A multivalent peptoid conjugate modulates androgen receptor transcriptional activity to inhibit therapy-resistant prostate cancer

Abstract

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPCs) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand binding mutations, and non-canonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared to dihydrotestosterone (DHT) and enzalutamide, a clinically used anti-androgen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR anti-proliferative gene expression program.

INTRODUCTION

Prostate cancer cells depend on the androgen receptor (AR) for survival and proliferation (1). Therefore, targeting the AR pathway by blocking androgen production (e.g., abiraterone) or utilizing AR antagonists (e.g., enzalutamide) to impede AR’s transcriptional activity forms the cornerstone of treatment for localized (2) and metastatic prostate cancer (3). Despite the initial efficacy of AR pathway inhibitors, the development of castration-resistant prostate cancer (CRPC) and subsequent relapse is inevitable, leading to significant mortality (4).

Treatment resistance in castration-resistant prostate cancer (CRPC) arises from various mechanisms. One major mechanism observed in approximately 50% of men with CRPC involves the amplification of the AR locus (5,6), resulting in increased AR expression. Additional resistance pathways include point mutations in the AR, enabling activation by drugs like enzalutamide (7,8), and the emergence of constitutively active AR variants (9,10). Treatment failure with AR-pathway inhibitors is also associated with prostate cancer variants displaying characteristics of small cell and neuroendocrine carcinomas (11), which arise from prostate adenocarcinoma (12). These variants, which remain enzalutamide-resistant and maintain nuclear AR expression, utilize a distinct AR transcriptional program to promote growth (13). Therefore, new strategies are needed to target the AR and induce an anti-proliferative response to overcome resistance to current AR inhibitors.

We created multivalent peptoid conjugates (MPCs) to regulate AR activity and inhibit prostate cancer cell growth. MPCs display copies of an AR ligand on a peptoid scaffold (14,15). Peptoids are N-substituted glycine polymers that have shown significant promise in drug discovery due to their superior proteolytic stability, solubility, and cell permeability characteristics compared to polypeptides (16). Ethisterone was chosen as the ligand due to its AR binding capacity, ability to influence AR transcriptional activity (17,18), and the presence of an ethynyl group to enable precise peptoid conjugation through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions (19,20). We previously reported a divalent ethisterone-peptoid conjugate, MPC6, that inhibited prostate cancer growth but lacked sufficient potency for clinical evaluation (21).

We performed a structure-activity relationship (SAR) study to improve MPC potency. This analysis revealed that MPC309, which features a trivalent ethisterone display, extended linker arms, and N-terminal acetylation, exhibited impressive potency against various CRPC cells in culture and in animal models. We examined the impact of MPC309 on AR-mediated gene expression, AR occupancy, chromatin accessibility, and AR’s interaction with coregulatory proteins. Additionally, we discovered that the cellular uptake of MPC309 relies on macropinocytosis, an endocytic process more prevalent in cancer cells than normal cells (22). Consequently, once inside the cell, MPC309 binds avidly to AR and promotes an AR-dependent anti-proliferative response to inhibit the growth of CRPC.

MATERIAL AND METHODS

Cell culture and reagents

LNCaP (ATCC, Cat#CRL-1740), 22Rv1 (ATCC, Cat#CRL-2505), and PC-3 (ATCC, Cat#CRL-1435) were cultured in RPMI-1640 supplemented with 10% FBS and 1% L-glutamine. LNCaP-abl cells (provided by Dr. Zoran Culig, University of Innsbruck, Austria) and LNCaP-95 cells (provided by Dr. John Isaacs and Dr. Jun Luo, Johns Hopkins University, Baltimore, MD) were maintained in RPMI-1640 medium supplemented with 10% charcoal-stripped fetal bovine serum (c-FBS, Hyclone) and 1% L-glutamine. DU145 cells (ATCC, Cat#HTB-81) and HEK293 cells (ATCC, Cat#CRL-1573) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. 16D, 42D, and 49F LNCaP-derived cells (from Dr. Amina Zoubeidi, Vancouver Prostate Center, Canada) were cultured in RPMI-1640 supplemented with 5% FBS and 1% glutamine, plus 10µM enzalutamide for 42D and 49F cell lines. All cell lines were mycoplasma-free (VenorTM GeM Mycoplasma Detection Kit, Sigma-Aldrich). 5α-Dihydrotestosterone (DHT) (Cat#D-073-1ML), Ethisterone (Cat#E1001-25G), and 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) (Cat#A3085-25MG) were purchased from Sigma-Aldrich. Enzalutamide (Cat#201821) was purchased from Medkoo. Bicalutamide (Cat#S1190) and Chlorpromazine (Cat#S5749) were purchased from Selleck Chemicals. SF1670 (#2774-1) was purchased from BioVision. The plasmid containing the Gal4 DBD (1–147) fused to the LxxLL fragment of SRC1a (1241–1441), the (GAL4)5TATA-Luc luciferase reporter plasmid, and the VP16 AD fused to AR LBD (640–919) plasmid are described in (23) and are gifts of Dr. Frank Claessens (KU Leuven, Belgium). The ARR3-tk-luciferase reporter construct consists of three copies of the rat probasin AREs (2244 to 296) ligated into the pT81 luciferase vector (24).

Mammalian Two-Hybrid for LBD Interaction with LxxLL

To study the interaction between the AR LBD and the LxxLL motif of SRC1a, HEK293 cells were seeded at 10,000 cells per 96-well plate in DMEM, 5% stripped serum, 1% Glutamax (Fisher Scientific, Cat# 10-569-010) and 1% Penicillin-Streptomycin (Fisher Scientific, Cat# 15-070-063). The next day, 100ng (Gal4) 5 TATA Luc reporter plasmid, 50ng of plasmid encoding Gal4 DBD-empty or Gal4 DBD-SRC1a (1241-1441), 10ng of a plasmid expressing VP16 AD-AR LBD (640-919) were co-transfected per 96-well using Lipofectamine^™ 3000 Transfection Reagent (Invitrogen, Cat#L3000001). The next day, cells were treated with increasing DHT or MPC309 (from 0.1nM to 1µM). Luciferase activity was measured with ONE-Glo^™ Luciferase Assay System (Promega) per the manufacturer’s protocol. Cell luminescence was measured using the SpectraMaxM5 Microplate Reader and analyzed with SoftMaxpro software (Molecular Devices, RRID: SCR_014240).

3D-culture of mouse prostate cancer organoids

Prostate of a mouse expressing transgenic cMyc and a floxed allele of p53 were infected with the GFP-AdCre virus to generate Myc⁺/p53^−/− (MP) organoids. These were then transduced with lentivirus with APC sgRNA or PTEN sgRNA and puromycin-selected to produce Myc⁺/p53^−/−/APC^−/− (MPA), Myc⁺/p53^−/−PTEN^−/− (MPP), respectively. Myc⁺/p53^−/− organoids were transduced with mouse AR cDNA to generate Myc⁺/p53^−/−AR^+/+ (MPAR+) organoids. These cells were cultured as 3D organoids using growth factor-reduced Matrigel (Corning, Cat#356231) and advanced-DMEM/F12 medium supplemented with 1% Glutamax, 1% HEPES, 2% B27, 1.25mM N-acetyl-L-cysteine, 0.05µg/ml EGF, 0.2µM A83-01, 0.1µg/ml Noggin, 0.5µg/ml Rspondin 1, and 1nM dihydrotestosterone (25). Upon cell passaging, the culture media was supplemented with 10 µM Y-27632 dihydrochloride, a ROCK inhibitor, B27 (Cat#17504-044), Glutamax (Cat#35050-061), and HEPES (Cat#15630-080) were purchased from Thermo Fisher Scientific, N-acetyl-l-cysteine (Cat#A9165) from Sigma and EGF (Cat#AF-100-15), Noggin (Cat#120-10C), A83-01 (Cat#9094360), Y-27632 dihydrochloride (Cat#1293823) and R-spondin (Cat#120-38) from Preprotech.

