Discovery of Small-Molecule Inhibitors Selectively Targeting the DNA

Jul 25, 2014 - The human androgen receptor (AR) is considered as a master regulator in the ... In this study, we identified a binding site on the DNA ...
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Discovery of Small-Molecule Inhibitors Selectively Targeting the DNA-Binding Domain of the Human Androgen Receptor Huifang Li, Fuqiang Ban, Kush Dalal, Eric Leblanc, Kate Frewin, Dennis Ma, Hans Adomat, Paul S. Rennie,‡ and Artem Cherkasov*,‡ Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia V6H 3Z6, Canada S Supporting Information *

ABSTRACT: The human androgen receptor (AR) is considered as a master regulator in the development and progression of prostate cancer (PCa). As resistance to clinically used anti-AR drugs remains a major challenge for the treatment of advanced PCa, there is a pressing need for new anti-AR therapeutic avenues. In this study, we identified a binding site on the DNA binding domain (DBD) of the receptor and utilized virtual screening to discover a set of micromolar hits for the target. Through further exploration of the most potent hit (1), a structural analogue (6) was identified demonstrating 10-fold improved anti-AR potency. Further optimization resulted in a more potent synthetic analogue (25) with anti-AR potency comparable to a newly FDAapproved drug Enzalutamide. Site-directed mutagenesis demonstrated that the developed inhibitors do interact with the intended target site. Importantly, the AR DBD inhibitors could effectively inhibit the growth of Enzalutamide-resistant cells as well as block the transcriptional activity of constitutively active AR splice variants, such as V7.

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interactions with the DNA.19,21 Previous studies have reported nonselective strategies for inhibiting the AR DBD−DNA interaction, including the use of androgen response element (ARE)-specific hairpin polyamides22 and noncompetitive smallmolecule inhibitors.23 None of these approaches were based on structural information on the AR DBD−DNA complex. On another hand, available crystal structure of AR DBD dimer bound to a direct repeat of two hexameric half-site AREs (PDB code: 1R4I.pdb),19 can provide a foundation for rational, structure-based discovery of small-molecule inhibitors targeting the AR DBD. In this study, we hypothesized that targeting the AR DBD with small-molecule inhibitors could interfere with DBD−ARE interactions for both full-length AR as well as ARVs, as they all contain the DBD region. With the aid of a computational program,24 a plausible binding site was detected underneath the P-box region of the AR DBD. Furthermore, using structurebased virtual screening, we identified initial hit compounds capable of inhibiting AR transcriptional activity at micromolar concentrations. Through extensive exploration of analogues of the most active hit 1, a potent 4-(4-phenylthiazol-2-yl)morpholine (6) was obtained with 10-fold improved anti-AR activity. With the preliminary structure−activity relationship (SAR) established for this chemical series, we designed and synthesized the next generation of compounds, including a lead 25 that exhibits anti-AR potency comparable to the latest FDA-

INTRODUCTION The androgen receptor (AR) is a ligand-dependent transcription factor which regulates a large repertoire of genes involved in the development and maintenance of male phenotype. Importantly, the AR is also viewed as a key regulator in the occurrence and progression of prostate cancer (PCa), including lethal castration-resistant PCa (CRPC).1−5 Notably, all clinically used antiandrogens target the androgen binding site (ABS) in the C-terminal ligand-binding domain (LBD) of the receptor and compete with endogenous androgens to antagonize the AR. The development of acquired resistance is a major challenge in the use of current antiandrogens.6−9 The identified resistance mechanisms involve gain-of-function mutations in the AR ABS and expression of constitutively active AR splice variants (ARVs) lacking the entire LBD segment, such as ARV7 and ARV567es.10−15 Thus, new AR antagonists acting beyond the LBD of the receptor are critically needed to circumvent the rising resistance to current antiandrogens. AR binding to the DNA via the receptor’s DNA-binding domain (DBD) is an essential step in the regulation of transcription of genes by both full-length AR and ARVs.16 Thus, targeting the DBD of the receptor (i.e., to directly interrupt its interaction with the DNA) could represent a powerful means to overcome antiandrogen resistance. The DBD is a functional domain composed of two α helices packed in perpendicular fashion.17−20 One α helix, called the “recognition helix”, consists of a P-box region, which inserts directly into the major groove of the DNA and makes key © 2014 American Chemical Society

