Journal: British Journal of Pharmacology

Article Title: A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant Btk C481S mutation in B‐cell malignancies

doi: 10.1111/bph.14809

Figure Lengend Snippet: Characterization of XMU‐MP‐3 as a BTK inhibitor. (a) The chemical structure of XMU‐MP‐3. (b) Dose–response curves of BTK‐transformed and parental Ba/F3 cells (2 × 104 cells per well) exposed to XMU‐MP‐3 for 48 hr. Cell viability was assessed with MTS. Data are presented as mean ± SEM (n = 3). Each concentration point was performed in triplicate. (c) Dose‐inhibition curve of XMU‐MP‐3 on BTK enzymatic activity in the presence of 10‐μM ATP in vitro. Each concentration point was performed in duplicate. (d) The selectivity profiling of XMU‐MP‐3 against a panel of 30 oncogenic protein TK‐transformed Ba/F3 cell lines. Cells (2 × 104 cells per well) were exposed to XMU‐MP‐3 for 48 hr. Cell viability was assessed with MTS. Each concentration point was performed in triplicate. The IC50 values were determined and reported in Table S2. (e) Effects of XMU‐MP‐3 on BTK‐mediated signalling pathway components in BTK‐transformed Ba/F3 cells. Cells were treated with either DMSO or XMU‐MP‐3 for 4 hr. Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK; p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot. (f) XMU‐MP‐3 inhibited BTK signalling in a time‐dependent manner. Representative blots from three independent experiments and the data of relative intensity are shown. (g) BTK‐transformed Ba/F3 cells were treated with the indicated dose of XMU‐MP‐3 for 24 hr. Induction of cell apoptosis was assessed by Annexin‐V‐FLUOS/PI double staining. CGI‐1746, AVL‐292, and ibrutinib were used as controls. Representative results from five independent experiments are shown. (h) Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test

Article Snippet: CGI‐1746, AVL‐292, and ibrutinib were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Transformation Assay, Concentration Assay, Inhibition, Activity Assay, In Vitro, Double Staining

Journal: British Journal of Pharmacology

Article Title: A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant Btk C481S mutation in B‐cell malignancies

doi: 10.1111/bph.14809

Figure Lengend Snippet: XMU‐MP‐3 is “on‐target” to BTK. (a) Establishment of stable BTK and BTK mutant‐transformed Ba/F3 cell lines. Mutations of BTK significantly increased the phosphorylation of BTK. Representative blots from three independent experiments are shown. (b) Dose–response curves of BTK(T474M)‐Ba/F3 cells (2 × 104 cells per well, 48‐hr treatment). The gatekeeper mutant BTK(T474M) significantly elevated cellular IC50 more than 200‐fold. Cell viability was assessed with MTS in triplicate. Data are presented as mean ± SEM (n = 3). (c) Effects of XMU‐MP‐3 on the BTK‐mediated signalling pathway in Ba/F3 cells expressing BTK(T474M) after 4 hr of drug treatment. The gatekeeper mutation, T474M, showed robust resistance to XMU‐MP‐3 treatment. Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK, p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot. (d) BTK(T474M)‐Ba/F3 cells were treated with DMSO or escalating doses of XMU‐MP‐3, CGI‐1746, and AVL‐292 for 24 hr. Induction of apoptosis was assayed with Annexin‐V‐FLUOS/PI double staining. Representative results from five independent experiments are shown. (e) The ratio of Annexin‐V‐FLUOS‐positive cells indicates that the gatekeeper mutation BTK(T474M) caused BTK to be significantly resistant to inhibition with XMU‐MP‐3. Data are presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test

Article Snippet: CGI‐1746, AVL‐292, and ibrutinib were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Mutagenesis, Transformation Assay, Expressing, Double Staining, Inhibition

Journal: British Journal of Pharmacology

Article Title: A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant Btk C481S mutation in B‐cell malignancies

doi: 10.1111/bph.14809

Figure Lengend Snippet: XMU‐MP‐3 inhibits the growth of malignant B‐cells. (a) Dose–response curves for XMU‐MP‐3 against BTK‐positive B‐cell malignancy cell lines JeKo‐1, Ramos, and NALM‐6 following 48 hr of treatment. Cell viability was assessed with MTS in triplicate. Data are presented as mean ± SEM (n = 3). (b) Anti‐colony formation of XMU‐MP‐3 on NALM‐6. Colony formation were performed in 14‐day soft agar assays from three independent experiments with 3 × 104 cells per well. Data are presented as mean ± SEM (n = 3). (c) XMU‐MP‐3 induced cell apoptosis in malignant B‐cells JeKo‐1, Ramos, and NALM‐6. Induction of cell apoptosis was assessed by Annexin‐V‐FLUOS/PI double staining. CGI‐1746 and AVL‐292 were used as controls. Representative results from five independent experiments are shown. Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test. (d) XMU‐MP‐3 induced significant Caspase 3/7 cleavage and PARP activation in JeKo‐1, Ramos, and NALM‐6 cell lines. Representative blots from three independent experiments are shown. The relative intensity of each band normalized to β‐actin is shown under each blot

Article Snippet: CGI‐1746, AVL‐292, and ibrutinib were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Double Staining, Activation Assay