• Konkle ME, Blobaum AL, Moth CW, Prusakiewicz JJ, Xu S, Ghebreselasie K, Akingbade D, Jacobs AT, Rouzer CA, Lybrand TP, Marnett LJ. Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics. Biochemistry. 2016 Jan 19;55(2). 348-59. PMID: 26704937 [PubMed]. PMCID: PMC4721528.

Abstract 

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.