Received: March 20, 2014 Published: July 25, 2014 6458

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The site is solvent exposed with specific residues predicted to play a key role in anchoring possible binding of small molecules. Thus, polar residues Ser579, Gln592, and Try594 around the periphery of the site could be characterized as available for hydrogen bonding, whereas Phe583 in the core of the site may provide additional hydrophobic interactions with potential binders. 2. In Silico Identification of Hit Compounds Targeting the AR DBD Site. The lead-like ZINC molecular database,25 containing approximately 3 million purchasable chemicals, was screened against the identified binding site on the AR DBD using docking program Glide (Maestro 9.3 suite, Schrödinger). After filtration by the generated docking score, ligand efficiency, and physicochemical properties, the top 5000 ranked structures were clustered using fingerprint method implemented by MOE, and a final selected set of 48 chemicals was purchased. The purchased compounds were then assessed using a nondestructive enhanced green fluorescent protein (eGFP) assay, which quantifies the AR-driven transcriptional activity in LNCaP cells.26 Compounds which inhibited AR activated transcription by more than 40% at 3 μM administration, were further evaluated to obtain corresponding IC50 values. The prostate specific antigen (PSA) is regulated by AR and is the most commonly used serum marker for PCa. To avoid possible false positive detection by the eGFP assay, the complementary PSA assay was employed for most active molecules to confirm their AR inhibitory effect. The lately FDA-approved anti-AR drug Enzalutamide was used as a standard for eGFP and PSA assays. As the result, five hits corresponding to three different chemical scaffolds were identified (Table 1) showing moderate

approved antiandrogen Enzalutamide. Importantly, it also exhibits effective inhibition of PCa cells which have already developed resistance to Enzalutamide and no longer respond to the drug. The site-directed mutagenesis experiment consequently confirmed that the identified AR DBD inhibitors indeed bind to the proposed target site. Furthermore, we demonstrated that compound 6 and 25 could effectively inhibit the activity of truncated ARVs, which play a critical role in the development of resistance to conventional antiandrogens and have not been previously targeted by any small molecules. These results indicate that the developed inhibitors possess a novel mechanism of suppressing the AR through its DBD function, which differentiates them from conventional PCa drugs that all target the receptor’s LBD part. These findings provide significant insights into the development of anti-AR therapies acting beyond the AR LBD and aiming to address the problem of rising drug resistance in PCa.

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RESULTS AND DISCUSSION 1. Identification of a Potentially Druggable Binding Site on the AR DBD Surface. The rat AR DBD dimer bound to two hexameric half-site AREs (PDB code: 1R4I) is the only crystal structure of the DBD region of the receptor available to date. As the sequences of rat and human AR DBDs are identical, the 1R4I structure was used as a template to build a homology model of the human AR DBD. The “hot spots” on the AR DBD dimer−ARE complex were predicted by a Site Finder module within the Molecular Operating Environment (MOE 2011) package. A cavity underneath the P-box region of the AR DBD was considered as a potential site for smallmolecule binding which may disrupt the AR DBD−ARE complex (Figure 1). This cavity is mainly enveloped by residues Ser579, Val582, Phe583, and Arg586 of the recognition helix, as well as by polar residues Gln592 and Tyr594 belonging to the lever arm loop, and residues Pro613 and Arg616 from the other α-helix, together with the loop residues Arg609 and Lys610.

Table 1. Initial Hit Compounds from Virtual Screening

AR and PSA inhibition with the corresponding IC50 values around 10 μM. Among these, compound 1 demonstrated the most potent inhibition of both AR transcription (eGFP IC50 = 3.17 ± 0.3 μM) and PSA expression (PSA IC50 = 3.91 μM) (Supporting Information, Figure S1) and was selected as the starting structural template for similarity search.

Figure 1. Predicted binding site on the human AR DBD homology model using the rat AR DBD (PDB code: 1R4I) as a template. The dummy atoms indicate the binding site, which was enveloped by residues Ser579, Val582, Phe583, Arg586, Gln592, Tyr594, Arg609, Lys610, and Pro613 in human AR DBD. The green and pink surfaces indicate hydrophobic and polar area, respectively. 6459

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Subsequent similarity search27 was conducted on a full version of the ZINC database, containing about 12 million entries at the time, and molecular docking was employed to position the identified analogues into the AR DBD binding site. The conducted hit-based similarity search resulted in the identification of a series of 4-(4-phenylthiazol-2-yl)morpholine analogues, including 19 active compounds with corresponding IC50 (eGFP) values below 10 μM, all listed in Table 2 (26

