Zhengfang Yi

Colorectal cancer (CRC) remains a major cause of cancer-related mortality worldwide, and selective molecular targets and corresponding inhibitors remain insufficiently developed. Starting from a phenotypic screening hit, we performed systematic structural optimization guided by metabolism-guided modification, leading to the discovery of B9. B9 selectively inhibited HCT116 CRC cells and showed much weaker inhibition of normal colonic epithelial NCM460 cells (IC50 = 0.052 vs 1.114 μM). B9 also suppressed CRC clonogenic growth, 3D spheroid formation, and migration- and invasion-associated phenotypes in vitro. B9 achieved significant tumor growth inhibition (TGI = 80.1%) in vivo without obvious toxicity. RPB12 was identified as a candidate cellular target of B9 by activity-based protein profiling (ABPP) and validated by shRNA knockdown, CETSA, DARTS, SPR, and molecular docking. These findings support RPB12 as a novel promising target for the treatment of CRC and B9 as the first RPB12-targeting inhibitor for further development in CRC therapy.

Figure S3. Quantitative ChIP assay was used to detect the enrichment of CDKN1A, CXCR4 and negative control region by NCOR in SUDHL4 cells exposed to 5 μM WK692 for 24 h. (*, P < 0.05 versus control)

Figure S1. Chemical structures of FX1, BI3802 and WK692.

Figure S4. Activation effect of BI3802, FX1 and WK692 on downstream gene of BCL6 at 10 µM. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control).

Figure S6. WK692 targets BCL6-BTB domain. (A) Computer docking assay predicted that WK692 bound to the BCL6-BTB domain. (B) The binding affinities were measured by SPR assay. (C) SUDHL4 shBCL6-1/shBCL6-2 cells were infected with lentivirus for re-expression of BCL6 (vector, WT, C53A) and overexpression efficacy was examined by Western blots. (D) WK692 effectiveness against SUDHL4 shBCL6-1/shBCL6-2 cells that were infected with the indicated lentivirus expression vectors, and cell viability was determined by MTS assay.

Figure S5. Viability of SUDHL4 and Farage cells transduced with shBCL6 or shScr was measured by CCK8 assay.

Supplementary Table 1. Main pharmacokinetic parameters of WK692 after oral administration or intravenous injection in rats (mean ± SD).

Figure S2. WK692 specifically inhibits BCL6 but not other BTB-ZF Proteins. Reporter assays were performed to test the activity of WK692 with BCL6, Kaiso and PLZF. (***, P < 0.001 versus control).

Figure S7. H&E staining of liver, spleen, lung and kidney from subcutaneous tumor xenografts mice treated with FX1 and WK692. Scale bars, 50 μm.