Casey E. Bohl

Structural basis for antagonism and resistance of bicalutamide in prostate cancer

Carcinoma of the prostate is the most commonly diagnosed cancer in men. The current pharmacological treatment of choice for progressive androgen-dependent prostate cancer is the nonsteroidal antiandrogen, bicalutamide, either as monotherapy or with adjuvant castration or luteinizing hormone-releasing hormone superagonists to block the synthesis of endogenous testosterone. To date, no nonsteroidal or antagonist-bound androgen receptor (AR) structure is available. We solved the x-ray crystal structure of the mutant W741L AR ligand-binding domain bound to R-bicalutamide at 1.8-Å resolution. This mutation confers agonist activity to bicalutamide and is likely involved in bicalutamide withdrawal syndrome. The three-dimensional structure demonstrates that the B ring of R-bicalutamide in the W741L mutant is accommodated at the location of the indole ring of Trp-741 in the WT AR bound to dihydrotestosterone. Knowledge of the binding mechanism for R-bicalutamide will provide molecular rationale for the development of new antiandrogens and selective AR modulators.

Nonsteroidal Selective Androgen Receptor Modulators (SARMs): Dissociating the anabolic and androgenic activities of the androgen receptor for therapeutic benefit

Interest in the development and therapeutic potential of nonsteroidal tissue-selective androgen receptor modulators (SARMs) has increased dramatically within the past decade. Rapidly expanding knowledge of nuclear hormone receptor structure and function and successful proof-of-principle clinical trials with SARMs have revived an almost dormant search for improved androgens. This Award Address attempts to chronicle the landmark discoveries (with emphasis on our work), organize the SARM landscape into clinically relevant bins, and provide insight into the clinical prospects for SARMs. 1.1. Origins of Androgen Use. An early (1889) and unusual experiment in androgen therapy was performed by Charles Edouard Brown-Sequard, age 72. He administered a testicular extract to himself and reported that he felt “increased vigor and capacity for work”. Despite retrospective suggestions that any effect was purely placebo, this report resulted in widespread use of testicular extracts throughout Europe and North America for several decades. Attempts to isolate the active components of testicular extract failed until 1935 when testosterone (17 hydroxy-4-andosten-3-one) was isolated from bull testes. Shortly thereafter, its synthesis was reported. In the same year, extracts of urine from males were shown to cause nitrogen retention, an indicator of anabolic metabolism. Testosterone was the first anabolic androgen to be used clinically, but its use is limited by its androgenicity and pharmacokinetic (PK) issues. 1 In the latter half of the 20th century, the chemical scaffold of testosterone was modified extensively, producing many

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

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 tissue-selective 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.

Selective androgen receptor modulators in preclinical and clinical development

Androgen receptor (AR) plays a critical role in the function of several organs including primary and accessory sexual organs, skeletal muscle, and bone, making it a desirable therapeutic target. Selective androgen receptor modulators (SARMs) bind to the AR and demonstrate osteo- and myo-anabolic activity; however, unlike testosterone and other anabolic steroids, these nonsteroidal agents produce less of a growth effect on prostate and other secondary sexual organs. SARMs provide therapeutic opportunities in a variety of diseases, including muscle wasting associated with burns, cancer, or end-stage renal disease, osteoporosis, frailty, and hypogonadism. This review summarizes the current standing of research and development of SARMs, crystallography of AR with SARMs, plausible mechanisms for their action and the potential therapeutic indications for this emerging class of drugs.

Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design

Cyproterone acetate (CPA) is a steroidal antiandrogen used clinically in the treatment of prostate cancer. Compared with steroidal agonists for the androgen receptor (AR) (e.g. dihydrotestosterone, R1881), CPA is bulkier in structure and therefore seemingly incompatible with the binding pockets observed in currently available x-ray crystal structures of the AR ligand-binding domain (LBD). We solved the x-ray crystal structure of the human AR LBD bound to CPA at 1.8Å in the T877A variant, a mutation known to increase the agonist activity of CPA and therefore facilitate purification and crystal formation of the receptor·drug complex. The structure demonstrates that bulk from the 17α-acetate group of CPA induces movement of the Leu-701 side chain, which results in partial unfolding of the C-terminal end of helix 11 and displacement of the loop between helices 11 and 12 in comparison to all other AR LBD crystal structures published to date. This structural alteration leads to an expansion of the AR binding cavity to include an additional pocket bordered by Leu-701, Leu-704, Ser-778, Met-780, Phe-876, and Leu-880. Further, we found that CPA invokes transcriptional activation in the L701A AR at low nanomolar concentrations similar to the T877A mutant. Analogous mutations in the glucocorticoid receptor (GR) and progesterone receptor were constructed, and we found that CPA was also converted into a potent agonist in the M560A GR. Altogether, these data offer information for structure-based drug design, elucidate flexible regions of the AR LBD, and provide insight as to how CPA antagonizes the AR and GR.

