PROTACs therapeutically target the polyglutamine androgen receptor in spinal and bulbar muscular atrophy models

Figures

• 

  Fig. 1. ARDs promote time- and dose-dependent clearance of polyQ AR. (a) Chemical structures of ARD-2128, ARD-2585, and ARD-1676. Image created in ChemDraw. (b, d, f) Tet-on AR112Q PC12 ​cells were incubated with 1ug/mL doxycycline for 24 ​h and then treated with varying concentrations of ARD-2128 (b), ARD-2585 (d), ARD-1676 (f), or vehicle (V) for 24 ​h. PolyQ AR expression was analyzed by Western blot. Quantified below. (c, e, h) Tet-on AR112Q PC12 ​cells were incubated with 1ug/mL doxycycline for 24 ​h and then treated with 100 ​nM ARD-2128 (c), ARD-2585 (e), ARD-1676 (g), or vehicle (V) for varying times. PolyQ AR expression was analyzed by Western blot. Quantified below. (g) Lysates from panel (f) were evaluated by ELISA for AR levels. Data are mean ​± ​SEM from n ​= ​3–4 replicates. ∗p ​< ​0.05; ∗∗p ​< ​0.01; ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001 by one-way ANOVA with Dunnett's post-hoc analysis (b, d, f) or two-way ANOVA with Bonferroni post-hoc analysis (c, e, g).

• 

  Fig. 2. ARD-1676 promotes proteasomal degradation of polyQ AR. (a, b) Tet-on AR112Q PC12 ​cells were incubated with 1 ​μg/mL doxycycline for 24 ​h and then treated with 100 ​nM ARD-1676 for indicated times. PolyQ AR expression was analyzed by Western blot. Quantified below. (c) Tet-on AR112Q PC12 ​cells were incubated with 1 ​μg/mL doxycycline for 24 ​h. Doxycycline was removed and cells were treated with varying concentrations of ARD-1676 or vehicle (V) for 24 ​h. PolyQ AR expression was analyzed by Western blot. Quantified below. (d) Tet-on AR112Q PC12 ​cells were incubated with 1 ​μg/mL doxycycline and 10 ​nM R1881 for 72 ​h to induce polyQ AR misfolding. Cells then received fresh media without doxycycline or R1881 for 48 ​h, followed by treatment with 100 ​nM ARD-1676 or vehicle (V) for 24 ​h. Cytoplasmic (C) and nuclear (N) fractions were isolated and analyzed by Western blot for polyQ AR expression. Quantified below. (e) Tet-on AR112Q PC12 ​cells were incubated with 1 ​μg/mL doxycycline for 24 ​h. Following pre-incubation with 10 ​μM ARD Inhibitor (ARI), 100 ​nM epoxomicin, or vehicle (V) for 30 ​min, cells were treated with 100 ​nM ARD-1676 or vehicle (V) for 6 ​h. PolyQ AR…

• 

  Fig. 3. ARD-1676 promotes AR clearance in control and SBMA induced human motor neurons. (a) HT179 (control) and SB3 (SBMA) human iPSCs stably expressing an inducible human NGN2/ISL1/LHX3 cassette were treated with 1 ​μg/mL doxycycline for 4 days and then stained for the expression of Tuj1, AR, Hb9 and Isl1 (red). Nuclei were stained with DAPI (blue). Scale bar ​= ​50 ​μm. (b–e) HT179 and SB3 iMNs were differentiated as in (A). iMNs were treated with vehicle (designated V or -) or 100 ​nM ARD-1676 (+) for 24 ​h (b) AR levels assessed by Western blot. (c) Quantification of (b). (d) AR mRNA levels relative to Actin mRNA by qPCR. (e) Cell viability as assessed by XTT assay. Data are shown as mean ​± ​SEM from n ​= ​3–5 replicates. n.s., not significant, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001 by unpaired Student's t-test.

• 

  Fig. 4. ARD-1676 promotes AR clearance in control and SBMA induced skeletal muscle. HT179 (control) and SB3 (SBMA) iPSC lines were differentiated into iSKM. (a) Schematic of differentiation protocol. (b–d)NANOG (b), MYOD (c), and DESMIN (d) mRNAs relative to CPSF2 mRNA in iPSCs and iSKM by qPCR. (e, f) HT179 (Control) and SB3 (SBMA) iSKMs stained for desmin (e) or AR (f). In (f), cells were treated with 10 ​nM R1881 or vehicle (−) for 48 ​h. Nuclei stained by DAPI. Scale bar ​= ​50 ​μm. (g–k) iSKMs were treated with either vehicle (V) or 100 ​nM ARD-1676 (1676) for 24 ​h. (g–i) HT179 (control) iSKM, (J–L) SB3 (SBMA) iSKM. (g, j) AR levels assessed by Western blot. (h, k) Quantification of (G, J). (i, l) AR mRNA levels relative to CPSF2 mRNA by qPCR. Data are shown as mean ​± ​SEM from n ​= ​3 replicates. ∗p ​< ​0.05; ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001 by unpaired Student's t-test.

• 

  Fig. 5. Target engagement and therapeutic efficiency. (a–c) AR113Q male mice at 8–10 weeks were administered 30 ​mg/kg ARD-1676 or vehicle (V) by oral gavage and analyzed after 6 ​h n ​= ​6 mice/group. (a) AR levels in levator ani/bulbocavernosus muscle assessed by Western blot. (b) Quantification of (a). (c) Ar mRNA levels in tibialis anterior muscle relative to Cpsf2 mRNA by qPCR. (d–f) HT179 (control) and SB3 (SBMA) iSKM were treated with or without R1881 for 24 ​h and then treated with ARD-1676 in the presence of R1881 for 24 ​h, as indicated. (d) AR levels assessed by Western blot. (e, f) Quantification of (d), for HT179 (e) and SB3 (f). n ​= ​3 replicates/group. (g–i) Control (HT179, HT180) and SBMA (SB3, SB17) derived iSKM were treated as in panel D, in triplicate for each cell line and condition. Bulk RNA-seq was performed to identify DE genes (adjusted p value ​< ​0.05 and |log2(fold-change)| ​> ​1). (g) Volcano plot of DE genes in SBMA vs control iSKMs. (h) Gene ontology (GO) enrichment analysis of DE genes for molecular function in SBMA vs control iSKMs. (i) Heat map showing log2 (fold-change) of DE genes (SBMA vs control) that were significantly rescued (adjusted p value ​<…