De Novo Design of an Androgen Receptor DNA Binding Domain-Targeted peptide PROTAC for Prostate Cancer Therapy

Abstract

Androgen receptor splice variant-7 (AR-V7), one of the major driving factors, is the most attractive drug target in castration-resistant prostate cancer (CRPC). Currently, no available drugs efficiently target AR-V7 in clinical practice. The DNA binding domain (DBD) is indispensable for the transcriptional activity of AR full length and AR splice variants, including AR-V7. Based on the homodimerization structure of the AR DBD, a novel peptide-based proteolysis-targeting chimera (PROTAC) drug is designed to induce AR and AR-V7 degradation in a DBD and MDM2-dependent manner, without showing any activity on other hormone receptors. To overcome the short half-life and poor cell penetrability of peptide PROTAC drugs, an ultrasmall gold (Au)-peptide complex platform to deliver the AR DBD PROTAC in vivo is developed. The obtained Au-AR pep-PROTAC effectively degrades AR and AR-V7 in prostate cancer cell lines, particularly in CWR22Rv1 cells with DC₅₀ values 48.8 and 79.2 nM, respectively. Au-AR pep-PROTAC results in suppression of AR levels and induces tumor regression in both enzalutamide sensitive and resistant prostate cancer animal models. Further optimization of the Au-AR pep-PROTAC can ultimately lead to a new therapy for AR-V7-positive CRPC.

Keywords: androgen receptor splice variant-7 (AR-V7); peptide drug; prostate cancer; proteolysis-targeting chimera (PROTAC).

Figure 1

Schematic diagram and in silico design of an AR DBD PROTAC drug. A) Schematic diagram of an AR DBD‐specific PROTAC drug hijacking the E3 ligase MDM2 to degrade full‐length AR as well as AR‐V7. B) The binding structure of the designed peptide ligand (red) and the homodimeric structure of the AR DBD (gray, Protein Data Bank ID: 1r4i). C) Sequence logos of predicted sequence mutations for AR DBD‐targeting peptides. The peptide sequences are arranged in a single letter pattern. N, amino terminus; C, carboxyl terminus. The score for each amino acid was determined using the all‐atom Rosetta scoring function; a larger score means a higher affinity for the AR DBD. D) The structure of the protein complex between a 12‐aa peptide ligand (rainbow) and the MDM2 NTD (purple, Protein Data Bank ID: 3LNZ). E) Sequence logos for predicted sequence mutations for the MDM2‐targeting peptide. The peptide sequences are arranged in a single letter pattern. N, amino terminus; C, carboxyl terminus. The score for each amino acid was determined using the all‐atom Rosetta scoring function; a larger score means a higher affinity for MDM2. F) The binding affinity between the AR pep‐PROTAC drug and AR‐V7 was measured by ITC. The dissociation constant for binding between the AR pep‐PROTAC drug and AR‐V7 was 49.5 nM. G) The binding affinity between the AR pep‐PROTAC drug and MDM2 was measured by ITC. The dissociation constant for binding between the AR pep‐PROTAC drug and MDM2 was 12.2 nM.