MPC synthesis

The synthetic procedure for the linear peptoid scaffold has been previously described in (15). Peptoid scaffolds on resin were taken up in 2-butanol/ DMF/pyridine (5:3:2 by vol.), and the alkyne groups were allowed to react with ethisterone in the presence of CuI, ascorbic acid, and DIPEA in a conical vial. The vial was sealed and vigorously shaken at 45°C for 18 h. The resin was washed with DMF, Cu scavenger cocktail (DMF/pyridine 6:5 v/v, ascorbic acid 0.02 g/mL), and DCM. The resin was cleaved from the solid support with a 95% TFA/H₂O solution and characterized by electrospray ionization mass spectroscopy (ESI-MS). Sequential conjugation reactions were performed as previously described (25). The material was purified by reverse-phase HPLC on a preparative C18 column. HPLC chromatograms were monitored at 230 nm. Linear gradients were conducted from 5 to 95% solvent B (0.1% TFA in HPLC grade acetonitrile) over solvent A (0.1%TFA in HPLC grade water) in 50 min with a flow rate of 5.0 mL/min. Every compound underwent purification exceeding 95% utilizing reverse-phase analytical HPLC and was validated via ESI-MS analysis.

Cell proliferation assays

To determine the IC₅₀, cells were plated at a density of 2,000 cells per well in 96-well plates. The cells were then exposed to a single dose of the designated compound at the concentration specified in the figure captions for 5 days. Cell proliferation was measured with CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Cat# G7570) per the manufacturer’s protocol. For cell proliferation assays, cells were seeded in 384-well plates at 1,000 cells or in 96-well plates at 2,000 cells per well. Cells were then treated with the specified compound at the concentration and for the duration indicated in the figure legends. Cell proliferation was measured by CyQUANT^® Cell Proliferation Assay (Fisher Scientific, Cat#C35011) or PrestoBlue Cell Viability Assay (Thermofisher Scientific, Cat#P50200) per the manufacturer’s instructions. For 3D cultures, cells were seeded in 96-well plates (30µl Matrigel droplets). The cells were exposed to a single dose of the designated compound at the concentration specified in the figure legends for a duration of 72 hours. Cell proliferation was assessed using the CellTiter-Glo^® 3D Cell Viability Assay (Promega, Cat#G7570) according to the manufacturer’s instructions. Cell fluorescence, absorbance, or luminescence were quantified with the SpectraMaxM5 Microplate Reader and SoftMaxpro software (Molecular Devices, RRID: SCR_014240).

Dextran uptake assay

Cells were plated at a density of 20,000 cells per well onto glass coverslips. As previously described (26), 72 hours after cell seeding, macropinosomes were marked utilizing a high molecular weight TMR-dextran (Invitrogen, Cat#D1818). Cells were incubated with TMR-dextran (1mg/mL) for 30 minutes at 37°C in serum-free media. At the end of the incubation period, cells were rinsed five times in cold PBS and immediately fixed in 3.7% formaldehyde. Cells were treated with DAPI to stain the nuclei and coverslips mounted onto slides using DAKO Mounting Media (Agilent DAKO, Cat#S302380-2). Images were captured using an EVOS M7000 inverted fluorescent microscope (Zeiss) and analyzed using the ‘Analyze Particles’ feature in ImageJ (NIH, RRID: SCR_003070). The total particle area per cell was determined from at least 3 fields randomly selected from different regions across each sample.

AR ligand binding assay

Competitive binding to the AR in vitro was assessed using the PolarScreen^™ Androgen Receptor Competitor Assay Kit, Green (Thermofisher Scientific, Cat#A15880), per the manufacturer’s protocol.

AR co-activator assay

Coactivator peptide recruitment to AR in vitro was assessed using the LanthaScreen^™ TR-FRET Androgen Receptor Coactivator Assay Kit in agonist mode (A15878 Thermofisher Scientific Cat# A15878) per the manufacturer’s protocol. The D11FxxLF peptide sequence is VESGSSRFMQLFMANDLLT (27).

AR expression and purification

The protocol was adapted from Zhou et al. (28). The 6His-GST-AR bacterial expression plasmid containing the AR DNA binding domain and LBD (amino acids 556–919) was transformed into BL21 (DE3) bacteria. Cultures (1 liter) were grown at 37°C and were induced with 0.1mM IPTG at an OD₆₀₀ of 0.6. At the same time, DHT and Zn acetate were added to concentrations of 10µM. All buffers contained 10µM DHT and Zn acetate. Cultures were incubated at 30°C for 8 hours. Bacteria were pelleted and re-suspended in 20ml 1X PBS. Lysis was carried out using a French Press. The lysate was incubated overnight with Glutathione conjugated beads. The beads were pelleted and loaded on a nickel-charged affinity resin Ni-NTA agarose column. The column was washed with 15ml of PBS and eluted with 20mM glutathione in PBS. The most concentrated fractions were dialyzed into a buffer containing 20mM Tris-HCL pH8.0, 200mM NaCl, 10% glycerol, 10µM Zn acetate, and 10µM DHT and stored at −80°C.

Electrophoretic Mobility Shift Assay

For electrophoretic mobility shift assay (EMSA), 10µg of the 6His-GST AR-CDE were incubated with DHT (10nM), Backbone (10µM), or MPC309 (10µM) for 4h at RT in 5x Gel Shift Binding buffer (20% glycerol, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 250 mmol/L NaCl, 50 mmol/L Tris-HCl). After incubation, 0.25 mg/mL poly(dI)-poly(dC) and IRDye 700-labeled ARE oligonucleotide (LI-COR, Lincoln, NE) were added and incubated for 30 minutes at room temperature. The specimens were placed onto a pre-run gel made of 4–20% Tris-Borate-EDTA (TBE), and electrophoresis was conducted for 90 minutes at a current of 30 mA. Subsequently, the signal was assessed and quantified using the LI-COR Odyssey infrared imaging system. In order to validate the specificity of the AR DNA-binding activity, a competition assay was conducted using wild-type and mutant competitors. The sequences of the LI-CORE labeled oligonucleotides were:

Wild-Type (WT) ARE: 5’-GAA GTC TGG TAC AGG GTG TTC TTT TTG-3’

Mutant ARE: 5’-GAA GTC TGC AAC AGG GTC ATC TTT TTG-3’

Protein extraction and Western Blot analysis

For subcellular protein extracts, cells were fractionated using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermofisher Scientific; Cat#78840), per the manufacturer’s protocol. The proteins were separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Cat# IPVH00010, Fisher Scientific). The membranes were blocked in a solution of PBS, 0.1% Tween 20, and 5% BSA for 1 hour. Following the blocking step, the membranes were incubated overnight at 4°C with one of the following antibodies: anti-AR (441, Santa Cruz Biotechnology; Cat# sc-7305), anti-Histone H3 (Cell Signaling; Cat# 9715), or anti-tubulin (Covance; Cat# MMS-489P). After three washes with PBS, 0.1% Tween 20, the membranes were incubated for 1 hour with either goat anti-mouse horseradish peroxidase-conjugated (HRP) IgG (Cat# 7076S, Cell Signaling) or donkey anti-rabbit HRP-conjugated IgG (Cat# NA9340, Amersham). The membranes were then extensively washed with PBS, 0.1% Tween 20, and the immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Cat#1705060, Bio-Rad) according to the manufacturer’s instructions.

