Zammit Charlotte M, Nadel Cory M, Lin Ying, Koirala Sajjan, Ahani Elnaz, Potts Patrick Ryan, Nomura Daniel K
Department of Chemistry, University of California, Berkeley, Berkeley, California 94720 United States.
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720 United States.
J Am Chem Soc. 2025 Jun 18;147(24):20512-20524. doi: 10.1021/jacs.5c02801. Epub 2025 Jun 9.
Androgen-independent prostate cancers, correlated with heightened aggressiveness and poor prognosis, are caused by mutations or deletions in the androgen receptor (AR) or the expression of truncated variants of AR that are constitutively activated. Currently, drugs and drug candidates against AR target the steroid-binding domain to antagonize or degrade AR. However, these compounds cannot therapeutically access largely intrinsically disordered truncated splice variants of AR, such as AR-V7, which only possess the N-terminal transactivation domain and DNA-binding domain and are missing the ligand-binding domain. Targeting intrinsically disordered regions within transcription factors has remained challenging and is considered "undruggable". Herein, we leverage a cysteine-reactive covalent ligand library in a cellular screen to identify the degraders of AR and AR-V7 in androgen-independent prostate cancer cells. We identified a covalent compound, EN1441, that selectively degrades AR and AR-V7 in a proteasome-dependent manner through direct covalent targeting of intrinsically disordered cysteine C125 in the N-terminal transactivation domain of AR and AR-V7. EN1441 causes significant and selective destabilization of AR and AR-V7, leading to the aggregation of AR/AR-V7 and subsequent proteasome-mediated degradation. Consistent with targeting both AR and AR-V7, we find that EN1441 completely inhibits total AR transcriptional activity in androgen-independent prostate cancer cells expressing both AR and AR-V7 compared with AR antagonists or degraders that only target the ligand-binding domain of full-length AR, such as enzalutamide and ARV-110. Our results put forth a pathfinder molecule EN1441 that targets an intrinsically disordered cysteine within AR to destabilize, degrade, and inhibit both AR and AR-V7 in androgen-independent prostate cancer cells and highlights the utility of covalent ligand discovery approaches in directly targeting, destabilizing, inhibiting, and degrading classically undruggable transcription factor targets.
雄激素非依赖性前列腺癌与侵袭性增强和预后不良相关，是由雄激素受体（AR）的突变或缺失或组成性激活的AR截短变体的表达引起的。目前，针对AR的药物和候选药物靶向类固醇结合域以拮抗或降解AR。然而，这些化合物无法在治疗上作用于AR的大量内在无序截短剪接变体，如AR-V7，其仅具有N端反式激活域和DNA结合域，而缺少配体结合域。靶向转录因子内的内在无序区域一直具有挑战性，被认为是“不可成药的”。在此，我们在细胞筛选中利用半胱氨酸反应性共价配体文库来鉴定雄激素非依赖性前列腺癌细胞中AR和AR-V7的降解剂。我们鉴定出一种共价化合物EN1441，它通过直接共价靶向AR和AR-V7的N端反式激活域中内在无序的半胱氨酸C125，以蛋白酶体依赖性方式选择性降解AR和AR-V7。EN1441导致AR和AR-V7显著且选择性地不稳定，导致AR/AR-V7聚集并随后被蛋白酶体介导降解。与靶向AR和AR-V7一致，我们发现与仅靶向全长AR配体结合域的AR拮抗剂或降解剂（如恩杂鲁胺和ARV-110）相比，EN1441在同时表达AR和AR-V7的雄激素非依赖性前列腺癌细胞中完全抑制总AR转录活性。我们的结果提出了一种先导分子EN1441，其靶向AR内的内在无序半胱氨酸，以使雄激素非依赖性前列腺癌细胞中的AR和AR-V7不稳定、降解并抑制它们，并突出了共价配体发现方法在直接靶向、使经典不可成药的转录因子靶点不稳定、抑制和降解方面的效用。