corresponding to a 10-fold improvement compared to the parental chemical 1. The PSA expression was reduced by compound 6 with the corresponding IC50 value of 0.28 μM (Figure 2B), confirming its potent AR inhibitory effect. 3. Structure−Activity Relationship (SAR) for 4-(4Phenylthiazol-2-yl)morpholines. The predicted binding mode of compound 6 inside the AR DBD binding site implied that the morpholine group could form hydrogen bond interaction with Tyr594, and the thiazole ring is engaged in hydrophobic contacts with Val582 above and Phe583 below. The pair of Arg609 and Lys610 forms a clamp-like shape, anchoring the phenyl ring through hydrophobic interactions by their aliphatic side chains (Figure 2C). The docking configuration of 4-(4-phenylthiazol-2-yl)morpholines in the AR DBD target site and the corresponding established in vitro activity allowed developing the preliminary structure−activity relationship (SAR) on this series. The anti-AR potency of compound 6 analogues appeared to be rather sensitive to structural changes on the phenyl ring, especially at its para-position. The activity was almost completely abolished by bulky and hydrophobic chemical groups, such as −C2H5, −OCHH3, and −SO2CH3 (59, 60, and 61 in Supporting Information, Table S1) on that position. According to the docked pose, the para-position substituents of the ligands are solvent-exposed, and unfavorable solvation of bulky and hydrophobic groups is likely to account for the activity loss. This trend also maintains for halogen substitutions in the para position. When adding −F, −Cl, and −Br to the para-position (11, 50, and 55 in Table 2 and Supporting Information, Table S2), the percentage of inhibition at 3 μM decreases in the series as the bulk and hydrophobicity of a substituent increases. Although the −F group was tolerated (11 in Table 2), the activity dropped about 2-fold compared to the unsubstituted 6. Bulky groups at the ortho- and meta-positions may form hydrophobic clamp with the Arg609 and Lys610, which seem to be tolerated by the ligands, although their activity also decreases somewhat (9, 10, 20, and 21 in Table 2). It was observed that the addition of substituents to the thiazole ring led to decreased activity (22 and 24 in Table 2). When the thiazole was replaced with other heterocyclic rings, such as thiophene, the resulting activity was reduced while still retaining in submicromolar level (7, 15, and 17 in Table 2), and the oxadiazole replacement abolished the activity completely (65 and 66 in Supporting Information, Table S2). According to the docked pose of these analogues, the morpholine ring forms a hydrogen bond with the hydroxyl group of Tyr594 in the active site (Figure 2C). To capitalize on this observation, a hydrogen bond acceptor was incorporated in the form of thiomorpholine and pyridine groups as an alternative to maintain H-bonding to Tyr594. Such replacement allowed retaining the activity of analogues in low- and submicromolar range (8 and 13 in Table 2), confirming the crucial role of Hbonding anchoring to the Tyr594 residue in the AR DBD target site. 4. Rational Design, Synthesis, and Evaluation of 4-(4Phenylthiazol-2-yl)morpholine Analogues. On the basis of the above SAR considerations, medicinal chemistry investigations were conducted using compound 6 as a parental structure. As modifications on the phenyl ring could remarkably change the activity, analogues of compound 6 were designed by modifying the phenyl ring or replacing phenyl ring with heteroaryl groups. A total of 34 analogues of 6 (listed in Table 3 and Supporting Information, Table S2) were ordered from

Table 2. Analogues from Hit-Based Similarity Search

additional chemicals with lower activities were also presented in Supporting Information Table S1). Among the identified similarity-based hits, compound 6 (4-(4-phenylthiazol-2-yl)morpholine) demonstrated complete inhibition of AR-mediated transcription at a dose of 3 μM. When subjected to a concentration-dependent titration, compound 6 exhibited an IC50 (eGFP) value of 0.33 ± 0.12 μM (see Figure 2A), 6460

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Figure 2. (A) The AR inhibitory activity of compound 6. LNCaP eGFP cells were treated with 6 and Enzalutamide at various concentrations (0.006−3.125 μM) for 3 days in the presence of 0.1 nM R1881, and the AR transcriptional inhibition of compounds was evaluated by measuring the fluorescence. (B) The PSA suppression by these compounds was evaluated by measuring the PSA secreted into the media using the same LNCaP eGFP cells. (C) The docked pose of compound 6 (magenta) in the homology model of human AR DBD, forming hydrogen bond (dash line in red) with Tyr594.

Life Chemicals (http://www.lifechemicals.com/) and Enamine (http://www.enamine.net) companies. The synthetic schemes (Scheme 1, Supporting Information, Schemes S1−S6) and procedures are in the Experimental Section and Supporting Information. These efforts resulted in the creation of 21 active AR inhibitors (eGFP IC50 < 10 μM), with six of them demonstrating superior anti-AR potency compared to the template compound 6. In particular, a synthetic analogue 25, bearing 4,5-dibromoimidazole, displayed 2-fold improved activities (eGFP IC50 = 0.12 ± 0.01 μM; PSA IC50 = 0.17 μM, Figure 3A,B) compared to compound 6, reaching the level of activity of the latest FDAapproved PCa drug, Enzalutamide (eGFP IC50 = 0.11 ± 0.01 μM; PSA IC50 = 0.12 μM). In addition, compound 25 and other halogen-containing analogues (32, 37) are fully stable in media (see Table S3 in Supporting Information). With such promising inhibitory activity toward AR transcription and PSA expression, we further evaluated the ability of 25 to inhibit the growth of AR-dependent PCa cells in the AR-positive LNCaP system. Following hormone activation of the AR (0.1 nM R1881), compound 25 elicited a concentration-dependent inhibition of the cell growth. A similar potency for cell-growth inhibition was achieved when 25 was evaluated against the newly developed Enzalutamide-resistant cell line, MR49F.28 Importantly, compound 25 is ineffective in inhibiting the proliferation of AR-negative PC3 cells, supporting the mechanism of its action through specific interaction with the AR rather than by means of generic toxicity (Figure 3C). Consistent with the initial docking hypothesis for compound 6, its analogue 25 adopted the same docking pose in the AR DBD model where it forms similar receptor−ligand interactions (Figure 3D). The morpholine group retained the hydrogen bond interaction with Tyr594, and the thiazole ring maintains the van der Waals interactions with Val582 and Phe583. The 4,5-dibromo-imidazole group intercalates into the clampshaped groove formed by Arg609 and Lys610, making hydrophobic interactions. This structural basis was further used to guide the site-directed mutagenesis experiment on key binding residues on the AR DBD. 5. Confirmation of Compounds 6 and 25 Binding to the Proposed Site on the AR DBD. To prove that the developed DBD inhibitors directly bind to the intended target site on the AR, we designed and constructed amino acid mutations that may alter the predicted binding site of the

