Oxidation of anti-thyroid drugs and their selenium analogs by ABTS radical cation.

Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.

Oxidation of baicalein by tyrosinase and by o-quinones.

Baicalein (5,6,7-trihydroxyflavone) has been previously described as an inhibitor of tyrosinase (Guo et al. Int. J. Biol. Macromol. 118 (2018) 57-68). However, long before this article was published this flavonoid had been shown to be a substrate of this enzyme and a catecholic cofactor partially abolishing the lag-phase during oxidation of l-tyrosine. Other compounds with a 1,2,3-triphenol moiety, such as pyrogallol, gallic acid and its esters are also oxidized by tyrosinase. Gallic acid was also shown to reduce tyrosinase-generated o-quinones. We have demonstrated that baicalein is also rapidly oxidized by o-quinones generated from catechols by tyrosinase or by treatment with sodium periodate. Smaller changes of absorbance at 475 nm during oxidation of l-dopa by tyrosinase in the presence of baicalein do not result from enzyme inhibition but from reduction of dopaquinone by baicalein. This reaction prevents formation of dopachrome giving an effect of inhibition, which is only apparent. The actual reaction rates did not decrease but increased in the presence of baicalein, which we demonstrated by measurements of oxygen consumption.

In vitro oxidation of hispidin and gallic acid by horseradish peroxidase.

Gallic acid and hispidin have been previously described by us as inhibitors of horseradish peroxidase (Benarous, K., Benali, F. Z., Bekhaoua, I. C., and Yousfi, M. Journal of Biomolecular Structure & Dynamics, (2021) 39(18), 7168-7180). However, additional experiments have demonstrated that under the applied assay conditions both compounds are rapidly oxidized by this enzyme. After oxidation, the components of the reaction mixture undergo complex reactions giving products with much weaker absorption at the detection wavelength. This was interpreted by us as enzyme inhibition, which, however, is only apparent. In fact, the activity of horseradish peroxidase is not affected by these compounds, which was demonstrated by measurements of hydrogen peroxide consumption.Communicated by Ramaswamy H. Sarma.

Rifampicin is not an inhibitor of tyrosinase.

Rifampicin has been previously described as an inhibitor of tyrosinase (Chai et al., Int. J. Biol. Macromol. 102 (2017) 425-430). However, rifampicin contains a p-diphenol group and compounds with such a moiety have been shown before to reduce tyrosinase-generated o-quinones. Rifampicin also shows strong absorption in a region completely overlapping with the visible absorption band of dopachrome, the oxidation product of L-tyrosine and L-dopa, whose concentration is measured spectrophotometrically in the standard enzymatic assay to monitor the activity of tyrosinase. We have demonstrated that rifampicin is also rapidly oxidized by o-quinones generated from catechols by tyrosinase or by treatment with sodium periodate. Smaller changes of absorbance at 475 nm during oxidation of L-dopa by tyrosinase in the presence of rifampicin do not result from enzyme inhibition but from oxidation of rifampicin by dopaquinone, which leads to rapid decrease of rifampicin absorption in this range. The actual reaction rates are not affected, which we have demonstrated by measurements of oxygen consumption. Rifampicin behaves therefore as other compounds with reducing properties, such as ascorbic acid, hydroquinone, hydrazine derivatives, and flavonoids, some of which have also been incorrectly described before as inhibitors of tyrosinase.

Comment on "Natural and synthetic flavonoid derivatives as new potential tyrosinase inhibitors: a systematic review" by R. Obaid, E. Mughal, N. Naeem, A. Sadiq, R. Alsantali, R. Jassas, Z. Moussa and S. Ahmed, RSC Advances, 2021, 11, 22159.

A review article has been published recently (RSC Advances, 2021, 11, 22159-22198) describing flavonoids as inhibitors of tyrosinase. However, many compounds included in this review have been previously shown to act as substrates of this enzymes or antioxidants reducing tyrosinase-generated o-quinones. Products of their oxidation absorb light in a range different than dopachrome, the oxidation product of l-tyrosine or l-dopa, whose concentration is measured spectrophotometrically in the standard enzymatic assay to monitor the activity of this enzyme. This effect is interpreted as enzyme inhibition, which, in fact, is only apparent and results from inadequate methodology.

Quercetin is a substrate not an inhibitor of tyrosinase - comments on "Quercetin as a tyrosinase inhibitor: Inhibitory activity, conformational change and mechanism" published by Fan et al. (2017).