A Selective Androgen Receptor Modulator for Hormonal Male Contraception

The recent discovery of nonsteroidal selective androgen receptor modulators (SARMs) provides a promising alternative for testosterone replacement therapies, including hormonal male contraception. The identification of an orally bioavailable SARM with the ability to mimic the central and peripheral androgenic and anabolic effects of testosterone would represent an important step toward the “male pill”. We characterized the in vitro and in vivo pharmacologic activity of (S)-3-(4-chloro-3-fluorophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl)propionamide (C-6), a novel SARM developed in our laboratories. C-6 was identified as an androgen receptor (AR) agonist with high AR binding affinity (Ki = 4.9 nM). C-6 showed tissue-selective pharmacologic activity with higher anabolic activity than androgenic activity in male rats. The doses required to maintain the weight of the prostate, seminal vesicles, and levator ani muscle to half the size of the maximum effects (i.e., ED50) were 0.78 ± 0.06, 0.88 ± 0.1, and 0.17 ± 0.04 mg/day, respectively. As opposed to other SARMs, gonadotropin levels in C-6-treated groups were significantly lower than control values. C-6 also significantly decreased serum testosterone concentration in intact rats after 2 weeks of treatment. Marked suppression of spermatogenesis was observed after 10 weeks of treatment with C-6 in intact male rats. Pharmacokinetic studies of C-6 in male rats revealed that C-6 was well absorbed after oral administration (bioavailability 76%), with a long (6.3 h) half-life at a dose of 10 mg/kg. These studies show that C-6 mimicked the in vivo pharmacologic and endocrine effects of testosterone while maintaining the oral bioavailability and tissue-selective actions of nonsteroidal SARMs.

CHARACTERIZATION OF THE IN VITRO METABOLISM OF SELECTIVE ANDROGEN RECEPTOR MODULATOR USING HUMAN, RAT, AND DOG LIVER ENZYME PREPARATIONS

Compound S4 [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] is a novel nonsteroidal selective androgen receptor modulator that demonstrates tissue-selective androgenic and anabolic effects. The purpose of this in vitro study was to identify the phase I metabolites, potential species differences in metabolism, and the cytochromes P450 (P450s) involved in the phase I metabolism of S4 using 14C-S4, recombinant P450s, and other liver enzyme preparations from human, rat, and dog. The major phase I metabolism pathways of S4 in humans were identified as deacetylation of the B-ring acetamide group, hydrolysis of the amide bond, reduction of the A-ring nitro group, and oxidation of the aromatic rings, with deacetylation being the predominant pathway observed with most of the enzyme preparations tested. Among the major human P450 enzymes tested, CYP3A4 appeared to be one of the major phase I enzymes that could be responsible for the phase I metabolism of S4 [Km = 16.1 μM, Vmax = 1.6 pmol/(pmol · min)] in humans and mainly catalyzed the deacetylation, hydrolysis, and oxidation of S4. In humans, the cytosolic enzymes mainly catalyzed the hydrolysis reaction, whereas the microsomal enzymes primarily catalyzed the deacetylation reactions. Similar phase I metabolic profiles were observed in rats and dogs as well, except that the amide bond hydrolysis seemed to occur more rapidly in rats. In summary, these results showed that the major phase I reaction of S4 in human, rat, and dog is acetamide group deacetylation.

Unexpected binding orientation of bulky-B-ring anti-androgens and implications for future drug targets.

Several new androgen receptor antagonists were synthesized and found to have varying activities across typically anti-androgen resistant mutants (Thr877 → Ala and Trp741 → Leu) and markedly improved potency over previously reported pan-antagonists. X-ray crystallography of a new anti-androgen in an androgen receptor mutant (Thr877 → Ala) shows that the receptor can accommodate the added bulk presented by phenyl to naphthyl substitution, casting doubt on previous reports of predicted binding orientation and the causes of antagonism in bulky-B-ring antagonists.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Discovery and Mechanistic Characterization of a Novel Selective Nuclear Androgen Receptor Exporter for the Treatment of Prostate Cancer

Despite the success of medical strategies to reduce androgen levels in the treatment of prostate cancer, this disease invariably relapses to a castrate-resistant state that is generally fatal. Although it had been thought that androgen-insensitive cancers no longer relied on the androgen receptor (AR) for growth and survival, it is now clear that this is not the case. Because relapses are known to occur by many mechanisms that keep the AR functionally active, strategies to block AR accumulation in the nucleus may be therapeutically useful. Here, we report the discovery of a selective nuclear androgen receptor exporter (SNARE) that functions to exclude AR from the nucleus. SNARE-1 binds wild-type and mutant ARs and efficiently inhibits their transactivation activity and ability to induce PSA gene expression. SNARE-1 inhibits the androgen-sensitive growth of LNCaP cells and tumor xenografts. Quantitative subcellular localization studies suggest that SNARE-1 inhibits nuclear translocation of AR, but also facilitates export of nuclear AR that has been translocated by an agonist. Mechanistic studies indicate that SNARE-1 rapidly phosphorylates p38 mitogen-activated protein kinase (MAPK) and Ser(650) of the AR. Additionally, SNARE-1 was found to promote ubiquitination of AR in LNCaP cells. Lastly, SNARE-1 functions as a tissue-selective AR inhibitor, as it fails to phosphorylate p38 MAPK in U2OS bone cells that are stably transfected with AR. In summary, SNARE-1 inhibits AR function by a mechanism that is distinct from clinically available antiandrogens, such that it might inform novel methods to block AR function in androgen-independent prostate cancer.