Flavoenzymes Inhibited by Indomethacin

Abstract

The effect of indomethacin on the activity of five different flavoenzymes, three dehydrogenases and six hydrosases, was determined. Indomethacin at concentration 1.0 mM inhibited the activity, in decreasing order of sensitivity, of the following flavoenzymes: D-amino acid oxidase (pig kidney), flavin-containing monooxygenases (pig liver microsomal), cyclohexanone monooxygenase (Acinetobacter), NADPH-quinone reductase (pig liver), and glutathione reductase (yeast), but it had no effect on the activity of glucose oxidase (Aspergillus) or liver microsomal NADPH-cytochrome P-450 reductase. Indomethacin was competitive with D-alanine for the D-amino acid oxidase (Ki=30 microM) and with NADPH for all other flavoenzymes sensitive to this compound (Kis 170-500 microM). While indomethacin also inhibited two of the three NAD(P)+-dependent dehydrogenases tested, the Kis were relatively high (<1, 500 microM), and of the six different hydrolases tested only one, liver microsomal esterase, was inhibited by indomethacin (Ki=600 microM). Indomethacin also inhibited aminopyrine demethylation catalyzed by the liver microsomal P-450 monooxygenase (Ki=1,000 microM). Although the exact mechanism for the inhibition of functionally different flavoenzymes sensitive to indomethacin is not known, the inhibition is probably not due to the detergent properties of this drug.

Thioredoxin Reductase and its Inhibitors

“…Inhibitors competing with NADPH are rare in the literature; an example is indomethacin that is reported to compete with NADPH in GR [ 41 ]. Possibly, these inhibitors are pharmacologically less interesting than competitive inhibitors of the oxidizing substrate because many enzymes use NADPH and thus target specificity is unlikely: indeed indomethacin inhibited all five flavoreductases tested by Chen and coworkers, with K I about 170-500 μM [ 41 ]. Another generic inhibitor of NADPH dependent reductases is the dye cibachron blue [ 42 ], that has the advantage of undergoing an absorbance change upon binding to the free enzyme.…”

Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as “Doorstop” for NADPH Entry

“…This may result in low specificity and toxicity in vivo , where the high content of thiols may give rise to multiple, undesirable cross-reactions, although very recently a specific irreversible inhibitor of human hTrxR 1 targeting the Sec residue has been found . Additionally, inhibitors that target the NADPH binding site are rare and, given the conservation of this site throughout the members of the family, are likely to be nonspecific as was previously demonstrated with indomethacin, an NADPH competitive inhibitor . This scenario is exacerbated by the lack of structural data for protein–inhibitor complexes.…”

“…5 Additionally, inhibitors that target the NADPH binding site are rare and, given the conservation of this site throughout the members of the family, are likely to be nonspecific as was previously demonstrated with indomethacin, an NADPH competitive inhibitor. 22 This scenario is exacerbated by the lack of structural data for protein−inhibitor complexes. In the protein data bank only two structures with metal inhibitors, i.e., TGR from Schistosoma mansoni (SmTGR) in complex with auranofin and human hTrxR I (HsTrxR) in complex with terpyridine-platinum, are present.…”