Quercetin was described as an inhibitor of tyrosinase in an article published in Food Research International. However, it was firmly established in the literature before that this compound was a substrate of this enzyme and an antioxidant reducing tyrosinase-generated o-quinones.

Synthesis of disparlure and monachalure enantiomers from 2,3-butanediacetals.

2,3-Butanediacetal derivatives were used for the stereoselective synthesis of unsymmetrically substituted cis-epoxides. The procedure was applied for the preparation of both enantiomers of disparlure and monachalure, the components of the sex pheromones of the gypsy moth (Lymantria dispar) and the nun moth (Lymantria monacha) using methyl (2S,3R,5R,6R)-3-ethylsulfanylcarbonyl-5,6-dimethoxy-5,6-dimethyl-1,4-dioxane-2-carboxylate as the starting material.

Mechanisms of interference of p-diphenols with the Trinder reaction.

p-Diphenols, such as homogentisic acid, gentisic acid, etamsylate, and calcium dobesilate, interfere with diagnostic tests utilizing the Trinder reaction but the mechanisms of these effects are not fully understood. We observed substantial differences both in oxidation of p-diphenols by horseradish peroxidase and their influence on oxidation of 4-aminoantipyrine and various phenolic substrates. Homogentisic acid was rapidly oxidized by the enzyme and completely blocked chromophore formation. Enzymatic oxidation of the remaining p-diphenols was slow and they only moderately inhibited chromophore formation. However, in the presence of standard substrates all tested p-diphenols were rapidly converted to p-quinones. Hydrogen peroxide consumption was significantly accelerated by homogentisic acid but not much affected by the other p-diphenols. The magnitude and mechanisms of interference caused by p-diphenols therefore depend on their structure which determines their electrochemical properties - while for homogentisic acid with an electron-donating substituent and a lower reduction potential both enzymatic oxidation and reduction of the peroxidase-generated radicals occur, for p-diphenols with electron-withdrawing substituents and higher reduction potentials only the second mechanism is significant. Correlation of the effects on the Trinder reaction with reduction potentials of interfering compounds allows prediction of such properties for a wide range of other reducing compounds based on this parameter. It also explains why compounds with very different structures but strong reducing properties show such effects.

Collisional mechanism of ligand release by Bombyxmori JHBP, a member of the TULIP / Takeout family of lipid transporters.

Juvenile hormones (JHs) regulate important processes in insects, such as postembryonic development and reproduction. In the hemolymph of Lepidoptera, these lipophilic sesquiterpenic hormones are transported from their site of synthesis to target tissues by high affinity carriers, the juvenile hormone binding proteins (JHBPs). Lepidopteran JHBPs belong to a recently uncovered, yet very ancient family of proteins sharing a common lipid fold (TULIP domain) and involved in shuttling various lipid ligands. One important, but poorly understood aspect of JHs action, is the mechanism of hormone transfer to or through the plasma membranes of target cells. Since many membrane-active peptides and proteins, such as the pore-forming bacterial toxins, are activated by low pH or interaction with phospholipid membranes, we have examined the effect of these factors on JH binding by JHBPs. The affinity of Bombyx mori and Manduca sexta JHBPs for JH III was determined by the DCC assay, equilibrium dialysis, and isothermal titration calorimetry, and found to be greatly reduced at low pH, in agreement with previous observations. Loss of binding was accompanied by changes in fluorescence and near-UV CD spectra, indicating significant changes in protein structure in the environment of aromatic residues. The apparent dissociation rate constant (koff) of the JHBP-JH III complex was greater at acidic pH, suggesting that low pH favors ligand release by opening of the binding pocket. The affinity of recombinant B. mori JHBP (rBmJHBP) was also decreased in the presence of anionic phospholipid vesicles. Measurements of steady-state fluorescence anisotropy with the lipophilic probe TMA-DPH demonstrated that rBmJHBP specifically interacts with anionic membranes. These results suggest the existence of a collisional mechanism for ligand release that may be important for delivery of JHs to the target cells, and could be relevant to the function of related members of this emerging family of lipid-transport proteins.

Interference of carbidopa and other catechols with reactions catalyzed by peroxidases.

