The prostaglandin-endoperoxide H synthase-1 (PGHS- 1) and prostaglandin-endoperoxide H synthase-2 (PGHS-2) are the targets of nonsteroidal anti-inflammatory drugs (NSAIDs). It appears that the high degree of selectivity for inhibition of PGHS-2 shown by certain compounds is the result of two mechanisms (time-dependent, time-independent inhibition), by which they interact with each isoform. Molecular models of the complexes formed by indomethacin, sulindac, fenamates, 2-phenylpropionic acids and selective cyclooxygenase-2 (COX-2) inhibitors with the cyclooxygenase active site of human PGHS-2 have been built, paying particular attention to water molecules that participate in the hydrogen-bonding network at the polar active site entrance. The stability of the complexes has been assessed by molecular dynamics simulations and interaction energy decomposition analysis, and their biological significance has been discussed in light of available X-ray crystallographic and kinetic results. The selective PGHS-2 inhibitors exploit the extra space of a side-pocket in the active site of PGHS-2 that is not found in PGHS-1. The results suggest that active site hydration together with residues Tyr355, Glu524, Arg120 and Arg513 are crucial to understand the time-dependent inhibition mechanism. A marked relationship between the isoform selectivity and tightly interactions with residues into the side pocket bordered by Val523 is also found.

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In the present study, we investigated the free radical-scavenging effects of eight Nonsteroidal Anti-Inflammatory Drugs (NSAID) using a Superoxide (O 2 .À ) and Nitric Oxide ( . NO) generating system in vitro. Indomethacine showed higher scavenging activity against O 2 .À , it being more potent than ibuprofen and nimesulide at the same concentration. Diclofenac showed a more relevant effect on the .NO scavenging activity than against O 2 .À . Comparison of the activities of eight compounds showed that the aryl-acetic compounds have higher scavenging activities than the diarylheterocyclic ones. We have also found that the O 2 scavenging activity correlates with the Next-Highest-Occupied pOrbital (NHOMO) levels and the Next-Lowest-Unoccupied p-Orbital (NLUMO)-NHOMO gaps correlate with Cyclooxygenase-1 (COX-1) inhibitory activity. These results suggest that both NHOMO and NHOMO-NLUMO gap energies can be of predictive value in the search for new candidate anti-inflammatory drugs.

PGHS-1 and PGHS-2 are the targets of nonsteroidal anti-inflammatory drugs (NSAIDs). It appears that the high degree of selectivity for inhibition of PGHS-2 shown by certain compounds is the result of two mechanisms (time-dependent and time-independent inhibition), by which they interact with each isoform. The fenamic acids can be divided into competitive inhibitors of substrate binding and competitive inhibitors that cause time-dependent losses of cyclooxygenase activity. The cyclooxygenase activity was measured by oxygen consumption following preincubation of the enzyme and the inhibitor for increasing periods of time. The rate constants associated with binding inhibition kinetics and structure-activity relationships were calculated for a large number of fenamates, diclofenac and indomethacin. The K1* values are similar but the individual rate constants are markedly different: K1 is two-fold lower, and k2 is six-fold slower for diclofenac than for indomethacin. All the active time-dependent compounds show MEPs with a negative conical surface, with their vertex on the minimum of the carboxyl group, which extends around the first aromatic ring to the central region. The conical surface keeps an open angle of 61 degrees or larger, and a close contact surface with the residues Ala527, Ileu523, Val349, and Ser530, in the zones surrounding the bridging amino group and the chlorine atoms for meclofenamate and diclofenac, or in the region around the carbonyl group for indomethacin. The K1* and IC50 values indicate that the interactions that promote the slow binding kinetics must be examined in relation to the reaction energies of formation (delta Hr) of an ionic bond between the deprotonated carboxylic acid group of acid NSAIDs with the monocationic guanidinum group of Arg120, the free energies of solvation in aqueous solution, and the molecular volumes measured. Presumably indomethacin, diclofenac and meclofenamate cause the enzyme to undergo a subtle conformational change to a form that binds compounds even more tightly, with some slight structural changes confined to reorientations of the Arg277 and Gln358 side chains. These results show that the model has reliably chosen regions of biological significance consistent with both the X-ray crystallographic and kinetic results.