Mouse xenograft studies

LNCaP-abl cells (1×107) were combined with an equal volume of Matrigel (50 µl) and injected beneath the skin on the right flank of castrated male NOD SCID gamma (NSG) mice aged 6 to 8 weeks. After the tumors grew to an average volume of 100 mm3, the mice were divided randomly into five groups of five. Subsequently, the mice in each group received treatment via intraperitoneal injection of either Vehicle (DMSO), MPC309 (at doses of 50 mg/kg, 10 mg/kg, or 5 mg/kg), or oral administration of Enzalutamide (at a dose of 30 mg/kg). These treatments were administered daily for a duration of 19 days. Mice were weighed, and tumor volume was measured every 4 days until sacrifice. The mice were kept in a facility free from pathogens and received proper care following the guidelines approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC) under protocol number IA16-01775. All essential precautions were implemented to ensure the ethical and compassionate treatment of the animals throughout the duration of the study.

Pharmacokinetic studies

CD-1 male mice were treated intraperitoneally with MPC309 (50mg/kg) for 24h. Blood was collected for tripotassium EDTA-plasma preparation. Samples were extracted by acetonitrile protein precipitation and injected into an LC-MS/MS. MPC309 levels were determined relative to a calibration curve. These experiments were performed by WuXi AppTec company (Shangai, China). C57BL/6 mice were treated intraperitoneally with Vehicle (DMSO) or MPC309 (50 mg/kg) for 7 days. Mice were weighed, and blood samples were collected from the submandibular (facial) vein for flow cytometry analysis. This study was performed at the NYU School of Medicine. The animal research was approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC), protocol number IA16-01775.

RNA-sequencing

RNA-sequencing experiments were performed as previously described (29). RNA extraction was performed using the RNeasy kit (Qiagen), and libraries were prepared for sequencing using the NEBNext Ultra II RNA Library Prep Kit for Illumina with polyA selection. The concentration of the libraries was determined using Qubit, and their quality was assessed using Tapestation. The libraries were sequenced on an Illumina NovaSeq 6000 SP100 flowcell. Data analysis was conducted using the ROSALIND^® platform (https://rosalind.onramp.bio/), which utilizes the HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA). Reads were trimmed using cutadapt. Quality scores were assessed using FastQC68. Reads were aligned to the Homo sapiens genome build hg19 using STAR3. Individual sample reads were quantified using HTseq and normalized via Relative Log Expression (RLE) using the DESeq2 R library. Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step using RSeQC. DEseq2 was also used to calculate fold changes and p values and perform optional covariate correction. The clustering of genes for the final Heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library https://cran.r-project.org/web/packages/fpc/index.html. Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. Enrichment analysis was conducted by referencing several databases, including Interpro, NCBI, MSigDB REACTOME, and WikiPathways. The enrichment analysis was performed relative to a set of background genes that were relevant to the specific experiment. Differential gene expression analysis was carried out using the Enrichr platform developed by the Ma’ayan Laboratory (30).

Chromatin immunoprecipitation (ChIP)-sequencing

ChIP experiments were performed as previously described in (29). To preserve protein-DNA and protein-protein interactions, cells were subjected to double crosslinking using formaldehyde and the bifunctional protein crosslinker disuccinimidyl glutarate (DSG). For AR ChIP, a combination of two antibodies was used: AR 441 mouse monoclonal antibody from Santa Cruz Biotechnology (Cat# sc-7305) and AR D6F11 rabbit monoclonal antibody from Cell Signaling Technology (Cat# 5153). This antibody mixture was chosen to maximize AR enrichment and minimize epitope masking. Inputs were included for normalization, and additional IgG controls were used to ensure that any low occupancy peaks observed were specific to AR and not background noise. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit, quantified on Qubit, pooled equimolarly, and sequenced on an Illumina NovaSeq 6000 SP100 flowcell. Data were analyzed by Rosalind (https://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA). Reads were trimmed using cutadapt. Quality scores were assessed using FastQC. Reads were aligned to the Homo sapiens genome build hg19 using bowtie2. Per-sample quality assessment plots were generated with HOMER and Mosaics. Peaks were called using MACS2 (with input controls background subtracted). Peak overlaps were analyzed using the DiffBind R library. Read distribution percentages, identity heatmaps, and FRiP plots were generated as part of the QC step using the ChIPQC R library and HOMER. HOMER was also used to generate known and de novo motifs and perform functional enrichment analysis of pathways, gene ontology, domain structure, and other ontologies.

ATAC-sequencing

Chromatin profiling was performed by ATAC-seq. Cells (1×10⁵) were washed in cold PBS and lysed in cold lysis buffer (10 mM Tris-HCl, pH = 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). The transposition reaction (Illumina) was performed at 42C for 45 min. After purification of the DNA with the MinElute PCR purification kit (QIAGEN), the material was amplified for 5 cycles using NEB Next Ultra DNA Library Prep (New England BioLabs), and size was verified on Tapestation. Normalized libraries were sequenced on a HiSeq2500 1T in a 50bp/50bp Paired-end run at the Genome Technology Center at NYU, using the TruSeq SBS Kit v3 (Illumina). Data were analyzed by Rosalind (https://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. (San Diego, CA). Reads were trimmed using cutadapt. Quality scores were assessed using FastQC. Reads were aligned to the Homo sapiens genome build hg19 using bowtie2. Per-sample quality assessment plots were generated with HOMER and Mosaics. Peaks were called using MACS2 (with input controls background subtracted). Peak overlaps were analyzed using the DiffBind R library. Read distribution percentages, identity heatmaps, and FRiP plots were generated using HOMER as part of the QC step. HOMER was also used to generate known and de novo motifs and perform functional enrichment analysis of pathways, gene ontology, domain structure, and other ontologies.

Rapid immunoprecipitation and mass spectrometry of endogenous proteins (RIME)

Treatments and immunoprecipitation for RIME (31) were carried out as described for ChIP-sequencing, except the beads with antibody-bound proteins were washed twice with 50 mM NH4HCO3 and resuspended in 50μl 50 mM NH₄HCO3. In-solution protein digestion was performed by reduction of samples with DTT at 57°C for 1 hour (2 µl of 0.2 M), alkylation with iodoacetamide at room temperature in the dark for 45 minutes (2 µl of 0.5 M) and addition of sequencing grade modified trypsin (Promega) overnight on a shaker at room temperature (500 ng). For protein extraction, samples were loaded onto equilibrated Ultra-Micro SpinColumns (Harvard Apparatus) using a microcentrifuge, rinsed three times with 0.1% TFA and washed with 0.5% acetic acid. Peptides were eluted with 40% acetonitrile in 0.5% acetic acid, followed by the addition of 80% acetonitrile in 0.5% acetic acid. Organic solvents were removed using a SpeedVac concentrator, and the samples were resuspended in 0.5% acetic acid. MS Analysis was performed by analyzing 2/15th of each sample individually. LC separation was performed online with MS using the autosampler of an EASY-nLC 1000 (Thermo Scientific). Peptides were gradient eluted from the column directly to the Q Exactive mass spectrometer using a 1 hr gradient (Thermo Scientific). High-resolution full MS spectra were acquired with a resolution of 70,000, an AGC target of 1e6, with a maximum ion time of 120 ms, and a scan range of 400 to 1500 m/z. Following each full MS, twenty data-dependent high-resolution HCD MS/MS spectra were acquired. All MS/MS spectra were collected using the following instrument parameters: resolution of 17,500, AGC target of 5e4, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, and NCE of 27. MS/MS spectra were searched against a Uniprot human database using Sequest within Proteome Discoverer 1.4. Peptides and proteins were then grouped and filtered. Similar levels of AR were found in all groups (AR being one of the most abundant proteins), which indicated successful pulldown. Common background proteins were removed from the analysis. Fold change of average PSMs (Peptide Spectral Matches) was calculated by adding +5 to include PSM values of 0. For analysis, IgG-bound peptides were subtracted from antibody-bound peptides, and then samples were normalized to the amount of AR pulled down in each replicate.