BACKGROUND: A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated. METHODS: Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of carbidopa, an anti-Parkinsonian drug, and other catechols, including l-dopa, were monitored spectrophotometrically and by measuring hydrogen peroxide consumption. RESULTS: Chromophore formation in all three enzyme/substrate systems was blocked in the presence of carbidopa and other catechols. However, the rates of hydrogen peroxide consumption were not much affected. Irreversible enzyme inhibition was also insignificant. CONCLUSIONS: Tested compounds reduced the oxidation products or intermediates of model substrates thus preventing chromophore formation. This interference may affect interpretation of results of diagnostic tests in samples from patients with Parkinson's disease treated with carbidopa and l-dopa. GENERAL SIGNIFICANCE: This mechanism allows prediction of interference in peroxidase-based diagnostic tests for other compounds, including drugs and natural products.

Reactions of Flavonoids with o-Quinones Interfere with the Spectrophotometric Assay of Tyrosinase Activity.

Flavonoids are important food components with antioxidant properties and many of them have been described as tyrosinase inhibitors. Oxidation of quercetin, kaempferol, morin, catechin, and naringenin by mushroom tyrosinase and their influence on the oxidation of l-dopa and l-tyrosine was studied. Reaction rates measured spectrophotometrically and by oxygen consumption differed substantially. All tested flavonoids reacted with 4-tert-butyl-o-benzoquinone and/or 4-methyl-o-benzoquinone, although at different rates. These reactions generated products whose UV-vis spectra either overlapped or did not overlap with the spectrum of dopachrome. They therefore strongly influence the kinetic analysis performed by measuring the absorbance at 475 nm during oxidation of l-dopa or l-tyrosine generating false inhibition or activation effects. This method is therefore inappropriate for monitoring the activity of this enzyme in the presence of flavonoids and other compounds possessing strong nucleophilic or reducing groups.

Synthesis of the aggregation pheromone of the Colorado potato beetle from its degradation product.

Incubation of the Colorado potato beetle aggregation pheromone, (S)-1,3-dihydroxy-3,7-dimethyl-6-octen-2-one, with antennal or leg extracts from this beetle gave 6-methyl-5-hepten-2-one as the major product. This ketone was used as a substrate in a stereoselective synthesis of the pheromone. It was attached to the butanediacetal of glycolic acid with good stereoselectivity and the desired isomer was further enriched by purification of the product of this reaction on silica gel.

Synthesis and analysis of activity of a potential anti-melanoma prodrug with a hydrazine linker.

A potential anti-melanoma prodrug containing a phenolic activator, a hydrazine linker, and a nitrogen mustard effector - (N-{4-[bis-(2-chloroethyl)amino]benzoyl}-N'-(4-hydroxybenzyl)hydrazine) has been synthesized in seven steps. Spectrophotometric measurements of its oxidation by tyrosinase showed a rapid increase of absorbance at 337 nm. HPLC analysis demonstrated that two major products were formed. However, during the reaction one of the products was converted into the other. The stable product with a maximum of absorption at 337 nm was isolated and identified as 5,6-dihydroxy-1H-indazol-1-yl 4-[bis-(2-chloroethyl)amino]benzoate. It was formed by a cyclization of the enzymatically generated o-quinone. This reaction was unexpected, since the acylated hydrazine nitrogen atom should not be sufficiently nucleophilic to attack the o-quinone ring. This cyclization prevented the effector release from the enzyme-activated prodrug. As a result, the prodrug showed only limited specificity for B16-F10 murine melanoma cells compared to reference cell lines. When applied in solid tumors in mice it showed slightly higher activity than the parent mustard drug (4-[bis-(2-chloroethyl)amino]benzoic cid), but significantly lower activity than melphalan, a commercial mustard drug with a structure resembling tyrosine, occasionally used in the treatment of melanoma.

Oxidation of carbidopa by tyrosinase and its effect on murine melanoma.

Oxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid and 6,7-dihydroxy-3-methylcinnoline. These results demonstrate that after oxidation of the catechol moiety to an o-quinone either a redox exchange with the hydrazine group or a cyclization reaction occur. The cyclization product underwent additional oxidation reactions leading to aromatization. The cyclization reaction is undesired in the case of hydrazine-containing anti-melanoma prodrugs and will have to be taken into account in designing such compounds. Carbidopa was tested against B16(F10) melanoma cells in culture and showed cytotoxicity significantly higher than either of its oxidation products and l-dopa. This effect, however, was not specific to this cell line.

Indirect oxidation of the antitumor agent procarbazine by tyrosinase--possible application in designing anti-melanoma prodrugs.

