深入了解PBRM（一种17β-羟类固醇脱氢酶的共价类固醇抑制剂）的作用方式和选择性。,BIOCHEMICAL PHARMACOLOGY

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深入了解PBRM（一种17β-羟类固醇脱氢酶的共价类固醇抑制剂）的作用方式和选择性。
BIOCHEMICAL PHARMACOLOGY ( IF 5.3 ) Pub Date : 2017-08-13 , DOI: 10.1016/j.bcp.2017.08.004
Alexandre Trottier ₁ , René Maltais ₁ , Diana Ayan ₁ , Xavier Barbeau ₂ , Jenny Roy ₁ , Martin Perreault ₁ , Richard Poulin ₁ , Patrick Lagüe ₃ , Donald Poirier ₄

Affiliation

17β-羟基类固醇脱氢酶1（17β-HSD1）参与雌二醇的生物合成，雌二醇是哺乳动物中主要的生物活性内源性雌激素，并构成了依赖雌激素的疾病的有趣治疗靶标。最近，甾族衍生物3-{[（（16β，17β）-3-（2-溴乙基）-17-羟基雌二醇1,3,5（10）-三烯-16-基]甲基}苯甲酰胺（PBRM）被描述为17β-HSD1的非雌激素，不可逆抑制剂。但是，该抑制剂的作用方式及其选择性特征尚未阐明。我们通过体外动力学测量评估了PBRM的效力。还通过种间比较（人，小鼠，猪和猴子），通过体外测定和计算机分析，研究了酶灭活的机制。确定了小鼠和人血浆蛋白与PBRM的结合，而使用多种潜在的脱靶靶标（例如GPCR，hERG，CYP等）研究了其选择性。用纯化的17β-HSD1测定PBRM的亲和常数（Ki = 368nM）和酶失活率（kinact = 0.087min-1）值。发现PBRM与该酶共价连接。用PBRM预处理乳腺癌和胎盘细胞系后，需要较长的延迟时间（即3-5天）才能恢复17β-HSD1活性。机理分析表明，17β-HSD1抑制的重要种间差异支持了灭活对PBRM效应的重要性。

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Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17β-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off-targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki=368nM) and the enzyme inactivation rate (kinact=0.087min-1) values for PBRM were determined with purified 17β-HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3-5days) is required to recover 17β-HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17β-HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non-estrogenic and steroidal covalent irreversible inhibitor of 17β-HSD1 warrant its further development as a potential drug candidate for estrogen-dependent disorders.

更新日期：2019-11-01

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