Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet:

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques:

Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet: (A) Chemical structural illustration of ULK1/2 small molecule inhibitor SBI-0206965 . The compound interacts with the ULK1/2 hinge region, occupying the hydrophobic pocket (highlighted in red) and extending to the solvent-facing region (highlighted in yellow). The pyrimidine scaffold of SBI-0206965 forms hydrogen bonds with the hinge region (blue). (B) General synthetic route for the preparation of ULK 1/2 inhibitors. Method 1 reagents and conditions: (a) ZnCl 2 (1.0 M solution in ether), CH 2 Cl 2 - t BuOH, 0 °C, 1 h; R 2 NH 2 , Et 3 N, 0- rt, 1.5 h (b) R 1 XH, DIPEA, ACN or DMF, µW, 100-130°C, 10-30 min. Method 2 reagents and conditions: (c) R 1 XH, DIPEA, ACN, µW, 100 °C, 10 min. (d) R 2 NH 2 , AcOH, µW, 120 °C, 10 min.

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques: Solvent

Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet: (A) Crystal structure of SBP-5147 bound to the ATP-binding site of ULK1. (B) SBP-5147 key interactions with the ULK1 kinase domain. (C) Schematic of the interaction profiles of SBP-5147 to the ULK1 kinase domain. (D) Crystal structure of SBP-5147 bound to the ATP-binding site of the ULK2 kinase domain. (E) SBP-5147 key interactions with the ULK2 kinase domain. Polar contact counterparts to those observed with ULK1 are retained in the structure of the kinase domain of ULK2. (F) Schematic of the interaction profiles of SBP-5147 to the ULK2 kinase domain.

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet: Western blot analyses (upper panels) and quantification (lower panels) of Beclin1 and Vps34 phosphorylation upon treatment with ULK1/2 inhibitors. HEK293T cells were transfected with Myc-tagged kinase inactive (KI) or wildtype (WT) ULK1 and WT Flag-tagged ( A ) Beclin1 or ( B ) Flag-tagged Vps34 for 24 h. Subsequently, cells were treated with DMSO or the indicated ULK1/2 inhibitors at 10 µM for 1 h. Cell lysates were immunoblotted with the indicated antibodies. The density of each band was quantified for DMSO and compound-treated samples. Data are expressed as the ratio of p-Beclin1/Beclin1 or p-VPS34/VPS34 normalized to GAPDH and DMSO-treated samples. Data represent the mean ± SEM of three independent experiments. *P<0.05 vs SBI-0206965 by unpaired t-test with Welch’s correction.

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques: Western Blot, Phospho-proteomics, Transfection

Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet: ( A ) A549 cells expressing the reporter pBABE-mCherry-EGFP-LC3B were used to evaluate autophagic flux. GFP exhibits increased sensitivity to the acidic environment of the autolysosome compared to mCherry. Under conditions of high autophagic flux, fusion of autophagosomes with the lysosome increases the mCherry/GFP ratio in the cell. ( B ) A549 cells were incubated in growth media (control), starvation media (EBSS), or starvation media plus ULK1/2 inhibitors at 10 μM for 18 h prior to flow cytometry analysis. The relative LC3 distribution was used as a measure of autophagic flux in the cells: low flux (green) cells under growth media compared to high flux (red). ( C ) Representative histogram of A549 cells treated with ULK1/2 inhibitors, showing populations of low (green), intermediate (grey), and high (red) autophagic flux. ( D ) Analysis of three independent experiments, data indicate the mean ± SEM, ****P≤0.0001, ns= P≥0.05, *P≤0.05, **P≤0.01 by two-tailed paired t-test, high autophagic flux population vs control.

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques: Expressing, Incubation, Control, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Synthesis and Characterization of ULK1/2 Kinase Inhibitors that Inhibit Autophagy and Upregulate Expression of Major Histocompatibility Complex I for the Treatment of Non-Small Cell Lung Cancer

doi: 10.1101/2025.09.05.674519

Figure Lengend Snippet: Mice were dosed orally with the indicated compounds (10 mg/kg). Blood samples were collected retro-orbitally and plasma was separated by centrifugation at the indicated time points post-dosing. The compound concentration in the plasma was determined by LC-MS/MS analysis and data were analyzed using PKSolver software. Data represent the geo mean ± geo SD of N=3, log10 scale. The IC 50 values of SBP-5147 and SBP-7501 in the ULK1 NanoBRET target engagement assay are indicated at the dotted lines.

