A genome-wide and dose-dependent inhibition map of androgen receptor binding by small molecules reveals its regulatory program upon antagonism

The androgen receptor plays a critical role throughout the progression of prostate cancer and is an important drug target for this disease. While chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) is becoming an essential tool in studying transcription and chromatin modification factors, it has rarely been employed in the context of drug discovery. Here we report the first publicly available genome-wide and dose-dependent inhibition landscape of AR binding by drug-like small molecules including correlation with binding strength using ChIP-Seq. Integration of sequence analysis, transcriptome profiling, cell viability assays and in vivo tumor inhibition studies enabled us to establish a direct cistrome-activity relationship for two novel potent AR antagonists. By selectively occupying the strongest binding sites, AR signaling remains active even when low androgen levels are low, a scenario characteristic of first-line androgen ablation therapy. Coupled cistrome and transcriptome profiling upon small molecule antagonism led to the identification of not only key direct downstream effectors of AR but also their mode of regulation: unbiased pathway mapping revealed that AR is a key modulator of steroid metabolism by forming a tightly controlled feedback loop with other nuclear receptor family members. Furthermore, we found AR has an extensive role in negative gene regulation and estrogen (related) receptor likely mediates its function as a transcriptional repressor. In conclusion, our study provides a global and dynamic view of AR’s regulatory program upon antagonism, which may serve as a molecular basis for deciphering and developing AR therapeutics. Overall design: GSM814034-GSM814044: Compartative study of the mRNA profiles of VCaP cells treated with small molecule inhibitor of AR and those treated with AR-siRNA GSM817346-GSM817354: Comparative study of AR binding in VCaP cells (1) in the presence and absence of the synthetic AR agonist metribolone (R1881) and (2) in the presence of R1881 and small molecule inhibitors of AR

雄激素受体（androgen receptor, AR）在前列腺癌的整个进展进程中发挥关键调控作用，同时也是该疾病的重要药物靶点。染色质免疫共沉淀结合高通量测序（chromatin immunoprecipitation coupled with massively parallel sequencing, ChIP-Seq）已成为研究转录与染色质修饰因子的核心实验技术，但该技术在药物研发场景中的应用却相对稀缺。本研究首次报道了首个公开可用的、由类药小分子介导的AR结合位点全基因组范围及剂量依赖性抑制图谱，其中包含基于ChIP-Seq数据的结合强度相关性分析。通过整合序列分析、转录组谱分析、细胞活力检测与体内肿瘤抑制实验，我们成功确立了两种新型强效AR拮抗剂的直接顺式调控组（cistrome）-活性关联。这类新型化合物可选择性占据最强结合位点，使得即使在雄激素水平较低的情况下AR信号通路仍保持激活——这一情景正是一线雄激素剥夺治疗的典型特征。通过对小分子拮抗处理下的顺式调控组与转录组进行联合分析，我们不仅鉴定出AR关键的直接下游效应因子，还阐明了其调控模式：无偏倚通路图谱分析显示，AR可通过与其他核受体家族成员形成严格受控的反馈环路，成为类固醇代谢的关键调控因子。此外，我们发现AR在基因负调控中具有广泛作用，而雌激素（相关）受体可能介导其作为转录抑制因子的功能。综上，本研究为拮抗作用下AR的调控程序提供了全局且动态的视角，可为解析与开发AR靶向治疗药物提供分子基础。整体实验设计：GSM814034-GSM814044：对经AR小分子抑制剂处理与AR小干扰RNA（AR-siRNA）处理的VCaP细胞的mRNA表达谱开展对比研究；GSM817346-GSM817354：针对VCaP细胞中AR结合情况的对比研究，具体包括(1) 合成型AR激动剂美曲勃龙（metribolone, R1881）存在与缺失条件下的对比；(2) 仅存在R1881与R1881联合AR小分子抑制剂条件下的对比。