Lactate dehydrogenase (LDH) release assay

To evaluate necrotic plasma membrane permeabilization, cells were plated at a density of 2,000 cells per well in 96-well plates. The cells were then exposed to a single dose of the designated compound at the concentration specified in the figure captions for a duration of 24 hours. Cell viability was assessed by lactate dehydrogenase (LDH) leakage in the culture medium with the CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit (Promega; Cat# G1780).).

Cell cycle analysis

Cell cycle distribution analysis based on DNA content was performed using one-parameter flow cytometry (32). Cells (1×10⁶) were harvested by centrifugation, followed by resuspension in ice-cold 70% ethanol for 30 minutes. After another centrifugation step, the cell pellets were washed twice with PBS and then resuspended in PBS containing 50 μg/ml propidium iodide (PI). The fluorescence signal (FL2) was measured using a Beckman Coulter CytoFLEX flow cytometer, and cell cycle phases were estimated using FlowJo software (v.10.8.1, RRID: SCR_008520).

RNA preparation and quantitative RT-PCR

Total RNA was extracted using the RNeasy kit (Qiagen) following the manufacturer’s instructions. Reverse transcription of RNA (1 µg) was performed using the Verso cDNA Synthesis Kit (ThermoFisher Scientific; Cat# AB1453A) according to the manufacturer’s instructions. Gene-specific cDNA was amplified in a 10 µL reaction containing Fast SYBR Green qPCR Master Mix (ThermoFisher Scientific; Cat# 4385612). Real-time PCR was conducted using the Applied Biosystems Quantstudio 6 Flex Real-Time PCR System, with each gene tested in triplicate. Data analysis was performed using the ΔΔCT method, with GAPDH as the control gene and normalization to control samples, which were arbitrarily set to a value of 1. The primer sequences used for real-time PCR are as follows:

GAPDH:

F: 5′-TCACCACCATGGAGAAGGC-3′ and R: 5′-GCTAAGCAGTTGGTGGTGCA-3′

KLK3:

F: 5’-GGTGGCTGTGTACAGTCATGGAT-3’ and R: 5’-TGTCTTCAGGCTCAAACAGGTTG-3’

FKBP5:

F: 5’-GCGAAGGAGAAGACCACGACAT-3’ and R: 5’-TAGGCTTCCCTGCCTCTCCAAA-3’

Luciferase Activity Assay

LNCaP-abl cells stably expressing the ARR3-tk-luciferase reporter were seeded in 96-well plates at 2,000 cells per well. Cells were treated with a single dose of the specified compound at the concentration indicated in the figure legends for 24 hours. Luciferase activities in cell lysates were measured using the ONE-Glo^™ Luciferase Assay System (Promega; Cat#E6110). The results are presented as fold induction relative to vehicle-treated cells.

Immunohistochemistry

Immunohistochemistry (IHC) was conducted on paraffin-embedded tissue sections. The sections were dewaxed in xylene, rehydrated, and washed in phosphate-buffered saline (pH 7.4). Antigen retrieval was achieved by heating the sections in a microwave oven (900 watts) for 5 minutes in 10 mM citrate buffer. Subsequently, the sections were treated with 3% H2O2 and blocked with 20% normal goat serum. Sections were incubated at room temperature for 2 hours with an antibody against AR (AR N-20, Santa Cruz Biotech; Cat# sc-816) at a dilution of 1:500. After washing, the sections were incubated for 1 hour with a biotinylated rabbit secondary antibody (dilution 1:1000; Vector Labs). An avidin-biotin complex was formed, and diaminobenzidine chromagen was used for development. Finally, a counterstain with hematoxylin was performed. Slides were scanned with a Leica SCN400 F whole slide scanner (BF40X) and analyzed with the QPath software (https://qupath.github.io, RRID: SCR_018257).

Peripheral blood mononuclear cell isolation and flow cytometry analysis

Mouse blood was collected and mixed with 0.5 M EDTA. Red blood cells were lysed with 1X RBC buffer (Biolegend; Cat#420301) for 15 minutes. Peripheral blood mononuclear cells (PBMCs) were spun down at 1,500 r.p.m. for 5 min at 4 °C (Eppendorf centrifuge 5810R). The cells were washed with 1X PBS (Gibco; Cat#10010049), re-suspended in FACS buffer (2% FBS and PBS 1X), and processed for flow cytometry analysis. Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies purchased from Biolegend: DX5 (clone DX5), Ly6G (clone 1A8), CD25 (clone PC61), F4/80 (clone BM8), CD4 (clone GK1.5), Ly6C (clone HK1.4), CD45 (clone 30-F11), CD8 (clone 53-6.7), CD3 (clone 17A2), CD11b (clone M1/70), CD11c (clone N418). Live cells were determined by LIVE/DEAD staining with DAPI (Biolegend; Cat#422801). Cells were analyzed using BD Fortessa with BD FACS Diva (BD Biosciences, v.9.0, RRID:SCR_013311) and FlowJo software (v.10.8.1, RRID: SCR_008520).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism software (RRID: SCR_002798). The data are presented as mean ± SEM (technical replicates for each experiment as indicated). The number of experiments and specific statistical tests performed are specified in the figure legends. A p-value <0.05 was considered statistically significant, and significance levels are denoted as *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001 unless otherwise stated.

Data Availability

Data generated or analyzed during this study will be available from the corresponding author upon reasonable request. The datasets generated and analyzed during the current study are available in the NCBI/GEO repository under GSE236287 (ChIP-seq); GSE236288 (ATAC-seq); GSE236286, and GSE236088 (RNA-seq).

RESULTS

Development of MPC309 to selectively inhibit prostate cancer cell proliferation

We conducted a SAR study to improve MPC potency. This involved: i) varying the valency (trivalent and tetravalent) of the ethisterone groups; ii) modifying the length of the side chain alkyl linker group (propyl versus hexyl) between the peptoid backbone and the ethisterone to present the ligand in the most favorable orientation, and iii) acetylating the N-terminus of the oligomer to enhance uptake and AR binding (Fig. 1A). A series of compounds (MPC303, MPC305, MPC306, MPC307, MPC309, and MPC310) were designed and evaluated for their impact on the proliferation of LNCaP-abl cells cultured in media with charcoal-stripped serum, thus representing a model for advanced androgen-independent prostate cancer (33) (see structures in Fig. S1). The effects of these compounds were compared to staurosporine, a non-selective protein kinase inhibitor, and two AR antagonists, enzalutamide and bicalutamide (Fig 1B).

Figure 1. A structure-activity study identifies more potent Multivalent Peptoid Conjugates (MPCs) that selectively inhibit prostate cancer cell proliferation.

