Circumventing anti-androgen resistance by molecular design

Article abstract of DOI:10.1021/ja0701154

Nuclear/steroid hormone receptors (NHRs) function as ligand-dependent transcriptional regulators of diverse sets of genes involved in development and homeostasis. Mutations to the androgen receptor (AR), a member of NHRs, have been linked to the failure o

Full text of DOI:10.1021/ja0701154

Published on Web 03/10/2007
Circumventing Anti-Androgen Resistance by Molecular Design
Paula L. McGinley and John T. Koh*
Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716
Received January 6, 2007; E-mail: johnkoh@udel.edu
Prostate cancer (PCa) remains the second leading cause of cancer
death in men. As prostate tissue is dependent on androgens for
growth, anti-androgens used alone or in conjunction with inhibitors
of testosterone biosynthesis have been used in the treatment of PCa;
however, often cancer cells escape such androgen blockade
therapies. Androgen receptor (AR) mutations have been identified
as one mechanism leading to anti-androgen resistance and often
lead to a clinical phenomenon known as anti-androgen withdrawal
syndrome wherein anti-androgen resistant patients show symptom-
atic improvement after cessation of anti-androgen treatment.1 It has
been proposed that anti-androgen withdrawal syndrome is likely
associated with AR mutations such as Thr877fAla, Trp741fLeu,
and Trp741fCys which cause the antagonists flutamide and
bicalutamide (Bic) to act as agonists.2,3 Anti-androgens are pre-
sumed to apply a selective pressure on cancer cell growth such
that 31% of metastases arising with flutamide treatment have been
observed to possess the identical Thr877fAla mutation.4 As part
of our program to rescue nuclear receptor mutations through ligand
design, we describe the development of AR “pan-antagonists” that
function with wild-type (AR(wt)) and mutant ARs associated with
anti-androgen resistance.
Figure 1. Comparison of modeled structures. (A) Bic/AR(W741L) from
X-ray (green) with Bic/AR(wt)-H12 (yellow); (B) PLM1/AR(W741L)-H12
(green) with Bic/AR(wt)-H12 (yellow); (C) PLM1/AR(W741L)-H12,
conformation 2 (yellow) with Bic/AR(W741L) (green). Agonist position
of H12 from X-ray is shown in red.
The family of nuclear/steroid hormone receptors (NHRs) are
ligand-dependent transcriptional regulators for diverse sets of genes
involved in development and homeostasis. In the prototypical model
for NHRs, ligand binding induces a conformational change in the
ligand-binding domain that reveals a co-activator dimerization
surface on the receptor composed of helices 3, 5, and 12. As the
ligand-binding site is adjacent to helix 12 (H12), NHR antagonists
have commonly been designed by appending molecular extensions
to the core structure of NHR agonists that interfere with the
placement of H12, thereby disrupting co-activator recruitment.5
Recently, the structure of Bic with the Bic-resistant mutant
AR(W741L) was solved in the receptor’s agonist conformation.6
It has also been shown that sequences that compete for AR’s co-
activator binding site have been identified in both the N-terminal
and C-terminal domains of the receptor and are believed to play a
role in the transactivation function of AR. Therefore, the structure
of AR in its antagonist-bound form remains largely unknown.7
In the Bic/AR(W741L) cocrystal structure, the 4-fluorophenyl
sulfone group of Bic is situated between residues of H12 and the
side chain of Leu741, suggesting that in the wild-type receptor the
larger Trp741 side chain would require Bic to push against H12.6
Based on the assumption that Bic functions similarly to other NHR
antagonists by blocking H12 from its agonist conformation, we
modeled the antagonist conformation of Bic in AR(wt) by deleting
H12 from an AR(W741L) site model and changing the Leu741
residue back to Trp. Given the general lack of mechanistic and
structural details of the antagonist form of the receptor, this model
seemed a reasonable albeit crude model of the antagonist-bound
form of AR. Molecular dynamics simulations of Bic into this site
model suggested that, in the absence of the Tryp741fLeu mutant,
Bic prefers to bind in a manner that places the 4-fluorophenyl ring
Figure 2. Structure of bicalutamide and analogues.
in the space otherwise occupied by H12 in its active conformation
(Figure 1A).
Based on this proposed model for Bic antagonism, we hypoth-
esized that derivatives with an expanded aryl sulfone core, such as
PLM1, would similarly interfere with H12’s ability to adopt an
agonist conformation but would be unable to be switched to an