Article Snippet: The reaction for the ULK1 ADP-Glo assay used 2 μg/mL of recombinant human ULK1 protein (1-649, SignalChem #U01-11G) and 80 μg/mL of myelin basic protein (MBP, Sigma-Aldrich #M1891).

Techniques: Clinical Proteomics, Centrifugation, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Software, Drug discovery

Journal: Cell Death & Disease

Article Title: Identification of KW-2449 as a dual inhibitor of ferroptosis and necroptosis reveals that autophagy is a targetable pathway for necroptosis inhibitors to prevent ferroptosis

doi: 10.1038/s41419-024-07157-9

Figure Lengend Snippet: A , B MEF cells were pre-treated with DMSO or KW-2449 for 30 min followed by treatment with RSL3 for the indicated time. Cells were lysed and immunoblotted with indicated antibodies. C HEK293T cells were transfected with empty vector or Flag-tagged human ULK1. After 16 h, cells were treated with KW-2449 at indicated concentrations for 8 h. Cells were then lysed and immunoblotted with indicated antibodies. D Quantification of ADP-Glo kinase assays performed with recombinant human ULK1 in the presence of increasing concentrations of KW-2449. E Overall binding model of ULK1 with KW-2449. KW-2449 is shown in blue. α - helix is shown in orange. β-sheet is shown in yellow. F Closeup view of KW-2449 bound to ULK1. The π−alkyl interactions were indicated by the black line. Key residues that contact KW-2449 are labeled. G Paired WT and ULK1-deficient MEF cells were pre-treated with DMSO or KW-2449 for 30 min. Then, WT MEF cells were treated with RSL3 for 6 h and ULK1-deficient MEF cells were treated with RSL3 for 24 h, respectively. Cell viability was analyzed by CellTiter-Lumi assay. H ULK1-deficient MEF cells were infected with the lentivirus containing the pLVX with Flag-tagged mouse ULK1 to reconstitute the expression of ULK1. The expression of ULK1 was examined by Western blotting with ULK1 antibody. I ULK1-deficient and ULK1-deficient with Flag-ULK1 expressed MEF cells were pre-treated with DMSO or KW-2449 for 30 min followed by treatment with RSL3 for 6 h. Cell viability was analyzed by CellTiter-Lumi assay. Bar graphs represent the mean ± SD from three independent experiments. All Western data are representative of three independent experiments. Statistical analysis was performed using a two-sided student’s t-test. The levels of significance were indicated by ***P < 0.001; ns not significant.

Article Snippet: The mammalian expression plasmids of Flag-tagged human ULK1 (Cat#HG12031-NF) were purchased from SinoBiological (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Recombinant, Binding Assay, Labeling, Infection, Expressing, Western Blot

Journal: Cell chemical biology

Article Title: Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 expression and inhibits autophagic flux

doi: 10.1016/j.chembiol.2023.06.016

Figure Lengend Snippet: All experiments performed with THP-1 cells treated for 72 h. IFN-γ: 50 ng/mL, Hyp: 40 mM. One-way ANOVA was used. Bars represent mean ± SEM. (A) Confocal microscopy analysis of THP-1 cells expressing a mCherry-EGFP-LC3B fusion protein. Baf-A1 (bafilomycin A1, 50 nM) was applied for 4 h. Representative images are shown. Scale bar = 10 μm. (B) Analysis of the overlap of mCherry signal and EGFP signal, from the experiment depicted in A. Each data point indicates an independent field. Mander’s Overlap coefficient (MOC) is plotted. (C) Western blot analysis of LC3B. Baf-A1 (500 nM) was added for the last 2 h of treatment. LC3-I (soluble form) and LC3-II (phosphatidylethanolamine conjugated form) are indicated. LC3-II intensity is quantified, normalized to the β-actin loading control, and relative to the +IFN-γ -Hyp +Baf-A1 sample. (D) Western blot analysis of ULK1 phosphorylated at S757. p-ULK1 (S757) normalized to total ULK1 and β-actin is quantified. Torin-1 (1 μM) was used as an assay control. (E) Western blot analysis of p62 expression in cells treated with IFN-γ and/or Hyp. p62 normalized to β-actin is quantified. (F) Flow cytometry analysis of autophagic flux in either non-targeting control or ULK1 KO THP-1 cells. Autophagic flux calculated as the ratio of mCherry fluorescence to EGFP fluorescence. Normalized to the vehicle treated, non-targeting control. (G) Flow cytometry analysis of PD-L1 expression on the same cells as F. (H) Western blot analysis of ULK1 and PD-L1 in ULK1 KO or non-targeting control cells. PD-L1 densitometry (all bands combined) normalized to the β-actin loading control, relative to the IFN-γ + vehicle, non-targeting control.

