Inactivation of prostaglandin endoperoxide synthase by acylating derivatives of indomethacin

Abstract

Derivatives of the potent antiinflammatory agent and cyclooxygenase inhibitor indomethacin were synthesized in which the carboxylic acid moiety was converted into reactive acylating agents. Indomethacin imidazole (indomethacin-IM) and indomethacin N-hydroxysuccinimide (indomethacin-NHS) inactivated both the cyclooxygenase and peroxidase activities when incubated with the apo form of purified prostaglandin endoperoxide synthase (PGH synthase) at a stoichiometry of 1:1. Treatment of the inactivated enzyme with hydroxylamine at neutral pH led to recovery of all peroxidase and about 50% of the cyclooxygenase activity. Hydroxylamine did not regenerate the cyclooxygenase activity of indomethacin-inactivated protein. Reconstitution of the apoprotein with heme protected against inactivation by indomethacin-NHS. Visible spectroscopy established that indomethacin-NHS-inactivated apoenzyme had a reduced capacity to bind heme. Indomethacin-NHS also substantially protected the apoenzyme from cleavage at the trypsin-sensitive Arg277 site. Incubation of [2-14C]indomethacin-NHS with PGH synthase led to incorporation of radioactivity into the protein, but no adduct was detected by reversed-phase HPLC, suggesting it was unstable to the chromatographic conditions. Incubation of indomethacin-NHS with apoprotein followed by HPLC analysis led to the formation of greater amounts of the hydrolysis product indomethacin than did similar treatment of holoprotein. The results suggest that indomethacin-IM and indomethacin-NHS covalently and selectively label PGH synthase near the heme binding site, leading to loss of both catalytic activities of the enzyme.

Supramolecular Interactions of Nonsteroidal Anti‐inflammatory Drug in Nanochannels of Molecular Containers: A Spectroscopic, Thermogravimetric and Microscopic Investigation

“…[51] According to this method, the 1:1 guest-host stoichiometry can be written as Equation (7): For the above equilibrium the 1:1 complexation can be written as Equation (8): tively. The standard Gibbs energy changes (DG) of the drug molecule in the excited state were determined by using Equation (6), and the values are tabulated in Table 1. The molecular diameter of IMC, calculated by using Edward's method, [55] was found to be 8.28 .…”

“…The drug molecule acts as an inhibitor of prostaglandin biosynthesis and as a strong tocolytic agent. [6,7] IMC is one of the most commonly used chemotherapeutic drugs. This drug molecule has been widely prescribed as an anti-inflammatory, antipyretic and analgesic agent and has been used in rheumatoid arthritis, gout and collagen disease.…”

Bromoacetamido Analogs of Indomethacin and Mefenamic Acid as Affinity-Labeling Agents and Mechanistic Probes for Prostaglandin H2 Synthase

“…Affinity labeling offers a complementary approach by which NSAID binding sites may be mapped. Recently, acetylating derivatives of indomethacin (indomethacin imidazole and indomethacin A-hydroxysuccinimide) were synthesized and found to be potent inactivators of PGH2 synthase that selectively and covalently modified only the apoenzyme (Wells & Mamett, 1993). These indomethacin derivatives were shown to prevent the binding of heme to the active site.…”

A New Remote Control for Traceless meta‐CH Olefination of Phenols

“…Indomethacin [IMC; N ‐( para ‐chlorobenzoyl)‐5‐methoxy‐2‐methylindole‐3‐acetic acid; Figure 1] is a potent, clinically useful nonsteroidal anti‐inflammatory drug (NSAID) that has received considerable interest from the scientific community because of its various applications in medicinal treatment. The drug molecule acts as an inhibitor of prostaglandin biosynthesis and as a strong tocolytic agent 6. 7 IMC is one of the most commonly used chemotherapeutic drugs.…”