The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system

Abstract

Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux is still missing. In this study, we develop a general chemical tool and platform technology termed AUTOphagy-TArgeting Chimera (AUTOTAC), which employs bifunctional molecules composed of target-binding ligands linked to autophagy-targeting ligands. AUTOTACs bind the ZZ domain of the otherwise dormant autophagy receptor p62/Sequestosome-1/SQSTM1, which is activated into oligomeric bodies in complex with targets for their sequestration and degradation. We use AUTOTACs to degrade various oncoproteins and degradation-resistant aggregates in neurodegeneration at nanomolar DC₅₀ values in vitro and in vivo. AUTOTAC provides a platform for selective proteolysis in basic research and drug development.

Fig. 1. Nt-Arg-mimicking p62-ZZ ligands activate p62-dependent selective macroautophagy.

a A model illustrating the mode of action of autophagy-targeting ligands. b Chemical structures of autophagy-targeting ligands. c In vitro pulldown assay in HEK293T cells of the 12-mer V-BiP peptide or autophagy-targeting ligands. d In vitro p62 oligomerization assay in HEK293T cells incubated with the p62-ZZ ligands in (b). e ICC of HeLa cells treated with p62-ZZ ligands in (b) (2.5 μM, 24 h). Scale bar, 10 μm. f, g Quantification of (e) for puncta formation and co-localization, respectively (n = 3 biologically independent experiments each counting 50 cells). h A schematic/formulae for autophagy flux index. i Autophagic flux assay in HeLa cells treated with YOK-1304 or YTK-105 (2.5 μM, 24 h) in the presence or absence of HCQ (10 µM, 24 h). Data are presented as mean values ± SD where relevant. P-values (from a two-sided unpaired t test): ***P < 0.000956 (for p62 punctate formation), ***P < 0.000539 (for LC3 punctate formation), ***P < 0.000925 (for p62-LC3 co-localization). Source data are provided with this paper.

Fig. 2. Targeted autophagic degradation of endogenous oncoproteins by AUTOTAC.

a A model illustrating oncoproteins-targeting AUTOTAC. b Chemical structures of oncoprotein-targeting AUTOTAC. c In vitro p62 oligomerization assay in HEK293T cells incubated with PHTPP-1304, VinclozolinM2-2204, or Fumagillin-105. d Western blot (WB) in HEK293T cells treated with PHTPP-1304 at the indicated concentrations (24 h). e Densitometry of (d) (n = 3). f, g WB in ACHN and MCF7 cells, respectively, treated with PHTPP-1304 at the indicated concentrations (24 h). h WB in MCF7 cells treated with PHTPP-1304, PHTPP or YOK-1304 (1 µM, 24 h). i WB in LNCaP cells treated with VinclozolinM2-2204 at the indicated concentrations. j Densitometry of (i) (n = 3). k WB in LNCaP cells treated with VinclozolinM2-2204, Vinclozolin or YOK-2204 (1 µM, 24 h). l WB in HEK293T cells treated with Fumagillin-105, Fumagillin or YTK-105 at the indicated concentrations (24 h). m Densitometry of (l) (n = 3). n WB in U87-MG cells treated with Fumagillin-105 at the indicated concentrations (24 h). o WB in U87-MG cells treated with Fumagilin-105, Fumagilin or YTK-105 (1 µM, 24 h). When indicated, n = 3 biologically independent experiments. Data are presented as mean values ± SD where relevant. Source data are provided with this paper.

Fig. 3. Targeted degradation using AUTOTAC inactivates oncogenic signaling.

a ICC of LNCaP cells treated with Vinclo.-2204 (2.5 µM, 24 h). Scale bar, 10 μm. b Quantification of (a) (n = 3 biologically independent experiments each counting 50 cells). c ICC of ACHN cells treated with PHTPP-1304 at the indicated concentrations and HCQ (10 μM) (24 h). Scale bar, 10 μm. d Quantification of (c) (n = 3 biologically independent experiments each counting 50 cells). e WB in U-87 MG cells treated with fumagillin-105 (1 μM, 24 h) with or without bafilomycin A1 (200 nM, 6 h). f WB in HEK293T cells treated with PHTPP-1304 (0.1 μM, 24 h) under siRNA-mediated knockdown of p62 and ATG5 (40 nM, 48 h). g WB in HeLa cells treated with fumagillin-105 (0.1 μM, 24 h) following RNA interference of Ubb (40 nM, 48 h). h Identical to (g), but with PHTPP-1304 for ERβ. i WB in LNCaP cells treated with vinclozolinM2-2204 (2.5 μM) or inclozolin (10 μM) with or without DHT (15 nM) (24 h). j Identical to (i) but with PHTPP-1304 (0.5 μM) or PHTPP (5 μM) with or without E2 (10 nM) (24 h). k Wound healing assay in ACHN cells treated with PHTPP-1304, PHTPP, or YOK-1304 (5 μM) at the indicated time points. l Quantification of (k) (n = 2 biologically independent experiments). Scale bar, 100 μm. m, n Cell viability assay of ACHN and LNCaP cells treated with the indicated compounds and concentrations (n = 2 biologically independent experiments). Data are presented as mean values ± SD where relevant. P-values (from a two-sided unpaired t test): ***P < 0.000276, **P < 0.00161. Source data are provided with this paper.