The interaction of tyrosinase with the anticancer drug procarbazine has been investigated. In the presence of the enzyme alone no oxidation of this dialkylhydrazine above the background level was observed. However, when phenolic substrates (4-tert-butylcatechol or N-acetyl-l-tyrosine) were included in the reaction mixture, procarbazine was rapidly degraded. Oxygen consumption measurements showed that in a mixture both the phenolic substrate and the drug were oxidized. The major product of procarbazine degradation was isolated and identified as azoprocarbazine, the first active metabolite of this drug detected in previous in vivo and in vitro studies. This indirect oxidation of the hydrazine group in this anticancer agent indicates possible application of a hydrazine linker in construction of tyrosinase-activated anti-melanoma prodrugs.

Indirect oxidation of amino acid phenylhydrazides by mushroom tyrosinase.

We have investigated oxidation of amino acid phenylhydrazides by mushroom tyrosinase in the presence of 4-tert-butylcatechol and N-acetyl-L-tyrosine. Spectrophotometric measurements showed gradual disappearance of 4-tert-butyl-o-benzoquinone, generated by oxidation of 4-tert-butylcatechol with sodium periodate, after addition of amino acid phenylhydrazides. However, the presence of the phenylhydrazides did not influence the concentration of 4-tert-butyl-o-benzoquinone formed during enzymatic oxidation. Oxygen consumption measurements demonstrated that in a mixture both compounds were oxidized but the reaction rate was proportional to the concentration of the catechol. In the oxidation of N-acetyl-L-tyrosine addition of phenylhydrazides shortened the lag period, indicating that they acted as reducing agents, converting N-acetyl-L-dopaquinone to N-acetyl-L-dopa. In HPLC analysis of the oxidation 4-tert-butylcatechol and the phenylhydrazide of Boc-tryptophan only the N-protected amino acid and 4-tert-butyl-o-benzoquinone were detected as final products. In the presence of the natural substrates the oxidation of amino acid phenylhydrazides required much smaller amounts of the enzyme and was up to 40 times faster than the reaction carried out without these compounds. These results demonstrate that tyrosinase can oxidize phenylhydrazides indirectly through o-quinones. This reaction explains the inhibitory effect of agaritine, a natural amino acid hydrazide, on melanin formation and the inhibitory effects of other hydrazine derivatives on tyrosinase described in the literature.

Interaction of mushroom tyrosinase with aromatic amines, o-diamines and o-aminophenols.

3-Amino-L-tyrosine was found to be a substrate of mushroom tyrosinase, contrary to what had previously been reported in the literature. A series of amino derivatives of benzoic acid were tested as substrates and inhibitors of the enzyme. 3-Amino-4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid and 3,4-diaminobenzoic acid were oxidized by this enzyme, as previously reported for Neurospora crassa tyrosinase, but 4-aminobenzoic acid and 3-aminobenzoic acid were not. Interestingly, 3-amino-4-hydroxybenzoic acid was oxidized five times faster than 4-amino-3-hydroxybenzoic acid, confirming the importance of proton transfer from the hydroxyl group at C-4 position. All compounds inhibited the monophenolase activity but their effect on the diphenolase activity was small or negligible. 3-Amino-4-hydroxybenzoic acid was a stronger inhibitor than 4-amino-3-hydroxybenzoic acid, indicating their different binding affinity to the oxy form of the enzyme. Both, however, were weaker inhibitors than 3-amino-L-tyrosine, 4-methoxy-o-phenylenediamine and 3,4-diaminobenzoic acid, which was the strongest inhibitor from among the compounds tested. These results show that the relative positioning of the amino group and the hydroxy group in o-aminophenols with respect to the side chain is important both for binding to the dicopper center and for catalysis.

Analysis of involvement of the 3'-untranslated regions in regulating mRNA stability for vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus.

The degradation of the 3'-untranslated regions (UTRs) of vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus, was analyzed in vitro. The degradation pattern was similar for all three RNAs, with a high degradation rate in non-diapausing adult insects and no degradation in the fifth instar nymphs and in diapausing adults, and was not correlated with the expression levels of these three proteins. Proteins binding to the 3'-UTRs were detected in polysomal and cytosolic extracts. These factors, however, were present in all developmental stages. The abundance of the polysomal factor showed little variation, but the cytosolic factor was enriched in adult insects. Cross-competition experiments demonstrated that the same factors bound to all three RNAs with similar affinity. The pattern of degradation, presence of the binding factors in all stages, and their inability to distinguish between the target sequences indicate that the 3'-UTRs do not participate in controlling the expression of these three proteins.