Cyclooxygenases (COXs) are the therapeutic targets of nonsteroidal antiinflammatory drugs. Indomethacin (INDO) was one of the first nonsteroidal antiinflammatory drugs to be characterized as a time-dependent, functionally irreversible inhibitor, but the molecular basis of this phenomenon is uncertain. In the crystal structure of INDO bound to COX-2, a small hydrophobic pocket was identified that surrounds the 2'-methyl group of INDO. The pocket is formed by the residues Ala-527, Val-349, Ser-530, and Leu-531. The contribution of this pocket to inhibition was evaluated by altering its volume by mutagenesis of Val-349. The V349A mutation expanded the pocket and increased the potency of INDO, whereas the V349L mutation reduced the size of the pocket and decreased the potency of INDO. Particularly striking was the reversibility of INDO inhibition of the V349L mutant. The 2'-des-methyl analogue of INDO (DM-INDO) was synthesized and tested against wild-type COX-1 and COX-2, as well as the Val-349 mutants. DM-INDO bound to all enzymes tested, but only inhibited wt mCOX-2 and the V349I enzyme. Without the 2'-methyl group anchoring DM-INDO in the active site, the compound was readily competed off of the enzyme by arachidonic acid. The kinetics of inhibition were comparable to the kinetics of binding as evaluated by fluorescence quenching. These results highlight binding of the 2'-methyl of INDO in the hydrophobic pocket as an important determinant of its time-dependent inhibition of COX enzymes.
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