Assays to Interrogate the Ability of Compounds to Inhibit the AF-2 or AF-1 Transactivation Domains of the Androgen Receptor

Abstract

Prostate cancer is the leading cause of cancer and second leading cause of cancer-related death in men in the United States. Twenty percent of patients receiving the standard of care androgen deprivation therapy (ADT) eventually progress to metastatic and incurable castration-resistant prostate cancer (CRPC). Current FDA-approved drugs for CRPC target androgen receptor (AR) binding or androgen production, but only provide a 2-to 5-month survival benefit due to the emergence of resistance. Overexpression of AR coactivators and the emergence of AR splice variants, both promote continued transcriptional activation under androgen-depleted conditions and represent drug resistance mechanisms that contribute to CRPC progression. The AR contains two transactivation domains, activation function 2 (AF-2) and activation function 1 (AF-1), which serve as binding surfaces for coactivators involved in the transcriptional activation of AR target genes. Full-length AR contains both AF-2 and AF-1 surfaces, whereas AR splice variants only have an AF-1 surface. We have recently prosecuted a high-content screening campaign to identify hit compounds that can inhibit or disrupt the protein-protein interactions (PPIs) between AR and transcriptional intermediary factor 2 (TIF2), one of the coactivators implicated in CRPC disease progression. Since an ideal inhibitor/ disruptor of AR-coactivator PPIs would target both the AF-2 and AF-1 surfaces, we describe here the development and validation of five AF-2-and three AF-1-focused assays to interrogate and prioritize hits that disrupt both transactivation surfaces. The assays were validated using a test set of seven known AR modulator compounds, including three AR antagonists and one androgen synthesis inhibitor that are FDA-approved ADTs, two investigational molecules that target the N-terminal domain of AR, and an inhibitor of the Hsp90 (heat shock protein) molecular chaperone.

The Role of Androgens and Androgen Receptor in Human Bladder Cancer

“…Fancher et al, 2019 also report androgen receptor coactivators that have elevated expression in patients with prostate cancer, such as transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator (SRC)-1, RAC3, p300, CBP, Tip60, and ARA70 [ 24 ].…”

Current and emerging approaches to noncompetitive AR inhibition

“…67 Johnston et al have since reported the development of five assays that target the AF-2 domain, although the structures of the hits have not been disclosed to date. 68 More recent work has utilized computational modeling to screen the ZINC database for chemical entities that interact with the AR AF-2. This led to the synthesis of an oxadiazole series of which LHJ-647 was the most potent (Figure 7A).…”

“…Of the 286 confirmed active compounds, approximately 60% were able to inhibit or disrupt AR‐TIF2 PPIs with an IC 50 <40 µM 67 . Johnston et al have since reported the development of five assays that target the AF‐2 domain, although the structures of the hits have not been disclosed to date 68 …”