DEVELOPMENT OF AFFINITY LABELING AGENTS BASED ON NONSTEROIDAL ANTIINFLAMMATORY DRUGS - LABELING OF THE NONSTEROIDAL ANTIINFLAMMATORY DRUG-BINDING SITE OF 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effect by inhibiting the target enzyme cyclooxygenase (prostaglandin H-2 synthase); however, little is known about the peptides comprising its NSAID binding site. Hydroxyprostaglandin dehydrogenases also bind NSAIDs, but their NSAID binding sites have not been well characterized. Using existing synthetic strategies, we have incorporated the bromoacetoxy affinity labeling moiety around the perimeter of two potent NSAIDs, indomethacin and mefenamate, a N-phenylanthranilate. The compounds synthesized were 1-(4-(bromoacetamido)benzyl)-5-methoxy-2-methylindole-3-acetic acid (1), 3-(2-(2-bromoacetoxy)ethyl)-1-(4-chlorobenzyl)-5-methoxy-2-methylindole (2), 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (3), N-(3-(bromoacetamido)phenyl)-anthranilic acid (4), and N-(4-(bromoacetamido)phenyl)anthranilic acid (5). To access whether these compounds have general utility in labeling NSAID binding sites, the compounds were evaluated as affinity labeling agents for 3-alpha-hydroxysteroid dehydrogenase (3-alpha-HSD) from rat liver cytosol. This enzyme displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity, is inhibited potently by NSAIDs, and is homologous to bovine lung prostaglandin F synthase. Compounds 1-5 were shown to affinity label the NSAID binding site of 3-alpha-HSD. They inactivated 3-alpha-HSD through an E.I complex in a time- and concentration-dependent manner with 11/2 values ranging from seconds to hours. Ligands that compete for the active site of 3-alpha-HSD (NAD+ and indomethacin) afforded protection against inactivation, and the inactivators could demonstrate competitive kinetics against 3-alpha-hydroxysteroid substrates by forming an E.NAD+.I complex. Further, when compounds 1-3 were radiolabeled with [C-14]bromoacetate, inactivation of 3-alpha-HSD was accompanied by a stoichiometric incorporation of inactivator, indicating the labeling of discrete amino acids at the enzyme active site. This analysis suggests that these NSAID analogues may have general utility in affinity labeling the drug binding site of NSAID target enzymes.