Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer

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Abstract

Castrate resistant prostate cancer (CRPC) is associated with increased androgen receptor (AR) signaling often brought about by elevated intratumoral androgen biosynthesis and AR amplification. Inhibition of androgen biosynthesis and/or AR antagonism should be efficacious in the treatment of CRPC. AKR1C3 catalyzes the formation of potent AR ligands from inactive precursors and is one of the most upregulated genes in CRPC. AKR1C3 inhibitors should not inhibit the related isoforms, AKR1C1 and AKR1C2 that are involved in 5α-dihydrotestosterone inactivation in the prostate. We have previously developed a series of flufenamic acid analogs as potent and selective AKR1C3 inhibitors [Adeniji, A. O. et al., J. Med. Chem. 2012, 55, 2311]. Here we report the X-ray crystal structure of one lead compound 3-((4-(trifluoromethyl)phenyl) amino)benzoic acid (1) in complex with AKR1C3. Compound 1 adopts a similar binding orientation as flufenamic acid, however, its phenylamino ring projects deeper into a subpocket and confers selectivity over the other AKR1C isoforms. We exploited the observation that some flufenamic acid analogs also act as AR antagonists and synthesized a second generation inhibitor, 3-((4-nitronaphthalen-1-yl)amino)benzoic acid (2). Compound 2 retained nanomolar potency and selective inhibition of AKR1C3 but also acted as an AR antagonist. It inhibited 5α-dihydrotestosterone stimulated AR reporter gene activity with an IC50 = 4.7 μM and produced a concentration dependent reduction in androgen receptor levels in prostate cancer cells. The in vitro and cell-based effects of compound 2 make it a promising lead for development of dual acting agent for CRPC. To illuminate the structural basis of AKR1C3 inhibition, we also report the crystal structure of the AKR1C3·NADP+·2 complex, which shows that compound 2 forms a unique double-decker structure with AKR1C3.

Graphical abstract

Superimposed structures of AKR1C3 in complex with two N-aryl(amino)benzoic acid analogs, compound 1 (magenta) and compound 2 (blue).

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Editors note

A provisional patent application based on these compounds has been submitted to the US patent office. US provisional patent applications No. 61/4754,091 filed April 13, 2011.

Acknowledgments

Supported by R01-CA90744, P30-ES013508, a Prostate Cancer Foundation Challenge Grant, and UL1RR024134 from the National Center for Research Resources (NCRR) from the National Institute of Health awarded to T.M.P. Grant GM-056838 awarded to D.W.C., and Grant F32DK089827 awarded to M.C., from the National Institutes of Health. The crystallography studies are based upon research conducted at beamline X25 and X29 of the National Synchrotron Light Source. Financial support for the National

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