Cyclooxygenase-2 (COX-2) is the key enzyme involved in the synthesis pathway of prostaglandin G2 (PGG2) by transformation of arachidonic acid (AA). Although COX-2 is one of the principal pharmacological targets by the implication of PGG2 in several human diseases, the classical all-radical mechanism proposed for COX-2 catalysis has never been validated at the molecular level. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were combined to analyze the six steps of the all-radical mechanism. The results show that O2 addition on C11 of AA can follow an antarafacial or suprafacial approach with respect to tyrosine 385, but only the antarafacial addition leads to the product with the correct 11R stereochemistry as established in the mechanistic proposal. Moreover, only the reaction pathway coming from the antarafacial intermediate describes a viable 8,12-cyclization to form the prostaglandin-like bicyclo endoperoxide that finally leads, by kinetic control, to PGG2 with the 15S stereochemistry found experimentally. The formation of the more stable trans ring isomer of natural PGG2 in an enzymatic environment is also explained. Our molecular analysis shows how COX-2 uses its relatively narrow channel in the active site to restrain certain conformational changes of AA and of the reaction intermediates, so that the PGG2 enzymatic synthesis turns out to be highly regiospecific and stereospecific. A more recent 10-step carbocation-based mechanistic proposal has been discarded.

Cyclooxygenase-2 (COX-2) catalyzes the formation of prostaglandin G2 (PGG 2 ) from arachidonic acid (AA). Its activity is substantially altered by aspirin, a nonsteroidal antiinflammatory drug that irreversibly inhibits COX-2 by acetylation of its Ser530 residue. So far, it has been widely accepted that in the aspirin-acetylated COX-2, PGG 2 synthesis is blocked, whereas the acetylated enzyme still catalyzes the formation of hydroxyeicosatetraenoic acid 15R-HETE as the main product. However, recent experiments detected residual COX activity in recombinant human acetylated COX-2 with formation of prostaglandins, a surprising result that could not be rationalized. In fact, a complete molecular understanding of aspirin action is still lacking, and contradictory explanations have been given for the formation of the highly controlled stereochemical products. We have combined here molecular dynamics simulations and quantum mechanics/molecular mechanics calculations to study each step of the aspirininhibited catalytic mechanism. Our results confirm that 15R-HPETE (then reduced to 15R-HETE) is the main product of the AA oxidation catalyzed by aspirin-acetylated COX-2, being clearly more favorable than the formation of 15S-HPETE. The 15R-HPETE synthesis is kinetically dominant and inhibits the formation of the (9R, 11R) bicyclo endoperoxides to give PGH 2 , which can still be obtained as a minor product, as detected experimentally. Instead, the synthesis of isoprostanes does not happen because it is not kinetically competitive at all with the formation of 15R-HETE.

Ebselen is a potent competitive inhibitor of the active form of rabbit 15-lipoxygenase, an enzyme involved in many inflammatory diseases. Light-induced Z-to-E isomerization of the ebselen-like 2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester (BODTCM) molecule was used to convert the weak (Z)-BOTDCM inhibitor into the (E)-isomer with much higher inhibitory capacity. In this study, the binding modes of ebselen, (E)-BOTDCM and (Z)-BOTDCM, have been analyzed to provide molecular insights on the inhibitory potency of ebselen and on the geometric-isomer specificity of (E)- and (Z)-BOTDCM inhibitors. The inhibitor-enzyme structures obtained from docking and molecular dynamics simulations as well as from QM/MM calculations show that the inhibitor molecules are not coordinated to the nonheme iron in the active site. Thermal motion allows ebselen and (E)-BOTDCM to visit a wide range of the configurational space competing with the polyunsaturated fatty acid for binding at the active site. Both molecules present similar MM/PBSA binding free energies. The energy penalty for the bigger geometric deformation undergone by (E)-BODTCM would explain its lower inhibitor potency. The (Z)-isomer is the weakest inhibitor because thermal motion moves it to a region very far from the first coordination sphere of Fe, where it could not compete with the fatty acid substrate.

Gly526Ser COX-2 does not produce prostaglandins but 8,9-11,12-diepoxy derivatives of arachidonic acid.

Specialized pro-resolving lipid mediators (SPMs) are natural bioactive agents actively involved in inflammation resolution. SPMs act when uncontrolled inflammatory processes are developed, for instance, in patients of COVID-19 or other...