Fig. 4. Targeted autophagic delivery and degradation of misfolded protein cargoes using AUTOTACs with aggregate-binding warhead.

a A model illustrating the mode of action of aggregate-targeting AUTOTAC. b Chemical structures of PBA-1105, PBA-1106, Anle138b-F105 and PBA-1105b. c In vitro p62 oligomerization assay in HEK293T cells incubated with the indicated compounds in (b). d WB in HEK293T cells treated with MG132 (1 μM, 24 h), HCQ (10 μM, 24 h), PBA-1105, YTK-1105, and PBA (1 μM, 24 h). e Identical to (d), but with Anle-F105. f ICC of HeLa cells treated with HCQ (10 μM, 24 h), MG132 (2 μM, 18 h) or both in the presence of PBA-1106, YOK-1106 or PBA (1 μM, 24 h). Scale bar, 10 μm. g Quantification of (f) (n = 3, 50 cells). h ICC of HeLa cells treated with Anle138b-F105 (1 μM, 24 h). Scale bar, 10 μm. i Quantification of (h) (n = 3, 50 cells). When indicated, n = 3 biologically independent experiments each counting 50 cells. Data are presented as mean values ± SD where relevant. P-values (from a two-sided unpaired t test): ***P < 7.66E−05. Source data are provided with this paper.

Fig. 5. Selective degradation of pathological aggregation-prone tau species by aggregate-binding AUTOTAC.

a–c WB in SH-SY5Y-tauP301L cells treated with PBA-1105, PBA, or YTK-1105 at the indicated concentrations. d Densitometry of (a, b, and c) (n = 3 biologically independent experiments). e–g Same as (a–c) but with Anle138b-F105, Anle138b, or YTK-105. (h) Densitometry of (e, f, and g) (n = 3 biologically independent experiments). i WB in SH-SY5Y-tauP301L cells treated with PBA-1105 (0.1 μM) at the indicated time points. j ICC of HeLa cells expressing recombinant TauP301L-GFP and treated with the indicated compounds (1 μM, 24 h) and HCQ (10 μM, 24 h). Scale bar, 10 μm. k Quantification of (j) (n = 3 biologically independent experiments each counting 50 cells). l In vivo oligomerization assay in SH-SY5Y-tauP301L cells treated with okadaic acid (15 nM, 24 h) and the indicated compounds (0.1 μM, 24 h). m Triton X-100-fractionation assay in SH-SY5Y-tauP301L cells treated with a combination of HCQ (10 μM, 24 h), okadaic acid (15 nM, 24 h) or PBA-1105 (0.1 μM, 24 h). Data are presented as mean values ± SD where relevant. P-values (from a two-sided unpaired t test): **P < 0.00821. Source data are provided with this paper.

Fig. 6. Chaperone-based AUTOTAC ameliorates mutant tau pathology in brain-specific murine model.

a Schematic of hTauP301L-BiFC murine model construction. b Injection timeline and details of PBA-1105 in hTauP301L-BiFC murine model. c, d RIPA-insoluble fractionation assay in brain tissues of hTauP301L-BiFC mice intraperitoneally injected with PBA-1105 (20 or 50 mg/kg). e, f Normalized densitometry of (c) and (d) for hTau levels, respectively (n = 5 mice). g, h Same as (e) and (f) but for mTau levels, respectively (n = 5 mice). i, j Immunohistochemistry of BiFC fluorescence for total hTau levels or AT8 fluorescence for total phosphorylated levels in hTauP301L-BiFC mice injected with PBA-1105 as outlined in (b). Scale bar, 100 μm. k, l Quantification of BiFC or AT8 punctate fluorescence signals in i and j, respectively (n = 7 mice). Data are presented as mean values ± SEM where relevant. P-values (from a two-sided unpaired t test): *P < 0.0111 (for insoluble hTau, 20 mg/kg), **P < 0.00105 (for insoluble hTau, 50 mg/kg), *P < 0.0442 (for insoluble mTau, 50 mg/kg). Source data are provided with this paper.

Fig. 7. Speculative model of AUTOTAC and its mechanism-of-action.

Connecting a protein-of-interest target-binding ligand (TBL) to a p62-binding autophagy-targeting ligand (ATL) using an intermediate linker generates the chimeric AUTOTAC degrader. Recognition of the target protein occurs in tandem with the binding and activation of p62 via its ZZ domain, which is self-polymerized in complex with cargoes. Additionally, such interaction initiates a macroautophagy induction cascade in a p62-dependent manner. AUTOTACs show sustained degradative efficacy post-lysosomal degradation of the target protein, suggesting that it is recycled.