The structural and electronical factors that contribute affinity for the time-dependent inhibition of PGHS-1 by indomethacin, diclofenac and fenamates

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 P GHS-2 shown by certain compounds is the result of two mechanisms (time-depe ndent and time-independent inhibition), by which they interact with each is oform. The fenamic acids can be divided into competitive inhibitors of subs trate binding and competitive inhibitors that cause time-dependent losses o f cyclooxygenase activity. The cyclooxygenase activity was measured by oxyg en consumption following preincubation of the enzyme and the inhibitor for increasing periods of time. The rate constants associated with binding inhi bition kinetics and structure-activity relationships were calculated for a large number of fenamates, diclofenac and indomethacin. The K-I* values are similar but the individual rate constants are markedly different: k(1) is two-fold lower, and k(2) is six-fold slower for diclofenac than for indomet hacin. All the active time-dependent compounds show MEPs with a negative co nical surface, with their vertex on the minimum of the carboxyl group, whic h 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 s urface with the residues Ala(527), Ileu(523), Val(349), and Ser(530), in th e zones surrounding the bridging amino group and the chlorine atoms for mec lofenamate and diclofenac, or in the region around the carbonyl group for i ndomethacin. The K-I* and IC50 values indicate that the interactions that p romote the slow binding kinetics must be examined in relation to the reacti on energies of formation (Delta H-r) of an ionic bond between the deprotona ted carboxylic acid group of acid NSAIDs with the monocationic guanidinum g roup of Arg(120), the free energies of solvation in aqueous solution, and t he molecular volumes measured. Presumably indomethacin, diclofenac and mecl ofenamate cause the enzyme to undergo a subtle conformational change to a f orm that binds compounds even more tightly, with some slight structural cha nges confined to reorientations of the Arg(277) and Gln(358) side chains. T hese results show that the model has reliably chosen regions of biological significance consistent with both the X-ray crystallographic and kinetic re sults.