Abstract

Prostaglandin synthase (PGHS) is a bifunctional enzyme of particular pharmaceutical interest, containing both cyclooxygenase (COX) and peroxidase (POX) active sites. Development of PGHS-2-selective POX inhibitors may reduce the toxic and carcinogenic effects of PGHS-2 xenobiotic activation, as well as further define the role of this activity in vivo . Knowledge of the contribution of specific contacts to binding will be crucial to drug design. The results of an investigation of the thermodynamics of binding of a series of aromatic hydroxamic acid analogues to the archetypical peroxidase horseradish peroxidase (HRP) revealed the importance of hydrogen bonds to the distal catalytic residues, in particular the distal arginine of HRP. The ability of peroxidases, including PGHS-2, to oxidize a wide variety of compounds makes the design of selective, reversible inhibitors very challenging. Therefore, the potential of aromatic hydrazides as mechanism-based peroxidase inhibitors was investigated here, again using HRP as a model peroxidase. The results suggest that benzhydrazide is a mechanism-based inhibitor of HRP. In general hydrazides were observed to be good inhibitors of PGHS-2 POX activity with IC 50 values in the 1-800 oM range and with 2-naphthoichydrazide (2-NZH) being the most potent (IC 50 = 1.6 oM. Comparison of the IC 50 values for PGHS-2 and HRP support the potential development of peroxidase-selective hydrazides. The COX activity of PGHS-2 is the target of nonsteroidal antiinflammatory drugs (NSAIDs). The effects of MAID binding on the structure and conformational stability of PGHS-2 were investigated here. Diclofenac was observed to cause the greatest reduction in the conformational flexibility of PGHS-2, possibly due to its strained (entropically unfavorable) but very enthalpically favorable binding conformation, in comparison with the other compounds of the fenamate family investigated. Both the COX and POX activities of PGHS-2 isozymes undergo rapid suicide inactivation. The mechanism of inactivation is not known, although it is likely radical-based. Stoichiometric incorporation of 14 C-AA was observed here on the time scale of inactivation, suggesting that the incorporation of activated substrate intermediates may be involved in COX inactivation. The negligible degree of H 2 O 2 -dependent intermolecular crosslinking observed here indicates that this is not a major mechanism of PGHS-2 inactivation.