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

Abstract

Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Surface Recognition by Synthetic Receptors: A Route to Novel Submicromolar Inhibitors for α-Chymotrypsin

“…and hence to obtain appropriate values for K i or K i *, we measured the dependence of k obs on the concentration of substrate. With receptor 1 , k obs was seen to decrease as the substrate concentration increased, which is behavior unique to the competitive mechanism of slow binding inhibition (Figure ). , Full data for inhibition of α-chymotrypsin by each receptor are summarized in Table . For 3 , 4 , and 5 , time dependent effects could not be observed due to the very weak inhibition (less than 20% inhibition after 5 h of incubation).…”

Mechanism of Free Radical Oxygenation of Polyunsaturated Fatty Acids by Cyclooxygenases

“…253,254 These researchers synthesized two affinity labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (inhibitor I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (inhibitor II), through modification of the NSAIDs, indomethacin and mefenamic acid, respectively (Figure 71 A). 253 Each inhibitor caused time-dependent, irreversible inactivation of both the cyclooxygenase and peroxidase activities of COX-1. Thus, these compounds behaved differently from most NSAIDs, which inhibit the cyclooxygenase activity only.…”

“…Therefore, some other mechanism must lead to tyrosyl radical formation. 253 In their exploration of alternative mechanisms of tyrosyl radical formation, Tang et al carried out comparative studies of the kinetics of compound I and compound II formation in COX-1 versus COX-2. 254 Using either 20:4 or EtOOH as substrate, they discovered that compound I and compound II formation in COX-1 was very rapid and preceded 20:4 oxygenation, as monitored simultaneously by oxygen electrode.…”

Synthesis and Anti‐HIV‐1 Evaluation of New Sonogashira‐Modified Emivirine (MKC‐442) Analogues

“…We have previously attempted to synthesize potential irreversible inhibitors of HIV-1 in our laboratory [28 -31]. Since bromoacetamides as nonsteroidal anti-inflammatory drugs (NSAID) analogues were employed successfully to map the active sites of hydroxysteroid dehydrogenases [32] [33], we saw a potential in this functionality. Encouraged by a recent report on benzyl halides Pd-coupling with terminal ethynes [34], we used the method to couple bromoacetylated 4-iodoaniline directly to compound 1a (Scheme 1).…”