Figure 5. Manumycin A also interacts with UBR7 and engages in molecular glue activities with TP53.

(a) Structure of manumycin A highlighting potentially reactive sites in red. (b) Gel-based ABPP analysis of manumycin A against pure human UBR7 protein. UBR7 protein was pre-incubated with DMSO vehicle or manumycin A for 30 min prior to IA-rhodamine labeling of UBR7 for 1 h at room temperature. Protein was resolved by SDS/PAGE and visualized by in-gel fluorescence and protein loading was assessed by silver staining. (c) p53 reporter activity reported as the ratio between luciferase reporter activity versus cell number control renilla levels in HEK293T cells expressing the p53 reporter construct treated with DMSO vehicle control, manumycin A, asukamycin, or doxorubicin (1 μM) for 6 h. (d) Protein levels of TP53 and FLAG-tagged proteins by Western blotting in 231MFP cells stably expressing FLAG-UBR7 treated with DMSO vehicle or manumycin A (50 μM) for 3 h, after which FLAG-UBR7 interacting proteins were subsequently enriched. Higher molecular weight TP53 and FLAG-UBR7 species are noted with a (*). (e) Gel-based ABPP analysis of manumycin A against pure human TP53 protein. TP53 protein was pre-incubated with DMSO vehicle or manumycin A for 30 min prior to IA-rhodamine labeling of TP53 for 1 h at room temperature. Data shown in (c) are shown as individual replicate values and average ± sem and are n=3 biologically independent samples/group. Gels shown in (b, d, and e) are representative blots from n=3 biologically independent samples/group. Statistical significance as calculated with two-tailed unpaired Student’s t-tests and are shown as *p<0.05 compared to vehicle-treated controls. Source data for cropped blots can be found in Source Data for Figure 5. Source data for bar graphs can be found in Source Data Tables for Figure 5.