Structural Basis for the Glucocorticoid Response in a Mutant Human Androgen Receptor (AR<sup>ccr</sup>) Derived from an Androgen-Independent Prostate Cancer

Matías, Pedro M.; Carrondo, Maria Arménia; Coelho, Ricardo; Thomaz, M.; Zhao, Xiao‐Fan; Wegg, Anja; Crusius, Kerstin; Egner, Ursula; Donner, Peter

doi:10.1021/jm011072j

Cited by 78 publications

(61 citation statements)

References 43 publications

(89 reference statements)

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“…Purification of AR LBD bound to agonist ligands is well documented and involves removal of contamination chaperones, dnak and groEL, through cation exchange chromatography (21). The AR LBD is not retained by cation exchange chromatography in the presence of antagonist compounds and co-elutes with groEL after anion exchange chromatography, suggesting that antagonist-bound AR LBD is tightly associated with groEL, possibly as a result of partial receptor unfolding (25)(26)(27)(28).…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Bohl

Miller

Chen

et al. 2005

Journal of Biological Chemistry

190

214

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The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissueselective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact with the wild type AR. We have shown that changes induced to the positions of Trp-741, Thr-877, and Met-895 allow for ligand accommodation within the AR binding pocket and that a water-mediated hydrogen bond to the backbone oxygen of Leu-873 and the ketone of hydroxyflutamide is present when bound to the T877A AR variant. Additionally, we demonstrated that R-bicalutamide stimulates transcriptional activation in AR harboring the M895T point mutation. As a whole, these studies provide critical new insight for receptor-based drug design of nonsteroidal AR agonists and antagonists. The androgen receptor (AR)3 is a ligand-inducible nuclear hormone receptor involved in regulation of prostate growth, muscle and bone mass, and spermatogenesis in males. Endogenous ligands for the AR include the steroid, testosterone, and its more potent metabolite, dihydrotestosterone (DHT). Agonist compounds for the AR provide therapeutic potential in the treatment of osteoporosis, cachexia, contraception, and androgen deficiency (1). Antagonist AR ligands are commonly used in the treatment of androgen-dependent prostate cancer. The clinically available nonsteroidal antiandrogens, flutamide and bicalutamide, are advantageous over steroidal drug treatments because of their oral bioavailability and lack of cross-reactivity with other steroid receptors. Mutations to the AR that cause resistance to antiandrogens by converting the compounds to agonists exist and represent a significant problem with currently prescribed prostate cancer drugs that target the AR (2-4). Different AR mutations cause agonism for hydroxyflutamide (HF, active form of flutamide) as compared with bicalutamide, suggesting that these ligands do not antagonize the AR by the same mechanism. Our recent report of the W741L-R-bicalutamide crystal structure (5) demonstrated that R-bicalutamide induces the same overall fold of the AR ligand binding domain (LBD) observed in steroidal androgen-bound AR LBD crystal structures when bound to a mutant AR associated with re...

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Section: Resultsmentioning

confidence: 99%

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Bohl

Miller

Chen

et al. 2005

Journal of Biological Chemistry

190

214

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“…To further validate the selected models, ligand poses, after redocking of each of the 24 known AR antagonists, were manually inspected, with special attention to interactions of the ligands with receptor amino acids previously shown to be important for high-affinity binding by the AR LBD (22,(26)(27)(28)(29)(30). In particular, the formation of a hydrogen bond between ligands and R752 is speculated to be essential for high-affinity binding.…”

Section: Resultsmentioning

confidence: 99%

“…Here, we describe and validate a computational method to identify secondary antiandrogen activity of known drugs. By capitalizing on several critical contributions to the understanding of the AR structure and function (20)(21)(22)(23)(24), multiple models for antagonist-bound conformations of the AR were developed, and suitable models for ligand screening were selected based on docking scores for known antagonists. These models were subsequently screened against marketed drugs to identify compounds likely to have secondary antiandrogen activity.…”

