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）抑制雄激素向 双氢睾酮（DHT），阻断雄激素与雄激素受体（AR）的结合或AR信号传导。虽然 这些治疗最初有效，但由于AR剪接变异体（AR-SV）等因素最终失败， AR对雄激素以外的刺激做出反应。一种有效控制发病和治疗的方法 去势抵抗性前列腺癌（CRPC）的目的是开发小分子， 或促进其降解。如果不存在AR，则将不存在AR信令，无论是否或如何 CRPC中存在大量雄激素或其它AR信号刺激物。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 体外和体内AR阳性（AR+：IC 50 ~35 µ M）而非AR阴性（AR-：IC 50 ~70 µM）CRPC 模型（50 mg/kg/体重）。由于需要更高浓度的UroA来抑制CRPC生长， 我们的目的是设计和开发有效的UroA类似物，可以选择性地抑制AR激活， 浓度（nM），是口服生物可利用的，并抑制CRPC的生长。后续发展 一系列的UroA类似物和构效关系（SAR）的研究导致了两个鉴定 新的先导化合物ASR-600和ASR-603（US临时62/941588），其表现出更好的 在CRPC细胞中抑制AR信号传导的功效（nM）高于UroA（ASR-600：>40倍和ASR-603：>12倍） 线分子研究表明这两个ASR通过泛素化抑制AR和AR-SV的表达 以及在胞质溶胶中的降解。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 结合AR的N-末端结构域（NTD）并阻断AR和AR-SV的活化。在生理 在可达到的浓度下，ASR-600消除AR+和AR-SV（C4-2B和22 Rv 1）肿瘤生长， 异种移植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来抑制和/或促进AR和AR-SV表达的降解 抑制CRPC的生长。我们将通过以下具体目标来检验这一假设。目的 1.研究ASR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表达的机制，并优化ASRs的剂量。目标3：确定治疗 ASR抑制原位和患者来源的异种移植物（PDX）模型中CRPC生长的功效。的 拟议的研究将阐明ASR的作用机制，并导致更好地了解 这些化合物对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.