Fig 1: PS-2 induces BTK degradation in cells. Mino cells were treated with indicated compounds at 0, 1.6, 8, 40, 200, and 1000 nM for 24 h, Ramos and A20 cells were incubated with PS-1, PS-2, PS-3, and DD-03-171 at 40, 200, and 1000 nM for 24 h, followed by Western blotting for BTK. CC-220 is a IKZF1/3 degrader developed by Celgene and used for comparison. In contrast to PS-1, PS-3 and CC-220, PS-2 potently induces BTK degradation in Mino cells. In Ramos and A20 cell lines, PS-1 and PS-3 elicit very weak or no BTK degradation, while PS-2 induces potent BTK degradation that is comparable to DD-03-171.
Fig 2: Toxicities of poseltinib-based BTK PROTACs in cells and their binding affinities to BTK. a-b. MOLM14 and Mino cells were treated with serially diluted poseltinib and PS-RC-1 to PS-RC-4 for 72 h, followed by Alarma Blue assay to quantify the cell viabilities. c. Three MCL cell lines, including Mino, Jeko-1, and Rec-R cells, were treated with serially diluted PS-RC-1 for 72 h, followed by Alarma Blue assay to quantify the cell viabilities. d. TR-FRET based binding kinetics assay between poseltinib and BTK. Serial dilutions of poseltinib mixed with 2 nM of His-BTK, 0.3 nM Tb-anti-His, and 150 nM of BTK-BODIPY tracer. e. BTK binding affinity assays for poseltinib-based PROTACs (PS-RC-1 to PS-RC-4), following the same protocol as described in d. After 2 h incubation, TR-FRET signals were measured. The IC50 values were listed in Table 1 f. PS-RC-1 serves as a molecular glue to inhibit growth in Mino cells. Mino cells were pre-treated with a large excess of PS-RC-Ctrl (2 µM or 10 µM), followed by PS-RC-1 incubation for 72 h. The cell viabilities were quantified using an Alarma Blue assay. For cell viability assays, data represent mean ± SD (n = 3) and the IC50 values are defined as compound concentrations that reduce cell viabilities by 50%. For BTK binding assays, data represent mean ± SD (n = 3) and the IC50 values are defined as compound concentrations that reduce tracer binding by 50%. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 3: Poseltinib-based reversible covalent BTK PROTACs cannot induce BTK degradation in cells. a-e. Mino cells were treated with indicated compounds at 0, 1.6, 8, 40, 200, and 1000 nM for 24 h, followed by Western blotting for BTK. PS-RC-1, PS-RC-2, PS-RC-3, and PS-RC-4 are poseltinib-based reversible covalent BTK PROTACs. DD-03-171 is a BTK degrader developed by the Gray group and used as a positive control. f. HEK-293T cells stably expressing a BTK-nLuc fusion protein were treated with indicated compounds (same as in a-e) for 24 h. The BTK degradation was determined by evaluating luminescence signals of NanoLuc. The DC50 (concentration of PROTACs required to achieve 50% degradation of the target protein) and Dmax (maximum level of target protein can be degraded by PROTACs) values obtained through this assay are listed in Table 1.
Supplier Page from DNASU for BTK (Homo sapiens) in pENTR223 (Gateway donor/master vector)