compound while not affecting the AR function. On the basis of the molecular modeling, residues Ser579, Val582, Phe583, Arg586, Gln592, Tyr594, and Lys610 in human AR DBD are embedded in the binding site and provide important coordinating interactions with the developed ligands. We created aspartic acid point mutations at these amino acid positions in the full-length human AR and co-transfected them with an ARR3tk-luciferase reporter into AR-negative PC3 cells.29 Among those, two mutants, Gln592Asp and Tyr594Asp, allowed maintaining of the AR transcriptional activity, while the remaining point mutations destroyed the AR function and were not considered further (Figure 4A). Notably, Western blots confirmed no change in AR expression levels for all the mutants regardless of their transcriptional activity or presence of our compounds.30 Compounds 6 and 25 were tested on the two active mutants and compared to the luciferase-detected activities obtained with the wild-type AR. As expected, the inhibitory effect of 6 and 25 on the AR was diminished by Gln592Asp and Tyr594Asp mutations, confirming that the compounds interact with these residues in the proposed AR DBD target site. We next sought to rule out possible interaction of 6 and 25 with known functional sites in the AR LBD including the ABS, activation function 2 (AF2), and binding function 3 (BF3) sites, which are known to interact with various small molecules.28,31−37 To perform this task, we conducted androgen displacement experiments with 6 and 25 (Supporting Information, Figure S2) and demonstrated that neither of the chemicals could displace bound androgens from the AR. To exclude binding to the other sites, an in vitro biolayer interferometry (BLI) assay using recombinant AR LBD was carried out and also demonstrated the lack of binding of 6 and 25 to the LBD, effectively excluding the possibility of the compounds binding to all known sites on this domain (Supporting Information, Figure S2). Together, these findings suggest that the identified compounds act beyond known binding sites in the C-terminal LBD of the receptor. In addition, we also used BLI to exclude the possibility of these compounds binding to the ARE. The corresponding experiments detected no direct interaction between compounds 6 and 25 and the DNA (data not shown). 6. The Developed AR DBD Binders Inhibit Truncated Splice Variants of the AR. As the developed AR DBD inhibitors have been confirmed to bind to their intended target 6461

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whether the developed chemicals can inhibit such ARVs, we cotransfected plasmids encoding for AR-V7 and the ARR3tkluciferase reporter into AR-negative PC3 cells and evaluated the effects of 6 and 25 on the corresponding luciferase expression. As anticipated, both 6 and 25 demonstrated effective inhibition of the AR-V7 activity in a concentration-dependent manner (Figure 4B), which further validated the developed compounds as effective AR DBD inhibitors. A more detailed study of the action of the identified inhibitors on AR splice variants and indepth discussion around the developed ARV assays will be presented in a specialized publication.30 7. Selectivity of the Developed AR DBD Binders. As the DBDs of steroid nuclear receptors, such as progesterone receptor (PR), glucocorticoid receptor (GR), and estrogen receptor (ER) are highly conserved,39−41 compounds targeting this domain may demonstrate poor selectivity. However, the sequence alignment of the steroid nuclear receptors reveals distinct amino acid differences highlighted in Supporting Information, Figure S3. The Gln592 residue in human AR located in the proposed DBD binding site is different from residues in any other members of the nuclear receptor family, which may determine the specificity of small-molecule inhibitors, such as 6 and 25, rationally designed to engage Gln592 into binding (as it has been confirmed by the mutation experiments). Accordingly, when tested against PR, GR, and ER, the developed inhibitors 6 and 25 displayed negligible inhibition of PR or GR and exhibited only weak cross-activity toward ER (see Supporting Information, Figure S4). 8. Pyrvinium Pamoate as a Potential AR DBD Binder. Since the beginning of our in silico investigation of the AR DBD crystal structure over two years ago, no bona fide smallmolecule inhibitors targeting the AR DBD have been reported. At the onset of this study, we examined the literature for AR inhibitors which might interrupt the AR DBD function and found that the drug pyrvinium pamoate (PP) was recently reported to interfere with the AR transcription initiation complex.23 In our system, PP also showed potent inhibition of PSA (IC50 = 0.4 μM, Supporting Information, Figure S5) but was toxic to LNCaP cells and AR-negative PC3 cells even at low concentrations. In addition, PP was reported to inhibit multiple kinases such as Wnt family members in the nanomolar range, which may explain the observed generic toxicity. Because of such toxicity and nonspecificity, PP was dropped off from our drug discovery pipeline. During the preparation of this manuscript, the same group reported results of their molecular modeling, predicting that PP inhibits AR via binding to the DBD.38 The details on how this binding site on the DBD was identified remain unclear, but the site is similar to the model reported in the current work. The similarities between our models prompted us to test if PP could inhibit the full-length AR bearing Gln592Asp and Tyr594Asp substitutions in the DBD region. As shown in Figure 4A, much like Enzalutamide, PP strongly inhibits the mutated AR, suggesting it is not likely to bind to the DBD in the proposed manner.

