Inhibiting Androgen Receptor Splice Variants With Cysteine-Selective Irreversible Covalent Inhibitors To Treat Prostate Cancer

Authors

Thirumagal Thiyagarajan
Suriyan Ponnusamy
Dong-Jin Hwang
Yali He
Sarah Asemota
Kirsten L Young
Daniel L Johnson
Vera Bocharova
Weidong Zhou
Abhinav K Jain
Emanuel F Petricoin
Zheng Yin
Lawrence M Pfeffer
Duane D Miller
Ramesh Narayanan

Publication Date

1-3-2023

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abstract

Androgen receptor (AR) and its splice variants (AR-SVs) promote prostate cancer (PCa) growth by orchestrating transcriptional reprogramming. Mechanisms by which the low complexity and intrinsically disordered primary transactivation domain (AF-1) of AR and AR-SVs regulate transcriptional programming in PCa remains poorly defined. Using omics, live and fixed fluorescent microscopy of cells, and purified AF-1 and AR-V7 recombinant proteins we show here that AF-1 and the AR-V7 splice variant form molecular condensates by liquid-liquid phase separation (LLPS) that exhibit disorder characteristics such as rapid intracellular mobility, coactivator interaction, and euchromatin induction. The LLPS and other disorder characteristics were reversed by a class of small-molecule-selective AR-irreversible covalent antagonists (SARICA) represented herein by UT-143 that covalently and selectively bind to C406 and C327 in the AF-1 region. Interfering with LLPS formation with UT-143 or mutagenesis resulted in chromatin condensation and dissociation of AR-V7 interactome, all culminating in a transcriptionally incompetent complex. Biochemical studies suggest that C327 and C406 in the AF-1 region are critical for condensate formation, AR-V7 function, and UT-143's irreversible AR inhibition. Therapeutically, UT-143 possesses drug-like pharmacokinetics and metabolism properties and inhibits PCa cell proliferation and tumor growth. Our work provides critical information suggesting that clinically important AR-V7 forms transcriptionally competent molecular condensates and covalently engaging C327 and C406 in AF-1, dissolves the condensates, and inhibits its function. The work also identifies a library of AF-1-binding AR and AR-SV-selective covalent inhibitors for the treatment of PCa.

Keywords

Male, Humans, Receptors, Androgen, Cysteine, Prostatic Neoplasms, Androgen Receptor Antagonists, Prostatic Neoplasms, Castration-Resistant, Cell Line, Tumor, Protein Isoforms

Comments

Supplementary Materials

PMID: 36577061