Titration of GFP-eIF2α BacMam.

<p>(A) U-2 OS cells were mixed with different amount of GFP-eIF2α BacMam and plated to a 384 well culture plate for overnight. Cells were then treated with either DMSO or 2 μM Tg for 1 h at 37°C 5% CO₂. LanthaScreen was performed to detect eIF2α phosphorylation. Data was the average of 4 repeats. (B) U-2 OS cells were transduced with GFP-eIF2α BacMam in a culture flask with the optimized MOT for overnight. After trypsinized and washed, cells were resuspended to different cell density and plated into a 384 well plate. After the treatment with Tg for 2.5 h at 37°C and 5% CO₂, LanthaScreen assay was performed to detect the phosphorylation of GFP-eIF2α. Data was the average of 3 repeats.</p

LanthaScreen assay signal was PERK dependent.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam under the optimized conditions for overnight. Cells were then trypsinized and plated into a 384 well plate. Different doses of a specific PERK inhibitor GSK2606414 (A) or an inactive analog (B) was incubated with cells for 30 min at 37°C and 5% CO₂. DMSO, 1 μM Tg or 5 μM Tn was then added and incubated for 2 h at 37°C 5% CO₂. Phosphorylation of GFP-eIF2α was analyzed with LanthaScreen assay. Data was the average of 2 repeats.</p

Compounds increase NQO-1 protein expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM compound for 1 h or 3 μM compound for 24 h at 37°C and 5% CO₂. NQO-1 protein level was determined by ELISA. Stars indicate p value ≤0.001 (n = 3). (B) NHBE cells were transfected 25nM siRNA for 48 h, treated with DMSO, 0.5μTg, 3 μM compound B or C for 24 h. NQO-1 mRNA level was then determined by Real-Time PCR and was normalized with DMSO control (n = 3). (C) NHBEs were transfected 25 nM siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B or C for 24 h. NQO-1 protein level was determined by Western blot. Data was representative of 3 experiments.</p

Compounds have insignificant activity in IRE1 branch.

<p>HT1080 cells pretransduced with XBP-1 splicing reporter gene were treated with compounds at different concentrations for 3 h at 37°C and 5% CO₂. Luciferase activity was measured with Steady Glo reagents after the treatment. Data were average of 8 repeats.</p

Compounds increase Heme Oxygenase-1 (HO-1) expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM of compound for 1 h or 3 μM of compound for 24 h at 37°C and 5% CO₂, respectively. Tg (0.5 μM) and hemin (3 μM) served as controls. HO-1 protein was then determined. (B) NHBE cells were transfected with 25 nM non-target siRNA, PERK siRNA or NRF2 siRNA for 48 h, then treated with compounds at 3 μM for 24 h. HO-1 mRNA level was then analyzed. (C) NHBE cells transfected with non-targeting, NRF2 or PERK siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B, C or hemin for 24 h. HO-1 protein level was determined. Data was representative of 3 experiments.</p

Robustness features of the LanthaScreen assay.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam for overnight in flasks. Cells were then trypsinized, washed and resuspended for plating into 384 well plates for compound treatment. The low control was DMSO only, the high control was 1 μM Tg. All the conditions were the optimized final conditions as described in results.</p><p>Robustness features of the LanthaScreen assay.</p

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2‑Related Factor 2 (KEAP1:NRF2) Protein–Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch–NRF2 interaction. X-ray crystallographic screening identified three distinct “hot-spots” for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and <i>in vivo</i> models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1–NRF2 interaction