Table 3. Synthesized Analogues by Modifying the Ring A

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CONCLUSIONS In this study, we report that the DBD part of nuclear receptors (AR in particular) represents a viable drug target for small molecules. The initial virtual screening was performed against a predicted target site on the AR DBD surface, and an initial hit compound 1 was identified that effectively inhibited AR transcription and PSA expression at micromolar levels. Subsequently, a hit-based similarity search resulted in the

area, they were also expected to inhibit the activity of truncated ARVs, which lack the entire AR LBD and thus are completely resistant to all currently used anti-androgens. To measure 6462

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Scheme 1. Schemes for the Synthesis of Heteroaryl Analogues 25, 27, 28, 31, 32, 34, 36, 37, 39, and 40a

a Reagents and conditions: (A) IPA, 100 °C, 17−38%; (B) THF, H2O, K2CO3, PdCl2(PPh2C6H4SO3Na-m)2, 80 °C, 54−73%; (C) toluene, PdAc2(PPh3)2, 110 °C, 10−62%.

Figure 3. (A) The AR inhibitory activity of compound 25 in comparison with Enzalutamide using LNCaP eGFP cells in the presence of 0.1 nM R1881 by measuring the fluorescence. (B) The PSA suppression by these compounds was evaluated by measuring the PSA secreted into the media using the same LNCaP eGFP cells. (C) The antiproliferative effect of 25 on LNCaP, MR49F (Enzalutamide-resistant) and PC3 cells using MTS assay. The LNCaP, MR49F, and PC3 cells were treated with the inhibitor at various concentrations for 3 days in the presence of 0.1 nM R1881. (D) Plausible binding mode of compound 25 (yellow) in the human AR DBD model.

binders can effectively inhibit the constitutive, androgenindependent activity of truncated ARVs.

identification of a series of AR DBD binders represented by 4(4-phenylthiazol-2-yl)morpholine 6 that exhibited nanomolar potency against the AR. On the basis of the SAR on an extensive set of structural analogues of 6, a number of synthetic analogues were created and evaluated and resulted in the development of lead compound 25 with IC50 values of 0.12 μM. Thus, the synergetic applications of in silico and experimental screening with synthesis allowed more than a 25-fold improvement in anti-AR activity within the identified series of compounds. The site-directed mutagenesis of critical amino acids in the proposed AR DBD binding site revealed that the identified compounds indeed bind to the intended target area. Further investigations confirmed that such AR DBD

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EXPERIMENTAL SECTION

In Silico Experiments. Protein and Ligand Preparation. The crystal structure of a rat AR DBD dimer in complex with a direct repeat of two half-sites separated by 3 bp spacers (DR3) was prepared using the Protein Preparation Wizard within Maestro 9.3 (Schrödinger, LLC). The hydrogens were added, bond orders were assigned, and missing side chains for some residues were added using Prime. The added hydrogens was subjected to energy minimization until the root-mean-square deviation (RMSD) relative to the starting geometry reached 0.3 Å. 6463

