Fig 2: Targeted protein degradation induced by PROTAC derived from LXH-3-71. (A, C) Chemical structures of compounds 37 and 45, respectively. (B, D) Cells were incubated with either compound 37 or compound 45 at specified concentrations for 24 h, followed by an assessment of targeted protein levels via Western blot analysis. (E) Chemical structure of compound 47. (F) Western blot analysis of EGFR protein level in MG-132 (10 μmol/L), compound 47 (10 μmol/L), Gefitinib (10 μmol/L), MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with compound 37 for 24 h. (G) Western blot analysis of CDK4 protein level in MG-132 (10 μmol/L), compound 47 (10 μmol/L), Ribociclib (10 μmol/L), MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with compound 45 for 24 h.

Fig 3: LXH-3-71 functions as a molecular glue facilitating PHGDH-DDB1 Interaction. (A) Chemical structure of compound 18. (B) Silver staining of immunoprecipitated proteins separated by SDS-PAGE. (C) Data pertaining to the DDB1-CUL4 ubiquitin ligase, derived from LC‒MS/MS analysis. (D, E) Co-IP assays were executed utilizing anti-PHGDH and anti-DDB1 antibodies, respectively, followed by Western blot analysis. (F) Western blot analysis of DDB1 protein level in HCT-116 cells transfected with siCON or siDDB1 oligos. (G) Western blot analysis of PHGDH protein level in MLN4924 (1 μmol/L) or DDB1 knockdown pre-treated HCT-116 cells followed by treatment with LXH-3-71 for 24 h. (H) A protein affinity pull-down assay, utilizing probe 18, was conducted with HCT-116 cell lysate and subsequently analyzed through a Western blotting assay. (I) Interaction studies between DDB1 and PHGDH (left) or PHGDH preincubated with LXH-3-71 (right) using microscale thermophoresis method. Data are represented as mean ± SD, n = 3.