Ouellet Marc, Aitken Susan M, English Ann M, Percival M David
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Que., Canada H9R 4P8.
Arch Biochem Biophys. 2004 Nov 1;431(1):107-18. doi: 10.1016/j.abb.2004.07.031.
The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.
双功能酶前列腺素H(2)合酶-2（PGHS-2）的环氧化酶活性是非甾体抗炎药的作用靶点。对PGHS过氧化物酶活性的抑制研究较少。利用索雷特吸收变化，检测了芳香异羟肟酸与PGHS-2过氧化物酶位点的结合，以研究抑制的结构决定因素。典型的哺乳动物过氧化物酶，苯甲异羟肟酸的解离常数（K(d)）为42mM，远大于水杨异羟肟酸的解离常数（475μM）。异羟肟酸替泊沙林（25μM）的结合仅导致索雷特吸收的微小变化。然而，替泊沙林是一种有效的还原性共底物，表明它是一种替代电子供体，而非过氧化物酶活性的抑制剂。芳香酰肼是过氧化物酶的代谢活化抑制剂。2-萘异羟肟酰肼（2-NZH）导致PGHS-2过氧化物酶和环氧化酶活性的时间和浓度依赖性抑制。两种PGHS-2活性的失活都需要过氧化氢，而吲哚美辛（结合于环氧化酶位点）不影响2-NZH的过氧化物酶抑制效力。发现一系列芳香酰肼是PGHS-2过氧化物酶活性的有效抑制剂，在所检测的18种酰肼中，有13种的半数抑制浓度（IC(50)）值在6-100μM范围内。某些环取代增加了PGHS-2相对于髓过氧化物酶和辣根过氧化物酶同工酶C的选择性抑制。特别是，酰肼部分对位的氯原子相对于髓过氧化物酶和辣根过氧化物酶同工酶C增加了PGHS-2的选择性。