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ligand efficiency was used to rank the compounds, and top ranked compounds were classified into clusters using Fingerprint cluster in MOE 2011, using a similarity and overlap threshold of 60%. Finally, the docked poses were visualized and compounds with favorable interactions were selected. All tested compounds were purchased from commercial vendors such as Enamine, Vitas-M, and Life Chemicals with purity ≥95%. Similarity Search. The Instant JChem 5.9.0 (ChemAxon) is a similarity search tool which supports query and sorting functionality and handles large volumes of data. The Zinc database was imported into the Instant JChem. The identified hits can be used as a query to identify analogues by structure search criteria such as similarity. Chemistry. All reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise stated. The reactions were monitored by thin layer chromatography (TLC) on precoated silica gel F254 plates (SigmaAldrich) with a UV indicator using ethyl acetate/hexane (1:2 v/v). Yields were of purified product and were not optimized. The purity of the newly synthesized compounds was determined by LCMS analysis. The proton nuclear resonance (1H NMR) spectra were performed on a Varian GEMINI 2000 NMR spectrometer system with working frequency 400 MHz. Chemical shifts δ are given in ppm, and the following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad singlet (br s). All LC/MS data were gathered on an Agilent 1100 LC system. The compound solution was injected into the ionization source operating positive and negative modes with a mobile phase acetonitrile/water/formic acid (50:50:0.1% v/v) at 1.0 mL/min. The instrument was externally calibrated for the mass range m/z 100 to m/z 650. All synthesized compounds have a purity >95%, determined by LCMS. Unless otherwise noted, chemicals were purchased from commercial suppliers and used without further purification. Synthetic Procedure for Compound 27 (Scheme 1A). A mixture of 10 mmol of thiourea (a) and 10 mmol of halogenated ketone (b) was dissolved in 10 mL of 2-propanol. The formed solution was refluxed for 4 h, and the organic solvent was removed under vacuum. The residue was treated with 20 mL of brine and extracted with 30 mL of ethyl acetate (2 times). Combined organic layer was dried with sodium sulfate, filtered, and evaporated. The obtained solid was purified by preparative HPLC (eluent EtOAc/hexane = 1/1) to give the final product as a solid. 4-(4-(4,5-Dimethylthiophen-3-yl)thiazol-2-yl)morpholine (27). 1H NMR (DMSO-d6, 400 MHz): δ 2.24 (3H, s), 2.36 (3H, s), 3.41−3.43 (4H, m), 3.73−3.76 (4H, m), 6.76 (1H, s), 7.33 (1H, s). MS (ESI): m/z (M + H)+ 281.1. Yield: 38%; purity 100% by LCMS. Synthetic Procedure for Compound 28 (Scheme 1B). A mixture of 9.1 mmol of bromthiazole (Scheme 1B, c), 11.8 mmol of corresponding boronic acid, 22.5 mmol of potassium carbonate, and 0.1 mmol of a catalyst PdCl2(PPh2C6H4SO3Na-m)2 was dissolved in 100 mL of tetrahydrofuran and 16 mL of water. Then the reaction mixture was refluxed for 20 h. After cooling, it was extracted with 100 mL of ethyl acetate. The organic layer was separated, dried with sodium sulfate, filtered, and evaporated. The obtained residue was purified by preparative HPLC (eluent EtOAc/hexane = 1/1) to give the final product as a solid. 4-(4-(Thiophen-3-yl)thiazol-2-yl)morpholine (28). 1H NMR (DMSO-d6, 400 MHz): δ 3.41−3.43 (4H, m), 3.72−3.74 (4H, m), 7.14 (1H, s), 7.49−7.55 (2H, m), 7.72−7.73 (1H, m). MS (ESI): m/z (M + H)+ 253.1. Yield: 54%; purity 95% by LCMS. General Procedure for the Synthesis of Compounds 25, 31, 32, 34, 36, 37, 39, and 40 (Scheme 1C). A mixture of 4 mmol of bromthiazole (c), 12 mmol of corresponding imidazole (d), 0.1 mmol of catalyst PdAc2(PPh3), and 50 mL of toluene was refluxed for 24 h. After cooling, the organic solvent was removed under vacuum. The resulting residue was purified by preparative HPLC (eluent EtOAc/ hexane = 1/1) to give the final product as a solid. Compounds were additionally purified by reverse phase HPLC (eluent acetonitrile/ water) with content of acetonitrile ranging from 20 to 80%. 4-(4-(4,5-Dibromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine (25). 1H NMR (DMSO-d6, 400 MHz): δ 3.39−3.42 (4H, m), 3.70−

