Rational optimization of a transcription factor activation domain inhibitor

Transcription factors are among the most attractive therapeutic targets but are considered largely undruggable. Here we provide evidence that small molecule-mediated partitioning of the androgen receptor, an oncogenic transcription factor, into phase-separated condensates has therapeutic effect in prostate cancer. We show that the phase separation capacity of the androgen receptor is driven by aromatic residues and short unstable helices in its intrinsically disordered activation domain. Based on this knowledge, we developed tool compounds that covalently attach aromatic moieties to cysteines in the receptors’ activation domain. The compounds enhanced partitioning of the receptor into condensates, facilitated degradation of the receptor, inhibited androgen receptor-dependent transcriptional programs, and had antitumorigenic effect in mouse models of prostate cancer and castration resistant prostate cancer. These results establish a generalizable framework to target the phase-separation capacity of intrinsically disordered regions in oncogenic transcription factors and other disease-associated proteins with therapeutic intent. To understand the transcriptomic effects of our lead and engineered AR condensate targeting molecules (EPI-001 and 1ae), we dosed the drugs directly on LNCaP cells in RPMI-1640 5% FBS, for 6 and 24 hours. Vehicle control (DMSO) was matched for each time point. Samples were prepared in three biological replicates for each experimental condition. RNA was then extracted using a Zymo DirectZol Micro kit (Zymo R2062) according to the manufacturer’s protocol. Total RNA-seq libraries were then prepared using 1 µg of resulting RNA from each sample and KAPA RNA HyperPrep Kit with RiboErase (Roche KR1351) according to the manufacturer’s protocol with 10 amplification cycles. Libraries were sequenced on a NovaSeq 6000 with paired-end reads of 100 base pairs, with a read depth of 50 million fragments / library. Three libraries from three corresponding biological replicates were prepared for each treatment (time, dosage, and compound).