Article Snippet: TrueGuide Synthetic sgRNA, human ULK1, Assay ID: CRISPR887528_SGM, Target DNA Sequence: GGAGAACTCGAACTTGC CCA , Thermo Fisher Scientific , Cat#A35533.

Techniques: Confocal Microscopy, Expressing, Western Blot, Control, Flow Cytometry, Fluorescence

Journal: Cell chemical biology

Article Title: Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 expression and inhibits autophagic flux

doi: 10.1016/j.chembiol.2023.06.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TrueGuide Synthetic sgRNA, human ULK1, Assay ID: CRISPR887528_SGM, Target DNA Sequence: GGAGAACTCGAACTTGC CCA , Thermo Fisher Scientific , Cat#A35533.

Techniques: Recombinant, Modification, Synthesized, Transfection, Sequencing, Negative Control, Software, Mass Spectrometry, Targeted Proteomics

Journal: Cell chemical biology

Article Title: Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 expression and inhibits autophagic flux

doi: 10.1016/j.chembiol.2023.06.016

Figure Lengend Snippet: All experiments performed with THP-1 cells treated for 72 h. IFN-γ: 50 ng/mL, Hyp: 40 mM. One-way ANOVA was used. Bars represent mean ± SEM. (A) Confocal microscopy analysis of THP-1 cells expressing a mCherry-EGFP-LC3B fusion protein. Baf-A1 (bafilomycin A1, 50 nM) was applied for 4 h. Representative images are shown. Scale bar = 10 μm. (B) Analysis of the overlap of mCherry signal and EGFP signal, from the experiment depicted in A. Each data point indicates an independent field. Mander’s Overlap coefficient (MOC) is plotted. (C) Western blot analysis of LC3B. Baf-A1 (500 nM) was added for the last 2 h of treatment. LC3-I (soluble form) and LC3-II (phosphatidylethanolamine conjugated form) are indicated. LC3-II intensity is quantified, normalized to the β-actin loading control, and relative to the +IFN-γ -Hyp +Baf-A1 sample. (D) Western blot analysis of ULK1 phosphorylated at S757. p-ULK1 (S757) normalized to total ULK1 and β-actin is quantified. Torin-1 (1 μM) was used as an assay control. (E) Western blot analysis of p62 expression in cells treated with IFN-γ and/or Hyp. p62 normalized to β-actin is quantified. (F) Flow cytometry analysis of autophagic flux in either non-targeting control or ULK1 KO THP-1 cells. Autophagic flux calculated as the ratio of mCherry fluorescence to EGFP fluorescence. Normalized to the vehicle treated, non-targeting control. (G) Flow cytometry analysis of PD-L1 expression on the same cells as F. (H) Western blot analysis of ULK1 and PD-L1 in ULK1 KO or non-targeting control cells. PD-L1 densitometry (all bands combined) normalized to the β-actin loading control, relative to the IFN-γ + vehicle, non-targeting control.

Article Snippet: 150 , 151 TrueGuide Synthetic sgRNA from Thermo Fisher targeting human ULK1 were used: CRISPR887528_SGM, CRISPR887531_SGM, and CRISPR 887533_SGM.

Techniques: Confocal Microscopy, Expressing, Western Blot, Control, Flow Cytometry, Fluorescence

Journal: Cell chemical biology

Article Title: Hydroxyproline metabolism enhances IFN-γ-induced PD-L1 expression and inhibits autophagic flux

doi: 10.1016/j.chembiol.2023.06.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 150 , 151 TrueGuide Synthetic sgRNA from Thermo Fisher targeting human ULK1 were used: CRISPR887528_SGM, CRISPR887531_SGM, and CRISPR 887533_SGM.

Techniques: Recombinant, Modification, Synthesized, Transfection, Sequencing, Negative Control, Software, Mass Spectrometry