(A) Structural components of an MPC. The peptoid backbone, a linker, an ethisterone moiety, and an optional acetyl group are circled. (B) Comparison of the IC₅₀ of a library of MPCs to standard anticancer drugs on LNCaP-abl cells cultured in media with charcoal-stripped serum using CellTiter-Glo^® Luminescent Cell Viability Assay conducted 5 days after treatment with a single dose of MPC at day 0 (n=2). The valency (number of ethisterone moieties), the ligand spacing (length of the linker between ethisterone and the peptoid), and the presence or absence of an N-terminal acetyl group are indicated for each MPC. The peptoid scaffold lacking the ethisterone moieties is termed the “backbone.” (C) Evaluation of MPC309 binding the AR in vitro. Left: Graphic representation of the AR in vitro ligand binding assay, where the AR-LBD is bound to a fluorescent androgen ligand, and a test competitor is added. Competitors displace the fluorescent ligand from the AR-LBD, resulting in a low polarization value. Right: Ligand binding to the AR-LBD was performed using competitors DHT, MPC309, and enzalutamide at the indicated concentrations, and fluorescence polarization was measured. Below are the IC₅₀ (μM). (n=2). (D) Comparison of MPC309’s antiproliferative activity in AR-positive cell lines (LNCaP-abl and LNCaP-95) using CyQUANT^® Cell Proliferation Assay after 3 days of treatment (n=3). Cells were cultured in media containing 10% charcoal-stripped FBS. (E) Comparison of MPC309’s antiproliferative activity in LNCaP-derived CRPC cell lines using CyQUANT^® Cell Proliferation Assay after 7 days of daily treatment (n=3). 16D cells were cultured in media containing 5% FBS, whereas 42D and 49F cell lines were cultured in media with 5% FBS and 10µM enzalutamide. (F) Evaluation of MPC309 effect on the growth of mouse 3D prostate organoid cultures MP (MYC⁺/p53^−/−), MPA (MYC⁺/p53^−/−APC⁻/⁻), MPP (MYC⁺/p53^−/−PTEN⁻/⁻) and MPAR+ (MYC⁺/p53^−/−AR⁺/⁺) using CellTiter-Glo^® 3D Cell Viability Assay after 3 days of treatment (n=3). See materials and methods for a description of the media used for organoid cultures. One-way ANOVA was used to test for statistical significance between the groups, and the difference was considered significant for P values <0.05 (*P<0.05, **P <0.01, ***P <0.001, ****P <0.0001).

MPC309 exhibited the highest potency among the compounds tested, with an IC₅₀ in the nanomolar range (~27 nM). MPC309 featured a trivalent ethisterone display, an extended hexyl linker group, and N-terminal acetylation of the peptoid (Fig. 1B and Fig. S1). In contrast, the trivalent ethisterone conjugates MPC303 and MPC305, which incorporated a shorter propyl linker between the ethisterone and the peptoid, were inactive in the cell proliferation assays (IC₅₀ >100 μM). This suggests that increasing the spacing between the peptoid and the ligand is crucial for the potency of MPC309. Given these promising results, MPC309 was chosen for subsequent studies.

We evaluated MPC309 binding to the AR using an in vitro ligand-binding assay (Fig. 1C). MPC309 exhibited strong binding to AR, with an IC₅₀ of 28.9nM consistent with the IC₅₀ observed in the cell proliferation assays (Fig. 1B). The unconjugated oligomer backbone alone lacking ethisterone groups did not bind to AR. These results established that the multivalent MPC309 exhibits high-affinity binding to AR.

MPC309 suppresses prostate cancer growth more effectively than enzalutamide

We next examined the antiproliferative effects of MPC309 on various prostate cancer cell lines. We compared the activity of MPC309 to the unconjugated peptoid backbone and to enzalutamide. MPC309 treatment significantly reduced the proliferation of AR-expressing, androgen-dependent LNCaP cells (Fig. S2A) and blocked the proliferation of androgen-independent LNCaP cell derivatives, LNCaP-abl (33) that expresses full-length AR (34) and LNCaP-95 (27), that harbor both full-length AR and the AR-V7 variant, a known marker of enzalutamide resistance (Fig. 1D). MPC309 was 250-fold more potent than enzalutamide at inhibiting cell proliferation in LNCaP-abl and LNCaP-95 cells. The backbone alone was inactive. AR-deficient PC3 and DU145 cells did not respond to MPC309 treatment, indicating that the antiproliferative activity of MPC309 is dependent on AR expression (Fig. S2A). Consistent with this, MPC309 induced canonical AR target genes KLK3 (PSA) and FKBP5 (Fig. S2B) and enhanced a synthetic AR-responsive luciferase reporter gene but with lower potency than DHT (Fig. S2C).

We also evaluated the potential cytotoxic effect of MPC309 by measuring lactate-dehydrogenase (LDH) in the media, which is released upon damage to the plasma membrane and is a surrogate for cytotoxicity. MPC309 treatment did not induce LDH release in LNCaP-abl cells (Fig. S2D). In addition, we examined the potential effect of MPC309 on cell cycle arrest by measuring Propidium Iodine (PI) incorporation in treated cells. A trend toward the accumulation of cells at the G2/M stage was observed 72 hours after MPC309 treatment (Fig. S2E).

MPC309 treatment was also effective in overcoming enzalutamide resistance in three different LNCaP sub-lines derived from xenografts with a CRPC phenotype, 16D cells (CRPC phenotype) and 49F and 42D (enzalutamide-resistant CRPC phenotype) (13). The latter two are cultured continually in media with 10μM enzalutamide. For 49F cells (AR+/PSA+), the AR ligand-binding domain harbors a mutation (F876L) that converts enzalutamide into an AR agonist and confers resistance to enzalutamide (35). For 42D cells (AR+/PSA-), a non-canonical AR gene expression program promoting proliferation is driven by the unconventional AR coactivator EZH2 and represents an AR-indifferent cell model (13). Remarkably, all three cell lines were sensitive to MPC309 treatment (Fig. 1E), suggesting that MPC309 can engage AR to override diverse clinically relevant mechanisms of castration-resistant and enzalutamide-resistant prostate cancers.

We next took advantage of a 3D-organoid model (36) based on mouse prostate cells harboring defined genetic alterations that recapitulate lethal prostate cancer subtypes. These include organoids with: i) cMyc overexpression (Myc⁺) and p53 deletion (p53^−/−) (MP); ii) Myc⁺/p53^−/− and APC deletion (MPA); iii) Myc⁺;p53^−/− and PTEN deletion (MPP); iv) Myc⁺;p53^−/− and AR overexpression (MPAR+). Loss of either APC or PTEN and overexpression of AR are known to promote more aggressive forms of prostate cancers (37). Treatment with MPC309, but not enzalutamide, markedly inhibited the growth of all four types of organoids, with MPP organoids being the most sensitive (Fig. 1F). Thus, the proliferation of various enzalutamide-resistant prostate cancer models is inhibited by MPC309.

MPC309 enters prostate cancer cells via macropinocytosis

Peptidomimetic compounds, similar to MPC309, have been found to enter cells through macropinocytosis (38), an endocytic mechanism of non-specific fluid uptake used by many cancer cells to scavenge extracellular nutrients for cell growth (22). Indeed, peptidomimetic compounds show efficient cellular uptake and endosomal escape into the cytoplasm in macropinocytic cells (38). We investigated the effect of endocytosis inhibitors on the antiproliferative response of MPC309 in LNCaP-abl cells. Low temperature is expected to inhibit macropinocytosis and clathrin-mediated endocytosis as they are energy-dependent processes (39). We observed that the antiproliferative effect of MPC309 was significantly decreased when cells were incubated at 4°C compared to 37°C before the addition of MPC309 (Fig. 2A). This suggests that MPC309 enters cells through an energy-dependent endocytosis mechanism. We next tested whether inhibitors of macropinocytic or clathrin pathways decreased the antiproliferative effect of MPC309. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not affect the antiproliferative response of MPC309, whereas 5-(N-ethyl-isopropyl) amiloride (EIPA), an inhibitor of macropinocytosis, abolished the antiproliferative effect of MPC309 in LNCaP-abl cells (Fig. 2A). The impact of EIPA was also observed in the MPP organoid model (Fig. S2F). Treatment of the cells with EIPA did not affect the antiproliferative activity of the small molecules ethisterone or enzalutamide in LNCaP-abl cells, which show a partial anti-proliferative response at high concentrations (Fig. 2B). These findings suggest that macropinocytosis plays a significant role in MPC309 cellular uptake.

