Design and characterization of a heterobifunctional degrader of KEAP1

Abstract

The Kelch-like ECH-associated protein 1 (KEAP1) - nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway senses reactive oxygen species and regulates cellular oxidative stress. Inhibiting KEAP1 to activate the NRF2 antioxidant response has been proposed as a promising strategy to treat chronic diseases caused by oxidative stress. Here, we developed a proteolysis targeting chimera (PROTAC) that depletes KEAP1 from cells through the ubiquitin-proteasome pathway. A previously developed KEAP1 inhibitor and thalidomide were incorporated in the heterobifunctional design of the PROTAC as ligands for KEAP1 and CRBN recruitment, respectively. Optimization of the chemical composition and linker length resulted in PROTAC 14 which exhibited potent KEAP1 degradation with low nanomolar DC₅₀ in HEK293T (11 nM) and BEAS-2B (<1 nM) cell lines. Furthermore, PROTAC 14 increased the expression of NRF2 regulated antioxidant proteins and prevented cell death induced by reactive oxygen species. Together, these results established a blueprint for further development of KEAP1-targeted heterobifunctional degraders and will facilitate the study of the biological consequences of KEAP1 removal from cells. This approach represents an alternative therapeutic strategy to existing treatments for diseases caused by oxidative stress.

Keywords: Antioxidant; KEAP1-NRF2 pathway; Oxidative stress; PROTAC; ROS.

Graphical abstract

Fig. 1

Design of a CRBN-based KEAP1 degrader. (A) The structure and activity of the KEAP1 inhibitor 1 reported by Davies et al. [20]. (B) Activity of the synthesized racemic KEAP1 inhibitor 2 incorporated in the KEAP1 degrader. (C) KEAP1 inhibitor 1 co-crystalized with the KEAP1-Kelch domain [20]. The red circle highlights the linker attachment site used in the design of a PROTAC. (D) Design and structure of CRBN based KEAP1 degraders used in this study. IC₅₀ (SD) values shown represent 3 independent experiments using recombinant KEAP1-Kelch domain in a HTRF biochemical assay. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Scheme 1

General synthetic pathway to generate PROTACs. Reaction conditions: (a) K₂CO₃, BnBr, DMF, 0–20 °C; (b) AcOH, NaOAc, Br₂, 20 °C; (c) NaH, MeI, DMF, 0–20 °C; (d) Fe, AcOH, 65 °C; (e) NaNO₂, 10% aq. H₂SO₄; (f) t-butyl acrylate, DIPEA, P(o-tolyl)₃, Pd(OAc)₂, DMF, 95 °C; (g) [RhCl(cod)]₂, (3-(hydroxymethyl)-4- methylphenyl)boronic acid, Et₃N, dioxane, H₂O, 95 °C; (h) PPh₃, DIAD, THF, 0–20 °C; (i) Pd/C, H₂, MeOH, 20 °C; (j) K₂CO₃, alkyl halide, DMF, 80 °C; (k) Pd/C, H₂, MeOH; (l) 40, HATU, DIPEA, DCM, 20 °C; (m) TFA, DCM, 20 °C; (n) 41, DIPEA, DMF 80 °C.

Fig. 2

Evaluation of PROTACs with different linkers on the degradation of KEAP1 in HEK293T cells. HEK293T cells were treated with DMSO vehicle control or 1 μM of compounds (A) at the indicated concentrations (B) for 5 and 24 h. Western blots are representative of 3 independent experiments. Repeats are shown in Figs. S1 and S2.

Fig. 3

A comparison of the KEAP1 degradation efficiency of compounds 8 and 14. (A) Structures of compounds 8 and 14. (B) HEK293T cells were treated with DMSO vehicle control or 8 and 14 at the indicated concentrations for 5, 24, 48, and 72 h. Immunoblots are representative of 3 independent experiments. Repeats are shown in Fig. S3.

Fig. 4

Characterization of the targeted KEAP1 degradation capacity of compound 14. (A) HEK293T cells were pre-treated with DMSO, 10 μM of thalidomide (TOM), 10 μM of proteasome inhibitor MG132, or 1 μM of inhibitor 2 for 1 h. Cells were then treated and incubated with 14 for 5 h. (B) HEK293T cells were treated with DMSO vehicle control or compound 14 at the indicated concentrations for 24 h. Immunoblots are representative of 3 independent repeats. Repeats are shown in Figs. S4 and S5.

Fig. 5

Impact of PROTAC 14 on activation of the KEAP1/NRF2 signaling pathway. (A) HEK293T, HCA7, BEAS-2B, and A549 cells were treated with compounds at the indicated concentrations for 48 h. Immunoblots are representative of 3 independent experiments. Repeats are shown in Fig. S7. (B) HepG2 ARE-luciferase reporter cells were treated with compounds at the indicated concentrations for 48 h and luminescence was measured. Fold change in induction is relative to the DMSO control group. (C) BEAS-2B cells were treated with 0.13 μM of compounds for 48 h. The expression levels of the antioxidant signature genes were analyzed by RT-qPCR. For B and C, data shown are from three independent experiments, each of which was performed with technical triplicates. Data shown represent mean and S.D. Unpaired t-test was performed. (*) p < 0.05; (****) p 0.0001.

Fig. 6

KEAP1 degradation or inhibition induces anti-oxidant protein expression. Volcano plots showing differentially expressed proteins following treatment with (A) PROTAC 14, and (B) inhibitor 2. BEAS-2B cells were treated with DMSO, PROTAC 14, or inhibitor 2 for 48 h, lysed and processed for proteomic analysis. Antioxidant proteins that were significantly changed following PROTAC 14 or inhibitor 2 treatment relative to the DMSO control, are highlighted as black dots (>1.5-fold abundance change and adjusted P value less than 0.01). Proteins that showed a significant change but were not directly related to antioxidant response are labeled with yellow dots. For B, antioxidant proteins that showed more than 1.5-fold change, but were not significant, are labeled as enlarged grey dots. KEAP1, ZFP91, and ABHD4 are labeled with green, orange, and purple colors, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

KEAP1 degradation or inhibition rescues BEAS-2B cells from ROS induced cell death. BEAS-2B cells were pre-treated with the indicated compounds at various concentrations for 24 h and then exposed to 50 μM tBHP for an additional 24 h. Cells were harvested and the % of viable cells was analyzed by PI staining and flow cytometry. Data shown are from three independent assays, with each assay shown as a different symbol. Each assay was performed with single data points. Error bars represent average and S.D. Unpaired t-test was performed. (*) p < 0.05, (***) p < 0.001, (****) p < 0.0001.