Discovery of a highly potent and orally available importin- β 1 inhibitor that overcomes enzalutamide-resistance in advanced prostate cancer

Abstract

Nuclear transporter importin-β1 is emerging as an attractive target by virtue of its prevalence in many cancers. However, the lack of druggable inhibitors restricts its therapeutic proof of concept. In the present work, we optimized a natural importin-β1 inhibitor DD1 to afford an improved analog DD1-Br with better tolerability (>25 folds) and oral bioavailability. DD1-Br inhibited the survival of castration-resistant prostate cancer (CRPC) cells with sub-nanomolar potency and completely prevented tumor growth in resistant CRPC models both in monotherapy (0.5 mg/kg) and in enzalutamide-combination therapy. Mechanistic study revealed that by targeting importin-β1, DD1-Br markedly inhibited the nuclear accumulation of multiple CRPC drivers, particularly AR-V7, a main contributor to enzalutamide resistance, leading to the integral suppression of downstream oncogenic signaling. This study provides a promising lead for CRPC and demonstrates the potential of overcoming drug resistance in advanced CRPC via targeting importin-β1.

Keywords: CRPC; Cancer; Daphnane diterpenoid; Drug discovery; Enzalutamide-resistance; Importin-β1; Natural product; Nuclear transporter.

Graphical abstract

Figure 1

Structural optimization of DD1 afforded an improved analogue DD1-Br with better tolerability and oral bioavailability. (A) Structures of DD1−DD14 and the synthesis of daphnane derivatives. (a) 36% HCl, THF, rt, 0.5 h. (b) PBr₃, CH₂Cl₂, −60 °C. (c) 1% NaOH in MeOH (m/v), rt, 0.5 h. (d) H₂, 10% Pd/C, MeOH, 0.5 h. (e) Acetic anhydride/benzoyl chlorides/2-thiophenecarbonyl, Pyr, rt/50 °C. IC₅₀s for DDs in C4-2B cells treated for 48 h. (B) Survival curve showing the survival of the mice (KM) after the indicated single dose treatments. The estimated LD₅₀ value was indicated. (C) SARs of DDs on anti-proliferative activity and toxicity. (D) ICR mice were treated p.o. or i.p. with 1 mg/kg of DD1 or DD1-Br for pharmacokinetic (PK) analysis. Plasma concentration of DD1 or DD1-Br was measured within 24 h after a single dose treatment. Results are shown as mean ± SD. n = 3 mice at each time point.

Figure 2

DD1-Br significantly inhibits the growth and survival of CRPC cells. (A) IC₅₀s of DD1, DD1-Br, ENZ, and DOX in AR-positive or AR-negative cell lines treated for 48 h were shown in visual heat map. (B) Dose-dependent inhibition of CRPC cell viability in DD1-Br treatment for 48 h. (C) Immunoblotting analysis of DD1-Br on expression levels of the designated proteins in C4-2B and VCaP-CRPC cells. Cells were incubated with different concentrations of DD1-Br for 24 h. (D) 22Rv1 and C4-2B cells were incubated with different concentrations of DD1-Br for 12 days, and then colony formation was assessed. (E) Patient-derived xenograft derived organoids (PDO) were incubated with various concentrations of DD1-Br for 6 days. Typical photographs were recorded by microscope and PDO cell viability was assessed. Scale bar = 200 μm. Data in (D) and (E) are presented as mean ± SD. of three independent experiments; ∗P < 0.05, ∗∗P < 0.01 vs control; Student's t test.

Figure 3

DD1-Br directly targets importin-β1 and its anti-CRPC activity is importin-β1 dependent. (A) Direct interaction between DD1-Br and importin-β1 in vitro was analyzed by SPR assay. (B) Melt curves of importin-β1 protein in DD1-Br (20 nmol/L for 1 h) treated C4-2B cells were measured by CETSA. (C) Endogenous competition assay. 22Rv1 cells were incubated with different concentrations of DD1-Br (0, 5 or 10 μmol/L), and meanwhile treated with PT2 (5 μmol/L) for 6 h. (D) The binding mode of DD1-Br and importin-β1 (ID: 2P8Q) according to docking simulation. (E) DD1-Br blocks importin-β1-mediated nuclear import in living cells. HEK-293T cells stably expressing NFAT-GFP were treated with DD1-Br (40 nmol/L) for 1 h before treatment with ionomycin for 30 min to induce NFAT-GFP nuclear accumulation. Representative images and quantification of the percentage of cells with nuclear NFAT-GFP. Scale bars = 40 μm. (F) Anti-proliferation assay. CRPC cells were transfected with scrambled siCON or KPNB1 siRNA for 2 days, followed by incubation with different concentrations of DD1-Br for 2 days (in C4-2B) or 3 days (in 22Rv1). (G) 22Rv1-EV (empty vector), 22Rv1-KPNB1^wt and 22Rv1-KPNB1^m1 cells were incubated with various concentrations of DD1-Br for 4 day, viable cells were counted. Data in (F) and (G) are represented as mean ± SD. of three independent experiments; ∗P < 0.05, ∗∗P < 0.01, n.s., not significant vs control; Student's t test.