Figure 2. MPC309 enters prostate cancer cells via macropinocytosis.

(A) Comparison of LNCaP-abl cell viability after pre-incubation with inhibitors of representative endocytosis pathways. LNCaP-abl cells were pre-incubated at 4 degrees or with the macropinocytosis inhibitor, EIPA (50µM), or the clathrin inhibitor chlorpromazine (50µM) for 30min or left untreated before a 1h pulse with MPC309 (10µM). Cell viability was measured using PrestoBlue^® HS cell viability reagent after 3 days of treatment (n=3). Data were normalized to vehicle. One-way ANOVA was used to test for statistical significance between the groups, and the difference was considered significant for P values <0.05 (***P <0.001, ****P <0.0001). (B) Evaluation of the effect of a macropinocytosis inhibitor on ethisterone and enzalutamide activity in LNCaP-abl cells. LNCaP-abl cells were pre-incubated with EIPA (50µM) for 30min or left untreated before the addition of enzalutamide or ethisterone (10µM). Cell viability was measured using PrestoBlue^® HS cell viability reagent after 3 days of treatment (n=2). One-way ANOVA was used to test for statistical significance between the groups and the difference was considered nonsignificant for P values >0.05. (C) Comparison of LNCaP-abl cells and 22Rv1 macropinocytic activity. 22Rv1 cells were pre-incubated with SF1670 (2µM) for 30min or left untreated before the assay. TMR-dextran was added to serum-free medium at a final concentration of 1 mg/mL for 30min. Cells were counterstained with DAPI for 5min. Images were captured using a fluorescent microscope and analyzed using ImageJ’s ‘Analyze Particles’ feature. The total particle area per cell was determined from at least 3 fields randomly selected from different regions across each sample. One-way ANOVA was used to test for statistical significance between the groups and the difference was considered significant for P values <0.05 (*P < 0.05, ***P <0.001). (D) Evaluation of the effect of a PTEN inhibitor SF1670 on MPC309 activity in 22Rv1 cells. Cells were pre-incubated with SF1670 (2µM) for 30min or left untreated before adding MPC309 at the indicated concentrations. Cell viability was measured using PrestoBlue^® HS cell viability reagent after 3 days of treatment (n=2). Treated cells were compared to Vehicle control (set to 100%). One-way ANOVA was used to test for statistical significance between each treatment and their vehicle control and the difference was considered significant for P values <0.05 (**P <0.01, ***P <0.001, ****P <0.0001).

PTEN loss promotes macropinocytosis in prostate cancer cells (40), consistent with PI3K signaling regulating macropinocytosis (22). PTEN-deficient cell lines LNCaP, LNCaP-abl, and LNCaP-95 were highly sensitive to MPC309 treatment (Fig. 1D, Fig. S2A), while the PTEN-proficient cell line 22Rv1 showed significantly lower sensitivity (Fig. S2A). This led us to investigate the link between PTEN status and sensitivity to MPC309. We confirmed the higher macropinocytic index in LNCaP-abl cells compared to 22Rv1 (Fig. 2C). We treated 22Rv1 cells with the PTEN inhibitor SF16170. We found enhanced macropinocytosis (Fig. 2C) (40) and increased antiproliferative effect of MPC309 (Fig. 2D). SF1670 did not affect MPC309 activity on PTEN-deficient LNCaP-abl cells, which confirms the specificity of the inhibitor (Fig. S2G). These findings suggest that MPC309 enters prostate cancer cells via macropinocytosis, a process that is increased by the loss of PTEN. This is particularly relevant as PTEN loss is observed in up to 60% of metastatic prostate cancers (41).

MPC309 stimulates AR chromatin occupancy and promotes a unique transcriptome

To identify the genes and pathways affected by MPC309, we performed RNA-seq from LNCaP-abl cells treated overnight with vehicle (DMSO), peptoid backbone, MPC309, DHT, enzalutamide, or ethisterone. We found distinct gene expression profiles (transcriptomes) for cells treated with each compound, suggesting that MPC309 is a novel AR ligand that modulates the AR transcriptional response (Fig. 3A and Fig. S3A). MPC309 treatment compared to vehicle control resulted in the upregulation of 405 genes and the downregulation of 251 genes, with little overlap between genes regulated by MPC309 and enzalutamide (Fig. 3A) or ethisterone (Fig. S3A). Additionally, while some genes were shared between DHT- and MPC309-treated cells (153 genes upregulated and 25 genes downregulated) (Fig. 3A), the majority were unique to one treatment. MPC309 treatment differentially expressed 422 genes (186 upregulated and 238 downregulated) compared to DHT-treated cells (Fig. 3B). These findings highlight MPC309’s unique ability to modulate the AR transcriptional response.

Figure 3. MPC309 promotes a unique transcriptome.

LNCaP-abl cells were cultured under androgen deprivation and treated with vehicle (DMSO), 1μM backbone, 1μM MPC309, 10nM DHT, or 10μM enzalutamide overnight, and RNA-seq performed. (A) Venn diagrams show the number of unique and common genes upregulated and downregulated by MPC309 (vs. vehicle and backbone controls) and by DHT or enzalutamide treatments (vs. vehicle control) (1.25-fold, p <0.05). (B) Heatmap of differentially expressed genes in MPC309-treated vs. DHT-treated cells. Pathway analysis was performed using the Molecular Signatures Database (MSigDB). (C) Pathways associated with specific genes downregulated by MPC309 vs. DHT.

To identify the pathways associated with the genes upregulated and downregulated in MPC309-treated cells compared to DHT-treated cells, we utilized several databases, including the Molecular Signatures Database (MSigDB) (42) and EnrichR (43). The top pathway associated with genes upregulated by MPC309 was myogenesis, indicating a potential differentiation phenotype (Fig. 3B). We also observed the upregulation of E2F targets and G2M checkpoint genes, which play crucial roles in cellular division. The top pathways associated with genes downregulated by MPC309 were hypoxia and glycolysis (Fig. 3B), including metabolic enzymes like Enolase 1 (ENO1), Aldolase, Fructose-Bisphosphate C (ALDOC), and Phosphoglycerate Kinase 1 (PGK1), which are known to promote tumorigenesis (Fig. 3C) (44–46). Androgen response was also downregulated compared to DHT, consistent with our qPCR data for AR target genes (Fig. S2B) and indicative of MPC309 acting as a novel AR modulator (Fig. 3B and Fig. 3C).

To determine whether the genes induced and repressed by MPC309 are direct targets of the AR, we performed ATAC-seq to determine changes in chromatin accessibility and ChIP-seq for AR from LNCaP-abl treated with vehicle (DMSO), DHT, MPC309, and enzalutamide. The majority of peaks (>46,000) in the ATAC-seq experiments were common across all treatment groups. (Fig. 4A and Fig.S3B). However, MPC309 did induce some chromatin remodeling with 8,750 unique open chromatin sites observed in cells treated with MPC309 compared to the vehicle-treated cells (Fig. 4A). Enrichment of transcription factor (TF) binding-motifs surrounding regions of open chromatin unique to MPC309-treated cells revealed NR-binding motifs (GRE, PRE, ARE, AR-half sites). This suggests that MPC309-bound AR associates with chromatin at AR-specific sites (Fig. S3C).

Figure 4. MPC309 regulates chromatin accessibility and AR occupancy.

