Segal Cancer Centre and Lady Davis Institute for Medical Research, Sir Mortimer B Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada.
Proteins. 2010 Feb 15;78(3):623-37. doi: 10.1002/prot.22592.
Antiandrogen flutamide, an antagonist of the wild-type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist-agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand-binding domain (AR-LBD), we have investigated the impact of the T877A mutation on ligand-induced helix-12 positioning by replica-exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR-LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild-type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non-agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan-antiandrogen effective against the wild-type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity.
雄激素拮抗剂氟他胺，一种野生型雄激素受体（AR）的拮抗剂，临床上用于治疗转移性前列腺癌。然而，T877A 突变的 AR 却被羟氟他胺（氟他胺的活性形式）反常地激活。尽管有晶体学研究，但 T877A 突变如何导致羟氟他胺的拮抗剂-激动剂转换仍然是一个谜。在这里，我们从 AR 配体结合域（AR-LBD）的apo 形式的结构模型出发，通过具有独特方案的 replica-exchange 分子动力学（REMD）模拟研究了 T877A 突变对配体诱导的螺旋-12 定位的影响，该方案能够模拟 H12 动力学并保持 AR-LBD 的主体不变。具体来说，（i）我们通过计算证明，在羟氟他胺结合时，T877A 突变体中的 apo 形式的 H12 重组成激动剂形式，而在野生型受体中重组成拮抗剂形式，阐明了羟氟他胺的激动/拮抗作用；（ii）通过 REMD 模拟，我们预测了非甾体抗雄激素化合物 SC184 不是 T877A 突变体的激动剂。这通过荧光素酶测定得到了证实；（iii）基于模拟中羟氟他胺和 SC184 的结合模式，我们设计了一种新的氟他胺衍生物称为 SC333，随后预测其为 T877A 突变体的纯拮抗剂。我们随后合成并通过实验证实，SC333 是一种有效的泛雄激素拮抗剂，对野生型和 T877A 和 W741C 突变的 AR 均有效，在 LNCaP 细胞中具有低微摩尔的细胞毒性。重要的是，我们证明了从 REMD 模拟中得到的 H12 构象分布与配体的激动/拮抗剂活性相关。
