Jean-Jacques Perie

Characterisation and Assay of Five Enzymatic Activities in the Stratum Corneum Using Tape-Strippings

The report describes a method for the assay of five enzymatic activities involved in establishing the stratum corneum permeability barrier: β-glucocerebrosidase, acid phosphatase, phospholipase A2 (PLA2) and two serine proteases: chymotrypsin and its activator in the stratum corneum, trypsin. The specific activities of these different enzymes have been determined along with their pH profiles and sensivities to specific inhibitors. It can be noted that only two presented a pH optimum similar to the pH of the stratum corneum. This could suggest that their activities are regulated by local variations in pH. The method was applied to a pathological situation, that of a non-eczematous dry atopic dermatitis. Atopic skin had significantly reduced trypsin activity, increased acid phosphatase and no change in the activities of three other studied enzymes. Understanding these activities can provide a tool for the characterization of skin pathologies and for the development of a certain number of applications in cosmetology and therapeutics.

Epidermal Enzymes: Their Role in Homeostasis and Their Relationships with Dermatoses

This review underlines the importance of different enzymes (β-glucocerebrosidase, phospholipase A2, proteases and cholesterol sulfatase) in the formation and maintenance of the epidermal barrier function. Certain diseases may be characterized by the lack or excess of one or more of these different enzyme activities, altering the homeostatic equilibrium of the epidermis. In addition to this, particular enzymes may show potential in the development of novel dermocosmetic strategies.

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

Synthesis, binding and structure–affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS)

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.

Enzymatic synthesis of phosphoric monoesters with alkaline phosphatase in reverse hydrolysis conditions

Abstract Title compounds were synthesized on a preparative scale using alkaline phosphatase, orthophosphoric monoester phosphohydrolase B.C. 3.1.3.1, in reverse hydrolysis conditions. Optimization for one of the 25 phosphoryl acceptors investigated (glycerol) shows that up to 55% synthesis yield can be obtained using a large excess of substrate, conditions in which the enzymatic activity remains high. From the results obtained with different phosphoryl group donors, phosphate, pyrophosphate and polyphosphates and with enzymes of different sources, it comes up that the best results are obtained with pyrophosphate and with the weakly purified calf intestine alkaline phosphatase. The extent of enzymatic hydrolysis of the donor can be reduced owing to the existence of two different pH optima for the two reactions, phosphorylation and hydrolysis. The synthesis can be also performed using inert co-solvents which allow to reduce the amount of acceptor used, as long as Zn++ is added to the reaction medium. The results are discussed in terms of the catalytic mechanism of alkaline phosphatase.

Variability of Enzyme Markers during Clinical Regression of Atopic Dermatitis

Skin surface enzyme activities were found to be significantly different in healthy and in skin with atopic dermatitis and, following appropriate treatment, a close correlation was observed between the clinical staging of the atopic dermatitis and the levels of the assayed marker enzymes. Samples were taken, by stripping with simple adhesive tapes, from a group of subjects on cure in a spa. The corneocytes were recovered from the first layers of the stratum corneum. Aqueous extracts of the strips were tested for their activity on chromophoric substrates which allow fluorescence spectrometry to be used to assay the trypsin-like, acid-phosphatase-like and phospholipase-A2-like activities. We show that the restoration of return to activities close to those of healthy subjects is related to the general condition of the patients, who showed a clearly improved SCORAD. Recovery of the trypsin-like activity and attenuation of the phospholipase-like activity, paralleled the regression of the dermatitis as assessed by a decrease in clinically evaluated parameters of xerosis and inflammation.

Slow reversible inhibitions of rabbit muscle aldolase with substrate analogues: synthesis, enzymatic kinetics and UV difference spectroscopy studies

Various dihydroxyacetone-phosphate (DHAP) analogues bearing an aromatic ring or beta-dicarbonyl structures were synthesized. Their capacity to form a stabilized iminium ion or conjugated enamine in the reaction catalyzed by rabbit muscle aldolase (EC 4.1.2.13) were investigated by enzymatic kinetics and UV difference spectroscopic techniques. Whereas the aromatic derivative led to competitive inhibition without detectable iminium ion formation, slow reversible inhibitions of aldolase by beta-dicarbonyl compounds was shown to have taken place. Conjugated enamine formation at the active site of the enzyme was detected by their specific absorbances close to 317 nm.

Large-scale enzymatic synthesis of glycerol 1-phosphate

A large-scale synthesis of glycerol 1-phosphate (75 g 1−1; 41%) has been developed using alkaline phosphatase immobilized on corn grits in reverse hydrolysis conditions. The reaction is highly regioselective at position 1 on glycerol but leads to a racemic product. This result is obtained by taking advantage of the difference in pH optima between forward and reverse reactions and the fact that the immobilized enzyme is only weakly inhibited by large concentrations of the two substrates, phosphate and glycerol, and by the product glycerol phosphate. The use of the immobilized enzyme in an embedded form in a flow system also contributes to this significant yield.

SYNTHESIS OF SUBSTRATE ANALOGUES AND INHIBITORS FOR THE PHOSPHOGLYCERATE MUTASE ENZYME

Abstract Several substrate analogues of the title enzyme were synthesized as well as with possible irreversible inhibitors, analogues of phosphoglycolate, the cofactor for this enzyme. The main routes involved an Arbuzov type reaction on functionalized halides, a reaction significantly improved by a sonication procedure and reaction of the diethylphosphite anion on tosylates. epoxides were obtained by quantitative oxidation of conjugate double bonds using DMDO. Preliminary assays indicated a promising activity in some of these compounds. Symbols: EM: phosphoglycerate mutase, 1,3-diPGA: 1,3-diphosphoglycerate, 2-PGA: 2-phosphoglycerate, 3-PGA: 3-phosphoglycerate, 2,3-diPGA: 2.3-diphosphoglycerate, PG: phosphoglycolate, DMDO: dimethyldioxirane, m-CPBA: meta-chloroperbenzoic acid

An improved synthesis of phosphonomethyl analogues of glyceraldehyde- 3-phosphate and dihydroxy-acetone phosphate

Compounds 1 and 2, phosphonate analogues of dihydroxy-acetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) respectively, are easily and quantitatively obtained from diethyl-4,4-diethoxy-3-hydroxybutyl-1 -phosphonate 3. Depending upon the acidic conditions utilised for the deprotection of the phosphonate and carbonyl groups, the aldol/ketol rearrangement allows the synthesis of either compound.