(A) LNCaP-abl cells were cultured under androgen deprivation and treated with vehicle (DMSO) or 10nM DHT, 1μM MPC309, or 10μM enzalutamide overnight, and ATAC-seq was performed to determine the regions of open chromatin. Binding Heatmap for each condition is shown with the number of peaks called listed under. Shared peaks between all treatment groups (46,369) are highlighted in brown. Regions of open chromatin specific to MPC309 treatment are circled. (B) LNCaP-abl cells were cultured under androgen deprivation and treated with vehicle (DMSO) or 10nM DHT, 1μM MPC309, or 10μM enzalutamide overnight. Chip-seq for AR was performed to determine the regions of AR occupancy. ChIP-seq for AR was performed using a mixture of two antibodies to maximize AR enrichment and minimize epitope masking. Peaks were called using MACS26 with IgG control background subtracted. Heatmaps for AR occupancy under the indicated treatments are shown with the number of AR peaks listed under each treatment. AR selective binding upon MPC309 treatment is circled. (C) Example of a gene (FKBP9) regulated by MPC309. ATAC-seq and ChIP-seq tracks and the RNA-seq expression levels in LNCaP-abl cells are shown.

In ChIP-seq experiments, AR occupancy was higher in cells treated with MPC309 and DHT than in controls and enzalutamide (Fig. 4B), confirming that MPC309 promotes differential AR binding to chromatin. Combining ATAC-seq and AR ChIP-seq profiles, we found that 45% of the 33,034 distinct AR binding sites upon MPC309 treatment were at accessible chromatin. Genes associated with AR occupancy showed similar GO terms to the RNA-seq experiments. Of the 251 genes downregulated by MPC309 compared to controls (vehicle and backbone), 246 (98%) had an AR binding site associated within 15 kb upstream or downstream of the promoter. For example, FKBP9, a peptidyl-prolyl isomerase associated with worse outcomes in prostate cancer patients (41), showed decreased expression with MPC309 treatment compared to enzalutamide and DHT, and a distinct AR occupancy pattern at the gene’s promoter (Fig. 4C). Likewise, among the 405 genes upregulated by MPC309, 387 (96%) had an AR binding site within 15 kb of the promoter. This suggests that MPC309-driven gene regulation results from AR binding to chromatin and differentially impacts gene expression compared to DHT or enzalutamide, thus suppressing prostate cancer cell growth.

MPC309 promotes AR nuclear localization and modulates co-activator peptide binding

We utilized a cellular fractionation approach to investigate whether MPC309 treatment affected AR nuclear localization in LNCaP-abl cells (Fig. S3D). AR was present in the absence of androgens in the cytoplasm and nucleus of LNCaP-abl cells. Androgen treatment significantly enriched AR in the nucleus and chromatin-bound fractions. Enzalutamide did not promote AR nuclear localization, whereas MPC309 treatment increased nuclear and chromatin-bound AR levels compared with all other conditions, including DHT (Fig. S3D).

Next, we investigated the effect of MPC309 on the interactions of the AR LBD with the LxxLL-motifs present in co-regulators (e.g., SRC1) and the FxxLL-motif found in the AR N-terminal domain and certain co-activators (e.g., NCOA2) (47). We employed a mammalian two-hybrid assay (23) and observed that MPC309 decreased interaction with the LxxLL-motif of SRC1a compared to DHT (Fig. 5A). Additionally, an in vitro AR interaction assay revealed that MPC309 promoted AR binding to an FxxLF-motif coactivator peptide to the same extent as DHT (Fig. 5B). MPC309 did not affect AR binding to a consensus AR-Response Element (ARE) in vitro, as determined using an Electrophoretic Mobility Shift Assay (EMSA) with recombinant AR (DBD-LBD) (Fig. S3E). These findings indicate that the major impact of MPC309 is to promote AR association with chromatin utilizing a conformation that alters some coactivator binding (LxxLL- containing) but not others (FxxLF-containing). This selective activity contrasts markedly with the mechanism of action of classical antagonists and is reminiscent of a Selective Androgen Response Modulator (SARM) (48).

Figure 5. MPC309 regulates AR interactions with coregulatory proteins on chromatin.

(A) Mammalian two-hybrid assay to detect the interaction between the AR LBD and the LxxLL motif. The Gal4-DBD fused to SRC1a 1241–1441 and VP16AD-LBD WT were co-transfected with the 5x Gal4 RE-Luc reporter (n=3). An unpaired t-test was used to test for statistical significance between the two groups and the difference was considered significant for P values <0.05 (**P <0.01, ***P<0.001). (B) Evaluation of the MPC309 effect on AR coactivator peptide recruitment, shown as a TR-FRET emission ratio. This experiment was performed using the LanthaScreen^® TR-FRET AR Coactivator Assay PolarScreen^™ Kit in agonist mode (n=2). An unpaired t-test was used to test for statistical significance between the two groups. (C) LNCaP-abl cells were cultured under androgen deprivation and treated with vehicle (DMSO) or 10nM DHT or 1μM MPC309 overnight. RIME was performed to identify co-regulators associated with AR on chromatin. Venn diagrams show the number of unique and common proteins associated with AR in Vehicle, DHT, and MPC309-treated cells. (D) Left: Top 25 proteins enriched in MPC309 compared to DHT-treated cells. Right: Top 25 proteins enriched in DHT compared to MPC309-treated cells. Fold-change was calculated by adding +5 to PSM counts, subtracting IgG, and normalizing to AR count in each sample. Common background proteins were removed from the analysis.

MPC309 regulates AR interactions with coregulatory proteins on chromatin

To gain a deeper understanding of how MPC309 modulates AR activity, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins for analysis of chromatin complexes (RIME) (31) on LNCaP-abl cells treated with vehicle, MPC309, or DHT. Data were collected from three replicate experiments, normalized to IgG and AR abundance in each sample, and ranked by average peptide abundance. We found that the three treatment groups had distinct interacting protein profiles (Fig. 5C). Specifically, we identified 41 proteins unique to MPC309 treatment (not identified in the vehicle or DHT treatments) and 88 proteins more abundant in MPC309-treated cells compared to DHT-treated cells (fold-change >1.25). Within these groups (129 proteins unique or enriched in MPC309-treated cells) were members of the SWI/SNF chromatin remodeling complex, such as SMARCD1/BAF60a and SMARCA2/BRM, and members of the DEAD-box RNA helicase family, such as DDX5 and DDX17 (Fig. 5D). Previous studies have shown that SMARCD1/BAF60a recruitment by the AR through its FxxFF motif is critical for selectively activating specific AR-driven promoters (49). In addition to their role in modulating AR activity (50), BRM and SMARCC1 have significant antiproliferative and antitumoral functions in prostate cancer (51–53). DDX5 and DDX17 are coregulator proteins known to participate in normal physiological processes and are closely related to tumorigenesis and tumor progression (54).

We also identified proteins associated with AR upon DHT treatment absent from the MPC309 treatment group, such as HMGB1, CDH4, and HOXB13. HMGB1 is a DNA-binding protein involved in DNA replication and plays a pivotal role in the development of prostate cancer (55). CDH4 is a central subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex and a known AR interactor that plays an important role in prostate cancer proliferation. (56). HOXB13 has been shown to play a crucial role in prostate cancer cell growth, particularly in CRPC cells expressing the AR-V7 variant (57). These findings suggest that MPC309 alters AR coregulator interactions compared to DHT, facilitating a growth-suppressive AR transcriptome.

