Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0385 Cancer Therapeutics, Targets, and Chemical Biology Research Multivalent Peptoid Conjugates Which Overcome Enzalutamide Resistance in Prostate Cancer Cells Yu Wang1, Dilani C. Dehigaspitiya2, Paul M. Levine2, Adam A. Profit3, Michael Haugbro2, Keren Imberg-Kazdan4, Susan K. Logan1,5, Kent Kirshenbaum2, and Michael J. Garabedian1,4 Abstract Development of resistance to antiandrogens for treating antiproliferative effect, suggesting that both ethisterone ligand pre- advanced prostate cancer is a growing concern and extends to sentation and scaffold characteristics contribute to MPC6 activity. recently developed therapeutics, including enzalutamide. Therefore, Mechanistically, MPC6 blocked AR coactivator–peptide interaction new strategies to block androgen receptor (AR) function in prostate and prevented AR intermolecular interactions. Protease sensitivity cancer are required. Here, we report the characterization of a assays suggested that the MPC6-bound AR induced a receptor multivalent conjugate presenting two bioactive ethisterone ligands conformation distinct from that of dihydrotestosterone- or enzalu- arrayed as spatially defined pendant groups on a peptoid oligomer. tamide-bound AR. Pharmacologic studies revealed that MPC6 was The conjugate, named Multivalent Peptoid Conjugate 6 (MPC6), metabolically stable and displayed a low plasma clearance rate. suppressed the proliferation of multiple AR-expressing prostate Notably, MPC6 treatment reduced tumor growth and decreased cancer cell lines including those that failed to respond to enzalu- Ki67 and AR expression in mouse xenograft models of enzaluta- tamide and ARN509. The structure–activity relationships of MPC6 mide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class variants were evaluated, revealing that increased spacing between of compounds with the potential to combat treatment-resistant ethisterone moieties and changes in peptoid topology eliminated its prostate cancer. Cancer Res; 76(17); 1–9. Ó2016 AACR. Introduction inhibiting translocation of AR into the nucleus (7). Although enzalutamide represents a breakthrough in treatment of met- The relationship between androgen receptor (AR) signaling astatic prostate cancer, patients who initially respond eventu- and prostate cancer is well established. Prostate cancer cells are ally acquire resistance (8). Finding effective new therapies for dependent on AR signaling, and for this reason, targeting AR is the enzalutamide-resistant prostate cancer is an emerging clinical mainstay of treatment for metastatic prostate cancer (1, 2). This challenge (9). usually involves either luteinizing hormone-releasing hormone We have developed a new approach for antagonizing AR (LHRH) agonists that prevent testicular androgen synthesis or AR function using multivalent peptidomimetic conjugates (MPC; antagonists, such as bicalutamide (Casodex), which block AR reviewed in ref. 10). MPCs site-specifically display diverse bioac- transcriptional activity (3). Although initial responses to these tive ligands along an N-substituted glycine oligomer scaffold, therapies are effective, they inevitably fail because castration- termed a peptoid. Peptoids offer distinct advantages over other resistant prostate cancer cells emerge exhibiting enhanced AR polymeric multivalent constructs because of the ability to control activity (4). The realization that castration-resistant prostate the exact number of conjugated ligands and their precise spacing, cancers maintain their reliance on androgen signaling (5, 6) thereby enhancing affinity and specificity toward the desired prompted the development of the second generation of anti- target receptor (11). Unlike peptides, peptoids are resistant to androgens, such as enzalutamide, which block AR action by proteases, thus providing improved pharmacologic stability. In addition, peptoids exhibit desirable water solubility and cell 1Department of Urology, New York University School of Medicine, New permeability characteristics (12). Previously, we developed a York, New York. 2Department of Chemistry, New York University, New versatile synthetic platform that allows the modular design of 3 York, New York. York College, Institute for Macromolecular Assem- diverse peptoid oligomer products with multiple spatially defined blies and the Graduate Center of the City University of New York, Jamaica, New York. 4Department of Microbiology, New York Univer- reactive side chains (13). We successfully ligated the AR targeting sity School of Medicine, New York, New York. 5Department of Bio- ligand ethisterone to the peptoid side chains to create a library of chemistry and