Targeting AR and AR-Variants in Castration-Resistant Prostate Cancer

项目概要/摘要 目前的 AR 抑制剂（恩杂鲁胺和阿比特龙）抑制雄激素转化为 二氢睾酮 (DHT) 阻断雄激素与雄激素受体 (AR) 的结合，或阻断 AR 信号传导。虽然 这些治疗最初有效，但由于 AR 剪接变异 (AR-SV) 等因素最终失败 AR 对雄激素以外的刺激作出反应。控制出现和治疗的有效方法 去势抵抗性前列腺癌 (CRPC) 的治疗方法是开发抑制 AR 表达的小分子 或促进其降解。如果没有 AR，就不会有 AR 信令，无论是否或如何 CRPC 中存在大量雄激素或 AR 信号传导的其他刺激物。我们的初步研究表明 天然存在的尿石素 A (UroA) 抑制 AR 信号传导并选择性抑制 AR 阳性 (AR : IC50 ~35 µM) 但非 AR 阴性 (AR-: IC50 ~70 µM) CRPC 体外和体内 模型（50 毫克/公斤/体重）。由于抑制 CRPC 生长需要更高浓度的 UroA， 我们的目标是设计和开发有效的 UroA 类似物，可以在较低的条件下选择性抑制 AR 激活 浓度（nM），具有口服生物利用度并抑制 CRPC 的生长。后续发展 一系列 UroA 类似物和构效关系 (SAR) 研究导致鉴定出两种 新型先导化合物 ASR-600 和 ASR-603（美国临时 62/941588）表现出更好的性能 在 CRPC 细胞中抑制 AR 信号传导的功效（nM）比 UroA（ASR-600：>40 倍和 ASR-603：>12 倍） 线。分子研究表明这两种 ASR 通过泛素化抑制 AR 和 AR-SV 的表达 以及细胞质内的降解。分子动力学（MD）系统模拟研究、生物物理分析 基于核磁共振（NMR）和热位移分析表明这些小分子 与 AR 的 N 端结构域 (NTD) 结合并阻断 AR 和 AR-SV 的激活。在生理上 在可达到的浓度下，ASR-600 消除了 AR 和 AR-SV（C4-2B 和 22Rv1）肿瘤生长 异种移植物。根据我们的初步结果，我们假设“合理设计的 ASR 将有效地 通过直接靶向 AR-NTD 抑制和/或促进 AR 和 AR-SV 表达的降解 并抑制CRPC的生长。我们将通过以下具体目标来检验这一假设。目标 1. 研究ASRs对AR CRPC细胞的作用机制。目标 2. 确定体内信号传导 AR 和 AR-SV 表达机制并优化 ASR 剂量。目标 3. 确定治疗方案 ASR 在原位和患者来源的异种移植 (PDX) 模型中抑制 CRPC 生长的功效。的 拟议的研究将阐明 ASR 的作用机制，并导致更好地理解 这些化合物针对 CRPC 的化疗特性。

Project Summary/Abstract Current AR inhibitors (enzalutamide and abiraterone) inhibit either the conversion of androgen to dihydrotestosterone (DHT), block androgen binding to androgen receptor (AR), or AR signaling. Although initially effective, these treatments eventually fail because of factors such as, AR-splice variants (AR-SVs) and AR responding to stimuli other than androgen. An effective way to control the emergence and treatment of castration-resistant prostate cancer (CRPC) is to develop small molecules that either inhibit AR expression or promote its degradation. If there is no AR, there would be no AR signaling, regardless of whether or how much androgen or other stimuli of AR signaling are present in CRPC. Our preliminary studies demonstrated that naturally occurring Urolithin A (UroA) inhibited the AR signaling and selectively suppressed the growth of AR-positive (AR+: IC50 ~35 M) but not AR-negative (AR-: IC50 ~70 µM) CRPC in both in vitro and in vivo models (50 mg/kg/body weight). As higher concentrations of UroA are required to suppress CRPC growth, we aimed to design and develop potent UroA analogs that could selectively inhibit AR activation at lower concentrations (nM), are orally bioavailable and suppress the growth of CRPC. The subsequent development of a series of UroA analogs and structure-activity relationship (SAR) studies led to the identification of two novel lead compounds ASR-600 and ASR-603 (US Provisional 62/941588) which demonstrated better efficacy (nM) than UroA (ASR-600: >40-fold and ASR-603: >12-fold) in inhibiting AR signaling in CRPC cell lines. Molecular studies suggest these two ASRs inhibit both AR and AR-SVs expression via ubiquitination and degradation within the cytosol. Molecular dynamic (MD) system simulation studies, biophysical analysis based on nuclear magnetic resonance (NMR), and thermal shift assays showed that these small molecules bind to the N-terminal domain (NTD) of AR and block the activation of both AR and AR-SV. At physiologically achievable concentrations, ASR-600 abrogated AR+ and AR-SV (C4-2B and 22Rv1) tumor growth in xenografts. Based on our preliminary results, we hypothesized that ‘Rationally designed ASRs will effectively inhibit and/or promote the degradation of both AR and AR-SV expression by directly targeting the AR-NTD and inhibit the growth of CRPC. We will test this hypothesis with the following specific aims. Aim 1. Investigate the mechanism of action of ASRs on AR+ CRPC cells. Aim 2. Determine in vivo signaling mechanism of AR and AR-SVs expression and optimize the dose of ASRs. Aim 3. Determine the therapeutic efficacy of the ASRs to inhibit CRPC growth in orthotopic and patient-derived xenograft (PDX) models. The proposed studies will elucidate the mechanism of action of the ASRs and lead to a better understanding of the chemotherapeutic properties of these compounds against CRPC.