Journal: ACS Chemical Biology

Article Title: Discovery and Development of a Potent LIMK2 Isoform-Specific Degrader

doi: 10.1021/acschembio.6c00137

Figure Lengend Snippet: Screening and initial assessment of the degradation potency of PROTACs in HuCCT1 Cells. (A) LIMK1/2 Western blot analysis following 6 h PROTAC treatment across an extended concentration range. Degradation of both LIMK isoforms was assessed, and levels of the LIMK substrate cofilin were also monitored as phosphorylated cofilin (pCFL) and total cofilin (CFL). GAPDH served as a loading control. (B) Quantitative global proteomics of 21b following 6 h treatment. A volcano plot of differential protein abundance versus the DMSO control revealed isoform-specific degradation of LIMK2. LIMKs are highlighted in purple, while other significantly downregulated proteins are highlighted in yellow. (C,D) Western blot analysis of PROTACs screened after 6 h at 100 nM and 1 μM. In this series, compound 22b (THNAN69) induced the most potent degradation of LIMK2, while LIMK1 protein levels remained unaffected. (E) Heat map of EGFP–LIMK2. Fluorescence data were normalized to DMSO/mCherry controls and visualized to show LIMK2 degradation across all PROTACs. PROTACs were screened at five concentrations, and LIMK2 degradation was monitored in live cells over 72 h. (F) Degradation kinetics of the thalidomide-based PROTAC 21b and the PD-based PROTAC 22b (THNAN69). EGFP–LIMK2 fluorescence was monitored over 72 h as triplicates, and mean degradation curves were fitted using a one-phase exponential decay model.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), while HUCCT1 cells (ATCC, CRL-1997) were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) antibiotic mix (all from Sigma-Aldrich, St. Louis, Missouri, USA).

Techniques: Western Blot, Concentration Assay, Control, Quantitative Proteomics, Fluorescence

Journal: ACS Chemical Biology

Article Title: Discovery and Development of a Potent LIMK2 Isoform-Specific Degrader

doi: 10.1021/acschembio.6c00137

Figure Lengend Snippet: Cellular Characterization of 21b and THNAN69.(A) Western blot analysis of HuCCT1 cells following 6 h PROTAC treatment. LIMK isoforms, CFL, and pCFL levels were monitored, with GAPDH as a loading control. (B) Quantification of LIMK2 degradation potency. Mean maximum degradation ( D max ) and DC 50 values were determined from the EGFP–LIMK2 kinetic assay data, as described by Riching et al. Data were fitted using a variable slope dose–response model. (C) Rescue of LIMK2 degradation by neddylation inhibition. CRBN-mediated cullin-RING ligase activity was assessed using the cellular EGFP–LIMK2 assay after cotreatment with three concentrations of 21b or THNAN69 and MLN4924 (1 μM). Fluorescence traces monitored remained stable over 72 h. (D) Validation of THNAN69-mediated endogenous LIMK2 degradation by Western blot (6 h). HuCCT1 cells were treated with THNAN69 (100 nM) ± MLN4924 (1 μM). THNAN69-NC (1 μM) was also included. LIMK2, pCFL, and GAPDH levels were monitored. (E) Summary of mean D max values across varying PROTAC concentrations measured in the EGFP–LIMK2 cell-based assay.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), while HUCCT1 cells (ATCC, CRL-1997) were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) antibiotic mix (all from Sigma-Aldrich, St. Louis, Missouri, USA).

Techniques: Western Blot, Control, Kinetic Assay, Inhibition, Activity Assay, Fluorescence, Biomarker Discovery, Cell Based Assay

Journal: ACS Chemical Biology

Article Title: Discovery and Development of a Potent LIMK2 Isoform-Specific Degrader

doi: 10.1021/acschembio.6c00137

Figure Lengend Snippet: Selectivity of THNAN69 and Effects of LIMK2 Depletion on Phosphorylation Signaling. (A,B) Quantitative global proteomics of THNAN69 and LIMKi3 treatments in HuCCT1 cells (6 h). (A) Volcano plot of differential protein levels after THNAN69 treatment, revealed isoform-specific degradation of LIMK2. LIMKs are highlighted in purple, and other significantly downregulated proteins are shown in yellow. (B) Volcano plot of a proteomics experiment following LIMKi3 treatment under the same conditions. (C) Phospho-proteomics analysis of THNAN69 in HuCCT1 cells (6 h). Volcano plot revealed that LIMK phospho-Ser 3 (S3) substrates CFL1/2 and DSTN were not significantly downregulated (purple labels). (D) PROTAC treatment upregulated small G-protein-activated signaling pathways. Left panel: Global gene ontology analysis revealed upregulation of several Rho signaling pathways associated with PAK-ROCK-LIMK signaling. Right panel: KSEA Z-score analysis indicated increased phosphorylation and activation of kinases, including LIMK2-activating kinases ROCK1/2 and PAK1/2 (cyan). Kinases with decreased Z-scores are highlighted in red.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), while HUCCT1 cells (ATCC, CRL-1997) were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) antibiotic mix (all from Sigma-Aldrich, St. Louis, Missouri, USA).

Techniques: Phospho-proteomics, Protein-Protein interactions, Activation Assay