 mm wheal conformed to the definition of 100 IR. The mixture of the two major species found in France is justified by the in vitro study results. Extracts revealed complementary allergenic composition: Cup sempervirens showed a wider diversity of allergens whereas Cup arizonica showed a higher content of the major 43 kDa allergen. Thus, according to in vivo analysis, we are able to produce a standardized extract of Cypress pollen expressed in IR.

Molecular modeling and inhibition of phospholipase A2 by polyhydroxy phenolic compounds.

Phospholipases A(2) are enzymes responsible for the hydrolysis of membrane phospholipids that release arachidonic acid, which serves as substrate for pro-inflammatory mediators, such as prostaglandins and leucotriens. The design of specific inhibitors for PLA(2) might help in the development of new anti-inflammatory drugs. Polyhydroxy phenolic compounds, such as flavonoids, vitamin E, rosmarinic acid and aristolochic acid, are able to inhibit PLA(2) from different sources. Herein, we have studied the kinetic behavior and the capacity of inhibiting edema formation induced by PLA(2) of five different polyhydroxy phenolic compounds (two phenolic derivatives and three acetophenone hydroxylated derivatives) extracted from the venom of Crotalus adamanteus. The results showed that compounds 1,3-dihydroxy benzene, 1,3,5-trihydroxy benzene and 2,4,6-trihydroxy acetophenone were the most efficient in the inhibition of the enzymatic activity and edema induction by PLA(2). It was also verified that the number of hydroxyls in each molecule is not a limiting factor for the inhibition capacity of these compounds. Molecular modeling studies indicated that the most active compounds are linked to the amino acid Asp 49 and that they destabilize the coordination of the calcium atom, which is essential to the catalytic activity. The study of potential surfaces showed that there are conditions in which the potential values must be adequate for enzyme complex formation with polyhydroxy phenolic compounds. When the potential over the hydroxyl surfaces is very high, formation of stable complexes does not occur and the enzyme does not act intensely. These results might be helpful in the design of a drug that specifically inhibits PLA(2).