MPC309 exhibits favorable pharmacological properties and blocks tumor growth

Before conducting in vivo studies on MPC309, we examined its pharmacokinetic properties. MPC309 exhibits high solubility in aqueous solutions, with a kinetic solubility of 637µg/mL in a 50mM phosphate buffer at pH 7.4. After administering a 50mg/kg intraperitoneal (IP) dose, in vivo plasma levels of MPC309 reached a peak concentration (Cmax) of 47,483ng/ml within 30 minutes and remained detectable for a minimum of 24 hours (Fig. S4A). At the 24-hour mark, the MPC309 concentration in serum was 476nM, which is well above the IC₅₀ of 27nM in LNCaP-abl cells (Fig. 1B), indicating that there is enough compound in the serum to enter cells and bind to AR after 24 hours. Importantly, no toxicity was observed with MPC309. Short-term treatment with MPC309 (50mg/kg, IP) did not impact the behavior or general health of three CD-1 male mice at 6 and 24 hours post-dose. Additionally, daily one-week treatment with MPC309 (50mg/kg, IP) did not alter immune cell populations in the peripheral blood of four non-castrated C57BL/6 male mice compared to the vehicle (Fig. S4B). There were no changes in T cell, myeloid, and NK populations and macrophage and granulocyte (neutrophil) numbers, and CD4+/CD8+ ratios remained consistent (Fig. S4B). Furthermore, chronic daily treatment with MPC309 for three weeks (5 to 50mg/kg, IP) did not cause morbidity or weight loss in castrated NSG male mice bearing LNCaP-abl tumors (Fig. 6C). Thus, MPC309 is metabolically stable, exhibits low plasma clearance, and is well-tolerated in vivo.

Figure 6. MPC309 blocks tumor growth in prostate cancer mouse models.

(A) Castrated NSG mice bearing LNCaP-abl xenografts were treated daily by intraperitoneal injection (i.p) with DMSO vehicle or 5mg/kg (207μM), 10mg/kg (414μM) or 50/mg/kg (2.1mM) MPC309 or by oral gavage (p.o) with 30mg/kg (9.7mM) enzalutamide for 19 days. Tumor volumes were measured on the indicated days using calipers (n=4). One-way ANOVA was used to test for statistical significance between the groups and the difference was considered significant for P values <0.05 (*P <0.05). (B) Dissected tumors are shown (left panel), and their sizes were determined (right panel). One of the tumors in the enzalutamide-treated cohort is not shown because the mouse died during the experiment for reasons unrelated to the treatment condition. One-way ANOVA was used to test for statistical significance between the groups and the difference was considered significant for P values <0.05 (*P <0.05). (C) Weight of the mice over the treatment period.

We next explored the therapeutic potential of MPC309 using a xenograft model. We investigated whether MPC309 could inhibit tumor growth in vivo compared to enzalutamide, using LNCaP-abl xenografts in castrated NSG mice. Mice were treated daily via IP injection with either vehicle (DMSO) or MPC309 at 5mg/kg, 10mg/kg, and 50mg/kg or with enzalutamide at 30mg/kg (administered orally). We selected this enzalutamide concentration based on its demonstrated maximum efficacy in LNCaP xenografts (58). Our findings revealed a significant decrease in tumor size with MPC309 treatment in the 10 mg/kg and 50 mg/kg groups compared to enzalutamide (Fig. 6A–B). As previously mentioned, mouse weight remained stable after MPC309 treatment, suggesting that its effects on tumor growth were not due to general toxicity (Fig. 6C). MPC309 treatment did not influence AR expression levels in the tumors (Fig. S4C). Combining MPC309 with enzalutamide did not enhance the anti-tumor growth inhibition of MPC309 (Fig. S4D). Therefore, MPC309 is more potent than enzalutamide in inhibiting tumor growth and is well-tolerated in vivo.

DISCUSSION

We have identified and characterized a powerful trivalent ethisterone peptoid conjugate, MPC309, which binds to AR with nanomolar affinity and effectively suppresses the proliferation of diverse prostate cancer cell lines representing various mechanisms of CRPC. This includes enzalutamide-resistant cells expressing AR splice variants and exhibiting canonical (AR+/PSA+) or non-canonical AR gene expression programs (AR+/PSA-; AR-indifferent). In xenograft studies, MPC309 demonstrated significantly greater tumor suppression compared to enzalutamide. MPC309 modulates AR activity and inhibits cell proliferation by inducing an alternative gene expression program, increasing transcripts involved in differentiation, and decreasing those promoting cell division. Interestingly, MPC309 exhibits partial agonist activity towards the AR and influences the expression of specific AR target genes, although its potency appears lower compared to DHT. This characteristic holds potential clinical benefits: MPC309, by maintaining AR activity within the physiological range, could minimize the adverse effects of androgen deprivation (e.g., sexual dysfunction, cardiovascular events, and alterations in skeletal and body composition) observed with drugs like enzalutamide and abiraterone and potentially with AR-degraders, which completely inhibit the AR axis. Further investigation is necessary to explore this potential in more depth.

MPC309 offers an additional advantage over traditional small molecule AR inhibitors by enhancing selective cancer cell uptake through macropinocytosis. In fact, pharmacological inhibition of macropinocytosis activity in LNCaP-abl cells reduced MPC309’s antiproliferative effect while increasing macropinocytosis in 22Rv1 cells enhanced its antiproliferative effect. The macropinocytosis-mediated uptake of MPCs, combined with AR specificity, suggests an innovative approach to target tumor cells while minimizing systemic toxicity. In line with this notion, we observed no difference in immune cell populations in the blood of wild-type mice treated daily for one week with MPC309 compared to control vehicle-treated mice. Therefore, MPCs possess numerous advantages over conventional AR small-molecule antagonists.

How does the AR function when bound to MPC309? Our findings suggest that MPC309 exerts its activity through the combined effect of multivalent ligands and the peptoid scaffold, thereby influencing AR conformation to selectively alter cofactor interactions and promote an anti-proliferative gene expression program (Fig. S5). This is consistent with the partial agonistic activity of MPC309 on the AR target gene PSA, its full agonistic activity on another AR target gene, FKBP5, and its ability to stimulate an AR reporter gene, although less effectively than DHT. MPC309 fosters unique interactions between the AR and coregulatory factors, resulting in either agonistic or antagonistic effects on the expression of AR-dependent genes. This is supported by our ChIP-Mass spec data, which shows that MPC309 initiates a distinct AR protein interaction pattern on chromatin compared to DHT. To understand the conformational alterations triggered by MPC309, future structural studies of the MPC309-AR complex will be essential (59).

While MPCs show promise as potential therapeutic agents for the treatment of prostate cancer due to their precise multivalent ligand display, stability, preferential uptake by tumor cells, and novel mode of action in targeting the AR pathway, there are limitations. This includes limited oral bioavailability (21), complex structural characteristics that make the scale-up process necessary for drug manufacturing challenging, and the lack of clinical trial data making it difficult to predict the safety, efficacy, and potential side effects of peptoid-based drugs. Moreover, the discrepancy between human and mouse physiology, particularly regarding the presence of the sex hormone-binding globulin (SHBG), a protein that controls the active portion of circulating sex steroids in humans, but is absent in mice, may impact the access of MPCs to target tissues. This could be investigated using transgenic mice that express the human SHBG (60).

We envision using MPC309 in patients resistant to enzalutamide and abiraterone therapy regardless of the underlying resistance mechanisms (AR LBD mutations, AR amplification, and AR variants). The ability of MPC309 to enter cells through macropinocytosis would be highly advantageous for patients. This unique uptake mechanism would enable the drug to selectively target tumor cells- especially those with PTEN loss- while preserving AR signaling in normal cells, thereby minimizing systemic toxicities (61). For tumors with low macropinocytotic activity, the effectiveness of MPC309 might be lower than in tumors with increased macropinocytotic activity, although this needs to be tested in vivo. Our results encourage further exploration of MPCs as a promising new class of compounds for treating CRPC.