Figure 2

Design and Characterization of the Au‐AR pep‐PROTAC Drug. A) Schematic depiction of the synthesis process of the positive surface Au‐AR pep‐PROTAC drug. HAuCl₄, chloroauric acid. We developed a stable peptide‐loaded Au nanoparticle system loaded with the AR pep‐PROTAC drug after a 20 min reaction by adding the AR pep‐PROTAC drug‐containing cysteine and branched PEI (MW 25 000 Da) to HAuCl₄. B) TEM analysis showed that the diameters of the Au nanoparticles and Au‐AR pep‐PROTAC drug were approximately 5.00 nm. C) Hydrodynamic diameter distributions of Au nanoparticles and Au‐AR pep‐PROTAC. The hydrodynamic diameter of the Au nanoparticles was 5.72 nm, and the hydrodynamic diameter of Au‐AR pep‐PROTAC was 6.26 nm. D) The surface zeta potential of the Au nanoparticles and Au‐AR pep‐PROTAC drug was measured in 20 mM Tris‐HCl at pH 7.4. The surface charge of the Au nanoparticles was +17.9 mV, and the surface charge of the Au‐AR pep‐PROTAC was + 21.4 mV. E) The serum resistance of the free AR pep‐PROTAC drug and Au‐AR DBD PROTAC drug was tested in PBS containing 10% serum. The half‐life of the free AR pep‐PROTAC drug was only 1.9 h, while the half‐life of the Au‐AR pep‐PROTAC was 25.1 h. The AR pep‐PROTAC drugs were quantified by HPLC. The “Residual Drug” means the AR pep‐PROTAC still binding to Au nanoparticles without degradation. F) Release of the AR pep‐PROTAC drug from Au‐AR pep‐PROTAC nanoparticles in 10 mM glutathione solution (pH 7.4) to mimic the intracellular redox environment. The AR pep‐PROTAC drug was quantified by HPLC. G) Confocal micrographs of C4‐2 cells incubated with 12.5 nM Au‐AR pep‐PROTAC drug labeled with rhodamine (red). Nuclei are stained with Hoechst 33 342 (blue). All images were acquired with the same excitation wavelength and detector gain settings (scale bar: 50 µm).

Figure 3

The Au‐AR pep‐PROTAC drug effectively induces the degradation of AR and AR‐V7 in vitro. A) Cell viability assay of C4‐2 cells after 48 h of treatment with varying concentrations of the Au‐ARpep‐PROTAC, Au nanoparticles, and Au‐Peptide Control. The Au nanoparticles and Au‐Peptide Control showed no toxicity in C4‐2 cells, while the Au‐AR pep‐PROTAC showed dose‐dependent growth inhibition in C4‐2 cells, with an IC₅₀ = 230.8 nM. B) Cell viability assay of LNCaP cells after 48 h of treatment with varying concentrations of the Au‐AR pep‐PROTAC, Au nanoparticles, and Au‐Peptide Control. The Au nanoparticles and Au‐Peptide Control showed no toxicity in LNCaP cells, while the Au‐AR DBD PROTAC showed dose‐dependent growth inhibition in LNCaP cells, with an IC₅₀ = 248.1 nM. C) Cell viability assay of CWR22Rv1 cells after 48 h of treatment with varying concentrations of the Au‐AR pep‐PROTAC, Au nanoparticles, and Au‐Peptide Control. The Au nanoparticles and Au‐Peptide Control showed no toxicity in CWR22Rv1 cells, while the Au‐AR DBD PROTAC showed dose‐dependent growth inhibition in CWR22Rv1 cells, with an IC₅₀ = 126.9 nM. D‐F) IB analysis of AR in C4‐2 (D), LNCaP (E), and CWR22Rv1 (F) cells after 24 h of treatment with the Au‐AR pep‐PROTAC drug. G) Immunofluorescence staining of AR (red) in C4‐2 cells after 24 h of Au‐AR pep‐PROTAC drug treatment. DAPI (blue) was used for nuclear staining. Scale bar: 50 µm. H, I) IB analysis of WCLs from C4‐2 cells (H) and LNCaP cells (I) treated with CHX for the indicated times, with or without 250 nM Au‐AR DBD PROTAC drug treatment. The signal intensity of AR normalized to that of Vinculin was quantified. J) IB analysis of AR and AR‐V7 in CWR22Rv1 cells treated with CHX for the indicated times, with or without 250 nM Au‐AR pep‐PROTAC drug treatment. The signal intensities of AR and AR‐V7 normalized to that of Vinculin were quantified separately.

