Hörnig Michael, Markoutsa Stavroula, Häfner Ann-Kathrin, George Sven, Wisniewska Joanna M, Rödl Carmen B, Hofmann Bettina, Maier Thorsten, Karas Michael, Werz Oliver, Steinhilber Dieter
Institute of Pharmaceutical Chemistry/ZAFES, University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
Biochim Biophys Acta. 2012 Feb;1821(2):279-86. doi: 10.1016/j.bbalip.2011.11.001. Epub 2011 Nov 23.
U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.
最初被鉴定为磷脂酶C抑制剂的U73122是纯化的5-脂氧合酶（5-LO）的一种强效直接抑制剂，其IC50值为30 nM。5-LO催化花生四烯酸（AA）转化为白三烯，白三烯是参与炎症和过敏反应以及宿主对微生物防御反应的介质。由于对人5-LO酶的有效抑制取决于U73122马来酰亚胺基团的硫醇反应性，我们利用这一特性来鉴定5-LO蛋白中对U73122抑制5-LO重要的半胱氨酸残基。我们通过基质辅助激光解吸电离质谱法（MALDI-MS）发现，U73122与半胱氨酸残基99、159、248、264、416和449共价结合。将Cys416突变为丝氨酸会强烈降低U73122对5-LO的抑制作用，并且靠近Cys416的另外三个半胱氨酸的额外突变会进一步削弱该化合物对5-LO的抑制作用。用U73122和5-LO进行的洗脱实验表明U73122存在不可逆结合。总之，我们的数据表明，靠近向活性位点的拟议AA进入通道的Cys416周围区域是开发新型5-LO抑制剂的一个有趣靶点。