Figure 4

DD1-Br alters the key CRPC oncoproteins nuclear accumulation and shuts down their downstream oncogenic signaling. (A, B) Immunoblot and confocal microscopy images detection of the designated proteins in cytoplasm and nucleoplasm in C4-2B cells. Cells were incubated with DD1-Br (10 nmol/L) for 6 h. Scale bars = 20 μm. (C) The enrichment analysis of down-regulated genes by DD1-Br was displayed by bubble chart. Down-regulated genes were obtained from RNA-seq data of C4-2B cells incubated with DD1-Br (20 nmol/L) for 24 h. (D) GSEA profiles for AR, E2F, and MYC pathway signature gene sets from RNA-seq data. (E) qRT-PCR analysis of AR, E2F, and MYC target genes. C4-2B cells were incubated with various concentrations of DD1-Br for 24 h. Data are presented as mean ± SD. of three independent experiments; ∗∗P < 0.01 vs control; Student's t test. (F and G) Venn diagram (F) showed genes that were regulated by DD1 (20 nmol/L, 24 h) and DD1-Br (20 nmol/L, 24 h) treatment of C4-2B cells from RNA-seq. GO analysis (G) on DD1-Br uniquely regulated genes in C4-2B cells.

Figure 5

DD1-Br is a potent, orally available, and tolerable anti-CRPC agent in vivo. (A) Antitumor activity of the designated treatments (DD1-Br, 0.5 mg/kg or 0.1 mg/kg, intraperitoneally i.p., 5 times a week, n = 6 mice) on the tumor growth of C4-2B xenografts. a (P = 0.0012); b (P = 0.00044). (B) Growth curve of PDX model (TM00298) treated with DD1-Br (0.1 mg/kg intraperitoneally i.p., 5 times a week, n = 7 mice). a (P = 0.013). (C) IHC quantification of AR, E2F1, MYC, and PARK7 nuclear localization in xenograft tumor tissue of mice bearing PDX treated with vehicle or DD1-Br. Data are presented as mean ± SD. ∗∗P < 0.01 vs vehicle; Student's t test. (D) Anti-Ki67 or c-caspase 3 IHC images and quantification of tumor section from PDX were shown. Scale bars = 50 μm. (E) Effects of the indicated treatments (DD1-Br, oral administrations p.o., daily, n = 7 mice) on the growth of 22Rv1 xenografts. a (P = 5.09 × 10⁻⁵); b (P = 1.63 × 10⁻⁸). (F) Tolerance test, expressed as percentage change of mice body weight in previous study (on Days 10 and 22 in 22Rv1 tumor models or on Days 15 and 30 in PDX models) for the indicated DD1 doses. Student's t test. (G) Tolerance test, expressed as percentage change of mice body weight in (B) (on Days 15 and 30) and (E) (on Days 9 and 18) for the indicated DD1-Br doses. Results in (A), (B), and (E) are presented as the mean tumor volume ± SEM.

Figure 6

DD1-Br inhibited AR-V7 signaling and sensitized resistant CRPC to ENZ. (A) Immunoblotting analysis of AR-V7 in nucleoplasm (Nucle) and cytoplasm (Cyto) of 22Rv1, C4-2B/ENZR, and 22Rv1-KPNB1^m1 cells treated with DD1-Br (10 nmol/L for 6 h in C4-2B/ENZR cells; 25 nmol/L for 24 h in 22Rv1 and 22Rv1-KPNB1^m1 cells). (B) GSEA of AR-V7 up-regulated genes set in C4-2B cells. Cells were incubated with DD1-Br (20 nmol/L) for 24 h. (C) The heatmap presentation of alterations in expression of AR-V7 up-regulated genes in C4-2B cells. Cells were incubated with DD1-Br (20 nmol/L), DD1 (20 nmol/L), or IPZ (20 μmol/L) for 24 h. Gene expression was performed by RNA-seq. (D) C4-2B/ENZR and 22Rv1 cells were incubated with designated concentrations of DD1-Br, or in combination with ENZ for 14 days. Colony formation images were taken (left), and colony numbers were counted (right), Data are presented as mean ± SD. n = 3; ∗∗P < 0.01 vs control; Student's t test. (E, F) Effects of the indicated treatments (DD1-Br, i.p., 0.1 mg/kg; ENZ, p.o., 10 mg/kg; DD1-Br and ENZ combined; 5 times a week) on the tumor growth of 22Rv1 xenografts. a (P = 0.213, ENZ vs vehicle); b (P = 9.71 × 10⁻⁵, DD1-Br vs vehicle, P = 0.0062, DD1-Br vs ENZ); c (P = 4.32 × 10⁻⁷, DD1-Br + ENZ vs vehicle, P = 5.74 × 10⁻⁵, DD1-Br + ENZ vs ENZ, P = 0.0014, DD1-Br + ENZ vsDD1-Br) determined by Student's t test at 18 day. Results are presented as the mean tumor volume ± s.e.m. Volumes (left) and weights (right) are shown. (G) Individual tumor growth trajectories in (E). (H) Effects of the indicated treatments (DD1-Br, i.p., 0.5 mg/kg; ENZ, p.o., 10 mg/kg; DD1-Br and ENZ combined; 5 times a week) on the tumor growth of VCaP-CRPC xenografts in castrated mice. a (P = 0.497, ENZ vs vehicle); b (P = 0.037, DD1-Br vs vehicle, P = 0.012, DD1-Br vs ENZ); c (P = 0.013, DD1-Br + ENZ vs vehicle, P = 0.0021, DD1-Br + ENZ vs ENZ, P = 0.021, DD1-Br + ENZ vsDD1-Br) determined by Student's t test at 30 day. (I) Establishment and treatment of VCaP-CRPC xenograft model. (J) Anti-Ki67 IHC images and quantification of tumor cells from (E) and (H). Scale bars = 50 μm.