Figure 4

The Au‐AR pep‐PROTAC Drug Degrades AR & AR‐V7 in an AR DBD‐ and MDM2‐Dependent Manner. A) IB analysis of WCLs and anti‐Flag immunoprecipitate (IP) from 293T cells transfected with the indicated plasmids, with or without Au‐AR pep‐PROTAC drug treatment. B) IB analysis of WCLs and Ni–NTA pulldown products derived from 293T cells transfected with the indicated plasmids, with or without Au‐AR pep‐PROTAC drug treatment. Where indicated, 20 µM MG132 was added for 6 h before harvesting the cells. C) IB analysis of WCLs and anti‐Flag immunoprecipitates from 293T cells transfected with the indicated plasmids, with or without Au‐AR pep‐PROTAC drug treatment. D) IB analysis of WCLs and Ni–NTA pulldown products from 293T cells transfected with the indicated plasmids, with or without Au‐AR pep‐PROTAC drug treatment. Where indicated, 20 µM MG132 was added for 6 h before harvesting the cells. E) IB analysis of WCLs from 293T cells transfected with the indicated plasmids and treated with CHX for the indicated times. The signal intensity of AR‐V7 or AR ΔDBD was normalized to that of Vinculin. F) IB analysis of WCLs from C4‐2 cells transfected with siRNAs targeting MDM2 or negative control (NC) siRNAs in the presence of the Au‐AR pep‐PROTAC drug. G) Cell viability assay of AR‐negative DU145 and PC‐3 cells after 48 h of treatment with varying concentrations of the Au‐AR pep‐PROTAC. The data are presented as the mean ± SD values (n = 3).

Figure 5

The Au‐AR pep‐PROTAC Drug Inhibits Prostate Tumor Growth In Vivo. A) Tumor growth curves of C4‐2 xenografts in nude mice treated as indicated (n = 5 per group). Statistical analysis was performed using the nonparametric Kruskal‐Wallis test; ****, p < 0.0001. The data are presented as the mean ± SD values (n = 5). B) Photos of C4‐2 tumors excised at the end of the experiment after different drug treatments. C) Average weight of tumors excised from each group of mice at the end of drug treatment. The data are presented as the mean ± SD values (n = 5). Statistical analysis was performed using the nonparametric Kruskal‐Wallis test; ***, p < 0.001; ns, not statistically significant. The data are presented as the mean ± SD values. D) Tumor growth curves of CWR22Rv1 xenografts in nude mice (n = 5 per group). Statistical analysis was performed using the nonparametric Kruskal‐Wallis test; ****, p < 0.0001. The data are presented as the mean ± SD values. E) Photos of CWR22Rv1 tumors excised at the end of the experiment after different drug treatments. F) Average weight of tumors excised from each group of mice at the end of drug treatment (n = 5). Statistical analysis was performed using the nonparametric Kruskal‐Wallis test; ***, p < 0.001; ns, not statistically significant. G) Histopathological analysis of the excised tumors using H&E staining and a TUNEL assay (scale bar: 10 µm). H) IHC analysis of AR in C4‐2 xenografts from each treatment group. IHC scores were determined with ImageJ: 4, highly positive; 3, positive; 2, minimally positive; 1, negative. I) IHC analysis of AR and AR‐V7 CWR22Rv1 xenografts. IHC scores were determined with ImageJ: 4, highly positive; 3, positive; 2, minimally positive; 1, negative. Statistical analysis was performed using the t‐test; ****, p < 0.0001.

Figure 6

The PK (pharmacokinetics) and biosafety analysis of Au‐AR pep‐PROTAC. A) Comparison of biodistribution between free AR pep‐PROTAC and Au‐AR pep‐PROTAC in CWR22RV1 xenograft models. B) Biodistribution of Au‐AR pep‐PROTAC at different time points in CWR22RV1 xenograft models. C) Pharmacokinetics analysis of Au‐AR pep‐PROTAC by ICP‐MS. D) The hERG tests of Au and Au‐AR pep‐PROTAC on cardiac muscle cells. E) Biosafety evaluation of Au‐AR pep‐PROTAC after 21‐ day treatment of Au‐AR pep‐PROTAC.