Figure 4. (A) Site-directed mutagenesis study on the predicted binding site residues indicates 6 and 25 bind directly to the site. Wildtype hAR and hAR mutant plasmids (S579D, V582D, F583D, R586D, Q592D, Y594D, and K610D) were transfected with a ARR3tkluciferase reporter into PC3 cells, and the transcriptional activities of tested compounds 6, 25, and Enzalutamide at 10 μM were measured based on the luminescence. (B) Compounds 6 and 25 inhibit the transcription of AR-V7 in a concentration-dependent manner. Wildtype hAR/ARV7 plasmids were co-transfected into PC3 cells with the ARR3tk-luciferase reporter and treated with compound 6, 25, and Enzalutamide. A library of 3 million commercially available molecules from the ZINC database25 were imported into Molecular Operating Environment (MOE 2011). All the molecules were protonated/deprotonated by a “wash” process, added partial charges and minimized with the MMFF94x force field to a gradient of 0.0001 kcal/mol Å. After the minimization, the database was exported as an sdf file. Identification of Plausible Binding Sites in AR DBD. Potential binding sites in the AR DBD dimer−DNA complex were predicted using Site Finder within MOE, which uses a geometric method based on Alpha Shapes. The Site Finder will first identify regions of tight atomic packing, filter out exposed sites, classify hydrophobic/ hydrophilic sites, and then calculate alpha spheres on the sites. Alpha spheres corresponding to an inaccessible region or exposed to solvent will be eliminated and the rest will be classified as hydrophobic/hydrophilic, clustered, and ranked using a propensity for ligand binding score. Detected sites were examined and compared based on parameters including size, shape, amino acid composition, and the volume of the pocket. Virtual Screening of Potential AR DBD Inhibitors. The docking program, Glide,42 in Maestro 9.3 was used for the virtual screening. The residues in the predicted binding site were used to define the active site for the virtual screening. For Glide docking, the grid was defined using a 20 Å box centered on the selected residues. No constraints were applied, and all the settings were kept as default. The ZINC database was docked using Glide SP mode, and the predicted binding pose of entire database was ranked by glidescore. Compounds with potentially low binding affinities (high glidescore values) were discarded, and remaining compounds were further narrowed down by applying physicochemical parameters, including the number of acceptor (≤ 5), donor (≤ 5), log P (≤ 5), molecular weight (≤ 300), rotatable bonds (≤ 10), charges (= 0), and ring (≤ 3).43−45 The 6464

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3.73 (4H, m), 7.14 (1H, s), 7.80 (1H, s). MS (ESI): m/z (M + H)+ 395.0. Yield: 51%; purity: 98% by LCMS; mp 116−118 °C. 4-(4-(5-Bromo-4-chloro-1H-imidazol-1-yl)thiazol-2-yl)morpholine (31). 1H NMR (DMSO-d6, 400 MHz): δ 3.41−3.43 (4H, m), 3.71−3.73 (4H, m), 7.15 (1H, s), 8.16 (1H, s). MS (ESI): m/z (M + H)+ 351.0. Yield: 14%; purity 96% by LCMS. 4-(4-(4,5-Dichloro-1H-imidazol-1-yl)thiazol-2-yl)morpholine (32). 1 H NMR (DMSO-d6, 400 MHz): δ 3.41−3.45 (4H, m), 3.71−3.73 (4H, m), 7.15 (1H, s), 8.14 (1H, s). MS (ESI): m/z (M + H)+ 305.1. Yield: 48%; purity 99% by LCMS. 4-(4-(4-Chloro-1H-imidazol-1-yl)thiazol-2-yl)morpholine (34). 1H NMR (DMSO-d6, 400 MHz): δ 3.42−3.44 (4H, m), 3.71−3.73 (4H, m), 6.97 (1H, s), 7.83 (1H, s), 8.17 (1H, s). MS (ESI): m/z (M + H)+ 271.1. Yield: 62%; purity 99% by LCMS. 4-(4-(4-Bromo-5-chloro-1H-imidazol-1-yl)thiazol-2-yl)morpholine (36). 1H NMR (DMSO-d6, 400 MHz): δ 3.41−3.43 (4H, t), 3.70−3.72 (4H, m), 7.14 (1H, m), 8.15 (1H, s). MS (ESI): m/z (M + H)+ 351.0. Yield: 27%; purity 96% by LCMS. 4-(4-(4,5-Diiodo-1H-imidazol-1-yl)thiazol-2-yl)morpholine (37). 1 H NMR (DMSO-d6, 400 MHz): δ 3.41−3.42 (4H, m), 3.71−3.73 (4H, m), 7.10 (1H, s), 8.10 (1H, s). MS (ESI): m/z (M + H)+ 488.9. Yield: 12%; purity 96% by LCMS. 4-(4-(4-Chloro-5-iodo-1H-imidazol-1-yl)thiazol-2-yl)morpholine (39). 1H NMR (DMSO-d6, 400 MHz): δ 3.41−3.42 (4H, m), 3.70− 3.71 (4H, m), 7.14 (1H, s), 8.14 (1H, s). MS (ESI): m/z (M + H)+ 397.5. Yield: 10%; purity 96% by LCMS. 4-(4-(4-Bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine (40). 1H NMR (DMSO-d6, 400 MHz): δ 3.42−3.43 (4H, m), 3.71−3.74 (4H, m), 6.98 (1H, s), 7.88 (1H, s), 8.18 (1H, s). MS (ESI): m/z (M + H)+ 316.1. Yield: 53%; purity 99% by LCMS. Synthesis procedures for the rest of the compounds (4-4-(3,4difluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (26), 2-fluoro-6(2-morpholinothiazol-4-yl)phenol (29), 4-(4-(4-fluoro-2methoxyphenyl)thiazol-2-yl)morpholine (30), 2,3-difluoro-6-(2-morpholinothiazol-4-yl)phenol (33), 5-fluoro-2-(2-morpholinothiazol-4yl)phenol (35), 4-(4-pyridin-2-yl)thiazol-2-yl)morpholine (38), 4-(4(pyridine-4-yl)thiazol-2-yl)morpholine (41), 3-fluoro-4-methoxyl-5(2-morpholinothiazol-4-yl)phenol (42), 4-(4-(3-fluoro-2-methanesulfonyl-phenyl)-thiazol-2-yl)morpholine (43), 4-(4-(4,5-dimethyl-1Himidazol-1-yl)thiazol-2-yl)morpholine (44), 4-(5-methyl-4-phenylthiazol-2-yl)morpholine (45), and inactive compounds (72−84) are in the Supporting Information. These compounds were synthesized by Enamine (http://www.enamine.net/). In Vitro Evaluation of Hit Compounds. eGFP Cellular AR Transcription Assay. The eGFP AR transcriptional activity was assayed as previously described.26 Briefly, the LNCaP human prostate cancer cells were stably transfected with a probasin-derived promoter (ARR2PB) fused with eGFP. The eGFP LNCaP cells were grown in phenol-red-free RPMI 1640 supplemented with 5% charcoal stripped serum (CSS) for 5 days. Then cells were seeded into a 96-well plate (35000 cells/well) for 24 h and treated with 0.1 nM R1881 and a single concentration or increasing concentrations (0−25 μM) of compounds for screening and measuring IC50, respectively. After cells were incubated for 3 days, the fluorescence was measured (excitation, 485 nm; emission, 535 nm). Prostate-Specific Antigen (PSA) Assay. The evaluation of PSA secreted into the media was performed in parallel with the eGFP assay using the same plate. The eGFP LNCaP cells were seeded and treated as performed in eGFP AR transcription assay. After incubation of 3 days, 150 μL of the media was taken and added to 150 μL of PBS. The PSA level was then evaluated using a Cobas e 411 analyzer instrument (Roche Diagnostics) according to the manufacturer’s instructions. Cell Viability Assay. The PC3, LNCaP, and MDV3100-resistant cells (MR49F) were seeded into a 96-well plate (3000 cells/well) in RPMI 1640 containing 5% CSS and treated with 0.1 nM R1881 and compounds (0−25 μM) for 4 days. Then the cell density was measured using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay according to the manufacturer’s protocol (CellTiter 961 Aqueous One Solution Reagent, Promega). The percentage of cell growth was normalized

