In silico discovery of potent small molecule inhibitors targeting the AR N-terminal domain for treating castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) remains an unsolved clinical challenge due to resistance mechanisms that occur upon treatment with androgen receptor pathway inhibitors (ARPI). Since the hallmarks of resistance are (i) AR amplification, (ii) the occurrence of splice variants like AR-V7 and (iii) mutations in the ligand binding domain (LBD), the novel concept of targeting the disordered N-terminal domain (NTD) of the androgen receptor is a compelling way of inhibiting AR in the context of castration-resistance. Here, we describe the discovery of small-molecule AR-NTD inhibitors by leveraging Peptone's HDX-MS and in silico platform. A virtual screen against a highly dynamic segment of the NTD yielded binders that were further developed into a series with potent in vivo activity. Binding was confirmed by multiple biophysical approaches, including a proprietary ultra-fast mixing hydrogen-deuterium exchange mass spectrometry (HDX-MS), NMR spectroscopy, spectral shift analysis, and cellular NanoBit assays, all of which validated direct interaction with the NTD. Our lead compounds exhibit potent cellular activity across a range of in vitro models. In AR full-length (FL-AR) and AR-V7 luciferase reporter assays, as well as LNCaP proliferation assays, potencies between 10-100 nM were obtained. Activity in the VCaP CRPC cell model was similarly strong, clearly outperforming standard-of-care compounds like Enzalutamide and Darolutamide and reaching equal potency to PROTACs. Mode of action (MoA) studies demonstrated that the lead compounds suppress the formation of FL-AR and AR-V7 homodimers (NanoBit assay), inhibit nuclear translocation of AR, and can efficiently downregulate AR-target genes in a dose-dependent manner in qPCR assays. The lead series displays drug-like physicochemical, ADME, and pharmacokinetic profiles, conducive to rapid preclinical and clinical development, while providing distinct differentiation over novel modalities such as PROTACs and RIPTACs. Our findings highlight the therapeutic promise of targeting the AR-NTD in CRPC via a unique, structure-driven approach, with small molecules that combine a novel mechanism with potent anti-tumor activity.