Molecular Pharmacology, New York University School MPCs (13, 14). Among these, we demonstrated that one of the of Medicine, New York, New York. divalent multivalent peptoid conjugates, called MPC6, competed Note: Supplementary data for this article are available at Cancer Research for androgen binding to the AR ligand binding domain (LBD) Online (http://cancerres.aacrjournals.org/). in vitro, and inhibited the proliferation and expression of AR Corresponding Author: Michael J. Garabedian, NYU School of Medicine, 550 target genes in androgen-independent LNCaP-abl cells (13, 14). First Avenue, New York, NY 10016. Phone: 212-263-7662; Fax: 212-263-8276; In this study, we determined the efficacy of MPC6 against a E-mail: [email protected] broad range of androgen-dependent and -independent prostate doi: 10.1158/0008-5472.CAN-16-0385 cancer lines, including enzalutamide-resistant variants, and tested Ó2016 American Association for Cancer Research. the effectiveness of MPC6 in vivo in mouse xenograft models. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0385 Wang et al. Using a chemical synthesis and screening platform, we also Fluorescent-Two Hybrid assay for AR N/C interaction performed structure-function analyses to determine the impact Fluorescent-Two Hybrid (F2H) assay for live-cell analysis of of ligand spacing and conformational flexibility on MPC6 func- AR N/C interactions was performed using the F2H-AR Kit (Chro- tion. We conducted preliminary pharmacokinetic and pharma- moTek) as per the manufacturer's instructions. The AR-N-termi- codynamic studies and also interrogated the mechanism of MPC6 nal-transcriptional domain (N) was fused to RFP and a DNA action. Our findings demonstrate that MPC6 is effective at block- binding domain linked AR-C-terminal LBD (C) fused to GFP were ing tumor growth in preclinical models of therapy-resistant pros- cotransfected into F2H-BHK cells containing an arrayed cognate tate cancer. DNA binding element. Cells were treated with vehicle, 10 nmol/L DHT, or 10 mmol/L MPC6 for 24 hours. The AR N/C interactions Materials and Methods were evaluated by confocal fluorescence microscopy (Zeiss). Cell culture and treatments LNCaP, 22Rv1, and PC-3 cells (ATCC), and C4-2 cells Western blotting (UroCor) were cultured in RPMI-1640 with 10% FBS. Western blotting was performed as previously described (18). LNCaP-abl cell (a gift from Dr. Zoran Culig, University of Mouse monoclonal anti-AR (441) (sc-7350) antibody was from Innsbruck, Austria; ref. 15) and LNCaP-95 cell (a gift from Santa Cruz Biotechnology. Rabbit monoclonal anti–AR-V7 anti- Dr. John Isaacs and Dr. Jun Luo, John Hopkins University body (ab198394) was from Abcam. Mouse monoclonal anti– Baltimore, MD; ref. 16) were maintained in RPMI-1640 sup- alpha-tubulin antibody (T9026) was from Sigma-Aldrich. plemented with 10% charcoal-stripped serum (CFBS). All cell lines were authenticated by short tandem repeat analysis Quantitative real-time PCR andfoundtobemycoplasmafree(Genetica).Celllineswere Total RNA was extracted by using a Qiagen RNeasy mini kit, and last authenticated January 5, 2016. Cycloheximide and 5a- 1 mg of RNA was reverse transcribed into cDNA using random Dihydrotestosterone (DHT) were purchased from Sigma-Aldrich. primers. cDNA amplification and quantification were done using ARN509 and enzalutamide were purchased from Medkoo. the SYBR Green Taq Ready Mix (USB-Affymetrix) and the Life Technologies QS6 real time PCR detection system. RPL19 gene MPC synthesis expression was used as the control. Primer sequences are listed in The synthesis procedure for 3-azido-1-aminopropane and Supplementary Table S2. linear peptoid scaffolds has been published previously (13). (S)-(þ)-1-Methoxy-2-propylamine (Alfa Aesar) was used as the Protease sensitivity assay chiral amine to synthesize compounds MPC13 and MPC14. Each LNCaP-abl cells were cultured in RPMI-1640 supplemented MPC candidate was purified to >95% by reverse phase analytical with 10% CFBS for 48 hours before being lysed with RIPA buffer. HPLC and verified by electrospray ionization mass spectrometry The lysate (250 mg) was incubated with vehicle control, 10 nmol/L performed on an Agilent LCMSD Trap XCT mass spectrometer. DHT, 10 mmol/L MPC6, or 10 mmol/L enzalutamide at 4C overnight. Samples were then digested with increasing concentra- Cell proliferation assay tions of elastase (1 ng/mL, 10 ng/mL, 100 ng/mL, or 1 mg/mL) for Cells were seeded in 96-well plates at a concentration of 3,000 5 minutes at room temperature. The digestion products were cells per well. Cells were then treated with a single dose of the loaded on SDS-PAGE gel, and AR peptides were detected by fi fi speci ed compound