agonist by a simple missense mutation of Trp741 (Figure 1B,C).
Simulations suggest that the lowest energy conformation of PLM1
in the AR(W741L)-H12 resembled the Bic/AR(wt)-H12 antagonist
conformation (Figure 1B), whereas energetically accessible con-
formations of PLM1, which place the proximal aryl ring in the
pocket created by the Trp741fLeu mutation, still extend into the
space occupied by H12 (Figure 1C). We synthesized PLM1 as well
as a series of expanded aryl sulfone analogues that were conceived
based upon the same design principle (Figure 2). Control com-
pounds C1-C3 were also made. These analogues can be accom-
modated within the full AR(W741L) site model without any
apparent clash with H12.
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J. AM. CHEM. SOC. 2007, 129, 3822-3823
10.1021/ja0701154 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. LNCaP cell proliferation (CyQuant) after 8 day ligand treatment
( R1881. Relative fluorescence ( SEM (R1881 ( ligand; P < 0.05, one-
way ANOVA).
Table 1. Cellular Activities and Competitive Binding Constants
(µM)
AR(wt)
IC50
AR(W741L)
IC50 Ki
AR(W741C)
IC50 Ki
AR(T877A)
IC50
ligand
Ki
PLM1
PLM2
PLM6
3.8
12.5
21.4
0.5
6.3
2.1
9.7
23.0
7.9
2.1
2.9
n.d.
3.3
11.6
7.4
5.9
2.8
n.d.
6.0
16.5
5.4
of acting as “pan-antagonists”, being potent antagonists of
AR(wt), AR(W741L), AR(W741C), and AR(T877A) (Table 1).
To further demonstrate that the cellular reporter gene assays are
reflective of inhibitory activities in prostate cell lines, cell growth
inhibition studies were conducted in LNCaP cells which contain
the AR(T877A) mutant. Consistent with reporter gene assays, PLM1
is able to repress androgen (R1881)-induced cell growth to levels
below media control similar to Bic (Figure 4).
These studies demonstrate that structure-based design can identify
design strategies that can be used to efficiently engineer second-
generation anti-androgens that have the potential to circumvent anti-
androgen resistance by complementing receptor mutations at the
molecular level. Because in principle there is a limited subset of
mutations that can cause an antagonist to function as an agonist, it
is conceivable that such approaches may ultimately lead to the
development of anti-androgens that resist mutations leading to anti-
androgen withdrawal syndrome.
Figure 3. Cellular reporter gene activities of analogues alone and in
competition with DHT: (A) AR(wt) ( 3 nM DHT; (B) AR(W741L) (
250 nM DHT; (C) AR(W741C) ( 200 nM DHT; and (D) AR(T877A) (
10 nM DHT. RLU ) relative light units ( SEM.
The analogues were evaluated alone and in competition with
dihydrotestosterone (DHT) by cellular reporter gene assays using
CV-1 cells transiently transfected with either wild-type or mutant
ARs (pSG5AR) and an AR responsive luciferase reporter gene
(ARE-luc). With AR(wt), all of the analogues have no agonist
activity, and in competition with 3 nM DHT, all of the ligands but
PLM7 and C3 antagonize AR(wt) by greater than 50% at 25 µM
(Figure 3A). In contrast, with the Bic-resistant mutant AR(W741L),
Bic, C1, and C2 are strong agonists and C3 has intermediate
activity; however, PLM1, PLM2, PLM6, and PLM7 are not
agonists, having activities at 10 µM comparable to Bic in AR(wt).
PLM3 and PLM4 have weak to partial agonist activity (27 and
40%, Figure 3B). Whereas Bic and the control compounds be-
come agonists with AR(W741L), half of the designed analogues
(3 of 6) retain potent antagonist activity with this mutant asso-
ciated with anti-androgen resistance. PLM3 no longer acts as an
antagonist, whereas PLM4 appears to be a weak antagonist/partial
agonist.
With AR(W741C), controls C1-C3 are all agonists with
activities similar to those observed with AR(W741L). PLM1,
PLM2, and PLM6 remain non-agonists and potent antagonists;
however, the meta-substituted aryl sulfones, PLM3 and PLM4, now
become potent agonists, suggesting that these modifications are
complemented by the larger binding pocket created by the Cys
mutant compared to the Leu mutant (Figure 3C). Additionally,
PLM1, PLM2, and PLM6 remain non-agonists/antagonists with the
flutamide-resistant mutant AR(T877A) (Figure 3D). Collectively,
these results suggest that these analogues have the unique property
Acknowledgment. We thank Professor Robert Sikes for helpful
discussions and advice. This work was supported by the National
Institutes of Health R01 DK054257.
Supporting Information Available: Complete experimental pro-
cedures, characterization and assay data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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