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Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain Inhibitor Pyrvinium
journal contribution
posted on 2019-02-01, 09:13 authored by Sumanta
K. Pal, Ben Yi Tew, Minyoung Lim, Brittany Stankavich, Miaoling He, Miles Pufall, Weidong Hu, Yuan Chen, Jeremy O. JonesPyrvinium
was identified as the first small molecule inhibitor
of the androgen receptor (AR) DNA-binding domain (DBD). It was also
among the first small molecules shown to directly inhibit the activity
of AR splice variants (ARVs), which has important clinical implications
in the treatment of castration-resistant prostate cancer. Important
questions about pyrvinium’s mechanism of action remain. Here,
we demonstrate through mutational analysis that amino acids 609 and
612 are important for pyrvinium action. Nuclear magnetic resonance
demonstrates a specific interaction between a soluble pyrvinium derivative
and the AR DBD homodimer–DNA complex. Chromatin immunoprecipitation
and electrophoretic mobility shift assay experiments demonstrate that,
despite an interaction with this complex, pyrvinium does not alter
the DNA-binding kinetics in either assay. AR immunoprecipitation followed
by mass spectrometry was used to identify proteins whose interaction
with AR is altered by pyrvinium. Several splicing factors, including
DDX17, had reduced interactions with AR in the presence of pyrvinium.
RNA sequencing of prostate cancer cells treated with pyrvinium demonstrated
changes in splicing, as well as in several other pathways. However,
pyrvinium did not alter the levels of ARVs in several prostate cancer
cell lines. Taken together, our new data pinpoint the direct interaction
between pyrvinium and AR DBD and shed light on the mechanism by which
it inhibits AR transcriptional activity.
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Keywords
androgen receptorDNA-binding domainChromatin immunoprecipitationImportant questionsDNA-binding kineticsprostate cancer cellsAR immunoprecipitationRNA sequencingmass spectrometryMechanistic InvestigationDDXacids 609interactionAR splice variantsmolecule inhibitorAR DBDAR transcriptional activityAndrogen Receptor DNA-Binding Domain Inhibitor Pyrvinium Pyrviniumpyrvinium actionelectrophoretic mobility shift assay experimentsprostate cancer cell linesARVcastration-resistant prostate cancer
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