Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade. BCR activation can contribute to the growth and survival of B-cell lymphoma or leukemia. The inhibition of the BCR signaling pathway is critical for blocking downstream events and treating B-cell lymphomas. Herein, we report potent and orally available proteolysis-targeting chimeras (PROTACs) that target BTK to inactivate BCR signaling. Of the PROTACs tested, UBX-382 showed superior degradation activity for wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration in diffuse large B-cell lymphoma cell line. UBX-382 was effective on 7 out of 8 known BTK mutants in in vitro experiments and was highly effective in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells over ibrutinib, ARQ-531, and MT-802. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. UBX-382 also provoked the cell type-dependent and selective degradation of cereblon neosubstrates in various hematological cancer cells. These results suggest that UBX-382 treatment is a promising therapeutic strategy for B-cell-related blood cancers with improved efficacy and diverse applicability.
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h4>Background: /h4> Bruton’s tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade. BCR activation can contribute to the growth and survival of B-cell lymphoma or leukemia. The inhibition of the BCR signaling pathway is critical for blocking downstream events and treating B-cell lymphomas. Herein, we report potent and orally available proteolysis-targeting chimeras (PROTACs) that target BTK to inactivate BCR signaling. Methods Fluorescence resonance energy transfer signals were used to assess BTK and cereblon (CRBN) ternary complex formation with Mab anti-6His-Eu cryptate and Mab anti-glutathione-S-transferase-XL665. BTK and CRBN binding activities were investigated using in vitro target binding assays. Subsequently, SDS-PAGE and western blotting of whole cell lysates was performed, followed by immunofluorescence analysis of TMD-8 cells. ELISA was performed in TMD-8 cells treated with 0.01 µM PROTAC, ibrutinib, acalabrutinib, ARQ-531, Binder, or MT-802 to measure CCL3 and CCL4 levels. Tandem ubiquitin-binding elements pull-down assay was used to characterize UBX-382 in Ramos cells. Various cells treated with inhibitor or PROTAC were assessed for proliferation and viability. Wild-type- or BTK-transfected cells were transplanted in CB17/SCID mice and the established tumors were assessed using immunohistochemistry. Results Of the PROTACs tested, UBX-382 showed superior degradation activity for wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of DC 50 in DLBCL cell line. UBX-382 was effective on seven out of eight known BTK mutants in in vitro experiment and was highly effective in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells over ibrutinib, ARQ-531, and MT-802. Remarkably, oral dosing of UBX-382 for less than 2-weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. UBX-382 also provoked the cell-type-dependent and selective degradation of CRBN neo-substrates in various hematological cancer cells. Conclusions These results suggest that UBX-382 treatment is a promising therapeutic strategy for B-cell-related blood cancers with improved efficacy and diverse applicability.