INHIBITION OF AROMATIC AMINO ACID DECARBOXYLASE BY A GROUP OF NEW POTENTIAL NONSTEROIDAL ANTI-INFLAMMATORY DRUGS WITH ANTILEUKOTRIENIC EFFECTS

Derivatives of phenylsulfanylbenzoic and phenylsulfanylphenylacetic acids were synthetized within the framework of research of anti-inflammatory drugs with a potential antileukotrienic mechanism of action (7). In in vitro experiments, some of the compounds showed a combined antileukotrienic effect via the inhibition of binding of LTD4 and LTB4 on the receptors. In connection with the found mechanism of action, the pharmacological properties of compound VÚFB 19363 (Quinlukast) as a representative of the group were evaluated as a potential antihistaminic drug. Studies of other effects, more or less specific, which may appear as enzyme inhibition or inactivation, belong to common investigations of structures synthesized as potential medicaments. We found such effects of drugs in the research of several groups of compounds prepared or already used in human therapy (8,9). On the other hand, in the case of phenylsulfanylbenzoic acid derivatives, a study with aminotransferases and glutamate decarboxylase showed very weak effects, which is favourable in view of their potential use as medicaments (5). Compounds originally prepared for a certain activity sometimes show properties which may contribute to their pharmacologically desirable effect by another mechanism. This was the case of the influence of anti-inflammatory drugs on aromatic amino acid decarboxylase, the enzyme participating in the formation of serotonin and dopamine (1). In the present study, a group of compounds with multiple antileukotrienic activities was examined for interaction with aromatic amino acid decarboxylase of the rat liver (AAD, EC 4.1.1.28).


Introduction
Derivatives of phenylsulfanylbenzoic and phenylsulfanylphenylacetic acids were synthetized within the framework of research of anti-inflammatory drugs with a potential antileukotrienic mechanism of action (7).In in vitro experiments, some of the compounds showed a combined antileukotrienic effect via the inhibition of binding of LTD 4 and LTB 4 on the receptors.In connection with the found mechanism of action, the pharmacological properties of compound VÚFB 19363 (Quinlukast) as a representative of the group were evaluated as a potential antihistaminic drug.
Studies of other effects, more or less specific, which may appear as enzyme inhibition or inactivation, belong to common investigations of structures synthesized as potential medicaments.We found such effects of drugs in the research of several groups of compounds prepared or already used in human therapy (8,9).On the other hand, in the case of phenylsulfanylbenzoic acid derivatives, a study with aminotransferases and glutamate decarboxylase showed very weak effects, which is favourable in view of their potential use as medicaments (5).
Compounds originally prepared for a certain activity sometimes show properties which may contribute to their pharmacologically desirable effect by another mechanism.This was the case of the influence of anti-inflammatory drugs on aromatic amino acid decarboxylase, the enzyme participating in the formation of serotonin and dopamine (1).
In the present study, a group of compounds with multiple antileukotrienic activities was examined for interaction with aromatic amino acid decarboxylase of the rat liver (AAD, EC 4.1.1.28).

Compounds studied
Phenylsulfanylbenzoic acid derivatives with potential antileukotrienic effects (VÚFB 19363, 19369, 19370, 19371, and 19760) were synthesized as described in (4).Their structural formulae are given in Table 1.Compouds were dissolved in 0.05M solution of NaOH, solutions were neutralized by 0.05 M HCl and buffered with 0.1 M phosphate buffer pH 7.2.

AAD preparation
Supernatant 20 000xg of the homogenate of rat liver as an organ which is rich in AAD activity was used as the source of the enzyme.The enzyme preparation was obtained as described previously (1).All experiments with rats were approved by the Animal Use and Care Committee of Charles University, Faculty of Pharmacy in Hradec Králové, Czech Republic.Specific AAD activity of the preparation (3), measured by the radiometric method (5), was 66.5 nkat per mg of protein.
Enzyme activity was determined radiometrically: The incubation mixture contained the AAD preparation, substrate 1-14 C-L-tyrosine (final concentrations 6x10 -5 -6x10 -4 M, specific activity 1.136 µCi.ml -1 ), pyridoxal-5'phosphate (1x10 -6 M in most experiments), the compounds under study and other components at different concentrations according to the aim of individual experiments, in 0.02 M Na-phosphate buffer, pH 7.2.The mixture was incubated at 37 °C for 30 min and the radioactivity of 14 CO 2 (liberated from the mixture by means of sulfuric acid and absorbed in 0,1 ml 30 % KOH) was measured in the scintillation cocktail (1) using the 1219 Rackbeta scintillation counter LKB Wallac.
AAD activities in individual samples were expressed in dpm and calculated as relative activities.Control samples without compounds under study were considered as 100 %.

Estimation of inhibition kinetics AAD inhibition by compounds VÚFB 19363 and 19369
was examined in experiments with constant concentrations of both AAD and the compound, and changing concentrations of the substrate.Reversibility of inhibition was evaluated graphically at changing concentrations of the enzyme.Type of inhibition was estimated using the Lineweaver and Burk (1/v versus 1/s) plot and the plot of enzyme concentration vs. velocity of reaction.1 summarizes AAD inhibition by individual compounds in vitro.Despite weak inhibition, two independent experiments with triplicate samples confirmed distinct differences between compounds.We tried to compare AAD inhibition with lipophilicity of compounds under study, using log P, obtained by means of HPLC, and with calculated distances of probable centers of interaction (values D).The results are summarized in Table 2.The results suggest a lack of correlation, which might be due to a low inhibitory effect, small differences in AAD inhibition, as well as due to using the equiconcentration condition instead of the equipotential one.The only exception in inhibition effi-ciency is compound 19369, which posesses lower lipophilicity and a shorter distance between the carboxy group and the hydrophobic part of the molecule.

Assessment of the influence of compounds under study on AAD activity in vitro Table
If we consider the results in view of the position of AAD in the synthesis of the biogenic amines serotonin and dopamine, the enzyme inhibition might be considered as a certain contribution to the anti-inflammatory effect.
Tab. 2: Comparison of AAD inhibition with lipophilicity and distances of centres of probable interaction of phenylsulfanylbenzoic acid derivatives.

Inhibition kinetics
In the cases of compounds VÚFB 19363 (the compound in the centre of interest) and 19369 (the most effective inhibitor of AAD), an attempt to evaluate the inhibition kinetics was made (Fig. 1 and Fig. 2).The parallel lines of inhibited and control reaction in the Lineweaver-Burk plot of results (Fig. 3) suggested the uncompetitive type of inhibition by VÚFB 19363, while it was not possible to distinguish between competitive and non-competitive inhibition in the case of VÚFB 19369 (Fig. 4).Graphical evaluation of reversibility of inhibition suggested full reversibility for VÚFB 19369 (Fig. 5).In a similar experiment, we did not succeed in evaluating reversibility of inhibition by VÚFB 19363 because of a too low inhibitory effect.

Tab. 1 :
Inhibition of AAD by phenylsulfanylbenzoic acid derivatives.The influence on the enzyme is expressed in % of control activity.Each number represents the mean of three measurements in two independent experiments.Solutions of individual compounds were prepared in concentrations respecting their low solubility in water.