to control wells with and without 0.1 nM R1881 and calculated as % growth = (sample − vehicle)/(0.1 nM R1881 − vehicle) × 100. Androgen Displacement Assay. The androgen displacement was assessed with the PolarScreen Androgen Receptor Competitor Green Assay Kit as per the instructions of the manufacturer (Life technologies). Site-Directed Mutagenesis Study. The residues in the predicted binding site were mutated using the Quickchange Site-Directed Mutagenesis Kit as per the instructions. The introduced mutations were verified by sequencing. Transient Transfection. The PC3 cells were starved in RPMI 1640 media (Gibco, USA) supplemented with 5% CSS for 5 days and then seeded into a 96-well plate (5000 cells/well). After 24 h, the wild-type human AR (50 ng/well), AR mutants, AR-V7, or other nuclear receptor plasmids, and 50 ng ARR3tk-luciferase plasmids were cotransfected into PC3 cells using 0.3 μL/well transfection reagent (TT20, Mirus). After the transfection for 48 h, cells were treated with compounds at various concentrations for 24 h. The AR, GR, and PR activation was stimulated with 0.1 nM R1881, 1 nM dexamethasone, and progesterone, respectively. ER-α transcriptional activity was measured with a MCF-7 cell line bearing stable transfection of an ERE-Luciferase reporter, with transcriptional activity stimulated by 1 nM estradiol. Cell lysis was carried out with 60 μL of 1× passive lysis buffer/well (Promega). Then 20 μL of cell lysate from each well were mixed with 50 μL of luciferase assay reagent (Promega), and luminescence was recorded on a TECAN M200pro plate reader.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary synthetic schemes and procedures, additional tables of identified inactive compounds and stability, figures illustrating the inhibition data, sequence alignment, selectivity data, purity and identity data of active compounds, and rankings of hit compounds (XLSX). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 604-875-4111. Fax: 604-875-5654. E-mail: artc@ interchange.ubc.ca. Author Contributions ‡

Dr. Cherkasov’s and Dr. Rennie’s laboratories made equal contributions to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Prostate Cancer Canada with generous support from Canada Safeway grant SP2013-02, an operating grant (no. 272111) and a proof-of-principle grant (no. 328186) from Canadian Institutes of Health Research, a Movember Discovery Program award, and a grant from Department of Defense (no. 11496001). We thank Dr. Barbara Lelj Garolla for the proofreading. H. Li is supported by 2013 PCF-BC grant-in-aid award and Evelyn Martin Memorial Fellowship. K. Dalal is supported by CIHR and MSFHR postdoctoral fellowships.

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