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confidence: 99%

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs

Bisson

Cheltsov

Bruey-Sédano

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. High-throughput screening is often plagued by low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we describe an efficient computational methodology to discover leads to a protein target from safe marketed drugs. We applied an in silico ''drug repurposing'' procedure for identification of nonsteroidal antagonists against the human androgen receptor (AR), using multiple predicted models of an antagonist-bound receptor. The library of marketed oral drugs was then docked into the best-performing models, and the 11 selected compounds with the highest docking score were tested in vitro for AR binding and antagonism of dihydrotestosterone-induced AR transactivation. The phenothiazine derivatives acetophenazine, fluphenazine, and periciazine, used clinically as antipsychotic drugs, were identified as weak AR antagonists. This in vitro biological activity correlated well with endocrine side effects observed in individuals taking these medications. Further computational optimization of phenothiazines, combined with in vitro screening, led to the identification of a nonsteroidal antiandrogen with improved AR antagonism and marked reduction in affinity for dopaminergic and serotonergic receptors that are the primary target of phenothiazine antipsychotics.androgen receptor ͉ drug design ͉ prostate cancer C urrent approaches for discovery of novel chemical leads against a molecular target rely heavily on high-throughput screening (HTS) and to a lesser extent on virtual ligand screening (VLS) techniques. HTS has provided rapid lead identification for numerous drug targets (1-8); however, HTS also has major drawbacks, including a significant level of false positives and false negatives and low hit rates for many targets (9). Successful leads from HTS can also suffer from poor bioavailability and unwanted toxicity profiles of compounds. These problems result partially from the nature of the chemical libraries used for HTS. Furthermore, because the pharmacological properties of most compound libraries are largely unknown, there is an additional high risk that optimization of hits identified with HTS will not be sufficient for their evolution into real drugs.In contrast, retrospective analysis of marketed drugs reveals that their physicochemical and structural properties are clustered around preferred values and scaffolds (10). In addition, some chemical motifs are associated with high biological activity and often confer activity against more than one target/receptor (11-16). These motifs have been referred to as ''privileged structures'' (11). These observations lead to an assumption that the chemical space of potential drugs is limited. Consequently, currently marketed...

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“…Human AR ccr ligand-binding domain was expressed in Escherichia coli and purified according to the procedure of Matias et al (29). Protein concentration was determined using the Bradford protein assay, and aliquots were stored until use at À80jC in 50 mmol/L Tris (pH 7.5) containing 0.8 mol/L NaCl, 10% glycerol, and 1 mmol/L DTT.…”

Section: Subcellular Distribution Of Lycopenementioning

confidence: 99%

Absorption and subcellular localization of lycopene in human prostate cancer cells

Liu

Pajkovic

Pang

et al. 2006

Molecular Cancer Therapeutics

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Lycopene, the red pigment of the tomato, is under investigation for the chemoprevention of prostate cancer. Because dietary lycopene has been reported to concentrate in the human prostate, its uptake and subcellular localization were investigated in the controlled environment of cell culture using the human prostate cancer cell lines LNCaP, PC-3, and DU145. After 24 hours of incubation with 1.48 Mmol/L lycopene, LNCaP cells accumulated 126.6 pmol lycopene/million cells, which was 2.5 times higher than PC-3 cells and 4.5 times higher than DU145 cells. Among these cell lines, only LNCaP cells express prostate-specific antigen and fully functional androgen receptor. Levels of prostate-specific antigen secreted into the incubation medium by LNCaP cells were reduced 55% as a result of lycopene treatment at 1.48 Mmol/L. The binding of lycopene to the ligand-binding domain of the human androgen receptor was carried out, but lycopene was not found to be a ligand for this receptor. Next, subcellular fractionation of LNCaP cells exposed to lycopene was carried out using centrifugation and followed by liquid chromatography-tandem mass spectrometry quantitative analysis to determine the specific cellular locations of lycopene. The majority of lycopene (55%) was localized to the nuclear membranes, followed by 26% in nuclear matrix, and then 19% in microsomes. No lycopene was detected in the cytosol. These data suggest that the rapid uptake of lycopene by LNCaP cells might be facilitated by a receptor or binding protein and that lycopene is stored selectively in the nucleus of LNCaP cells.

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Structural Basis for the Glucocorticoid Response in a Mutant Human Androgen Receptor (AR^ccr) Derived from an Androgen-Independent Prostate Cancer

Cited by 78 publications

References 43 publications

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs

Absorption and subcellular localization of lycopene in human prostate cancer cells

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Structural Basis for the Glucocorticoid Response in a Mutant Human Androgen Receptor (ARccr) Derived from an Androgen-Independent Prostate Cancer

Cited by 78 publications

References 43 publications

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs

Absorption and subcellular localization of lycopene in human prostate cancer cells

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Product

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Structural Basis for the Glucocorticoid Response in a Mutant Human Androgen Receptor (AR^ccr) Derived from an Androgen-Independent Prostate Cancer