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Manumycin polyketides act as molecular glues between UBR7 and P53

Abstract

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.

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Fig. 1: Asukamycin targets C374 of UBR7 in breast cancer cells.
Fig. 2: Asukamycin impairs breast cancer cell proliferation through targeting UBR7.
Fig. 3: Mapping molecular glue interactions of asukamycin and UBR7.
Fig. 4: Asukamycin acts as a molecular glue between UBR7 and TP53 and activates TP53 transcriptional activity in a UBR7-dependent manner.
Fig. 5: Manumycin A also interacts with UBR7 and engages in molecular glue activities with TP53.
Fig. 6: Comparing manumycin D with manumycin A.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Source data for Figs. 16 and Extended Data Figs. 110 are provided with the paper.

Code availability

Data processing and statistical analysis algorithms from our lab can be found on our lab’s Github site: https://github.com/NomuraRG, and we can make any further code from this study available at reasonable request.

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Acknowledgements

We thank the members of the Nomura Research Group, Maimone Research Group and Novartis Institutes for BioMedical Research for critical reading of the manuscript. The present study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Center for Proteomics and Chemistry Technologies. We thank F. Mapa, P. Aspesi, J. Ambrose, J. Oborski, Z. B. Kang, M. Shum and B. Cornett for their work profiling asukamycin in a panel of cancer cell lines. This work was also supported by the Nomura Research Group and the Mark Foundation for Cancer Research and Chordoma Foundation ASPIRE Award (to D.K.N. and Y.I.), and by a grant from the National Institutes of Health (grant no. R01CA240981 to D.K.N., T.J.M. and Y.I.). Y.I. and M.O. were also supported by the Japanese Society for the Promotion of Science postdoctoral fellowships.

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Authors and Affiliations

Authors

Contributions

Y.I., D.K.N. and T.J.M. conceived the project and wrote the paper. Y.I., J.A.T., J.M.K., L.M., M.S., S.M.B., M.D.J., X.L., W.F., T.J.M. and D.K.N. provided intellectual contributions and insights into project direction. Y.I., T.J.M., S.M.B. and D.K.N. designed the experiments. Y.I., M.O., R.W., S.M.B., X.L. and D.K.N. performed the experiments and analyzed the data. Y.I., J.A.T., J.M.K., L.M., M.S., S.M.B., M.D.J., X.L., W.F., T.J.M. and D.K.N. edited the paper.

Corresponding authors

Correspondence to Thomas J. Maimone or Daniel K. Nomura.

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Competing interests

J.A.T., J.M.K., L.M., M.S., S.M.B., M.D.J., X.L. and W.F. are employees of Novartis Institutes for BioMedical Research. D.K.N. is a co-founder, shareholder and adviser for Artris Therapeutics and Frontier Medicines.

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Extended data

Extended Data Fig. 1 Testing asukamycin growth inhibitory effects across 250 cancer cell lines.

a, GI50 values for asukamycin across 250 cancer cell lines by cancer tissue origin. The individual cell line data can be found in Source Data Tables for Extended Data Fig. 1. The box plot shows median, standard deviation, and minimum and maximum ranges. For bladder cancer, there were 2 cell lines; for bone cancer, 3 cell lines; breast cancer, 19 cell lines; CNS/glioma cancers, 4 cell lines; colorectal cancer, 28 cell lines; endometrial cancer, 4 cell lines; eye cancers, 3 cell lines; gastric cancers, 12 cell lines; kidney cancer, 3 cell lines; liver cancer, 8 cell lines; liver cancer, 8 cell lines; lung cancer, 73 cell lines; esophageal cancer, 13 cell lines, ovarian cancer, 10 cell lines; pancreatic cancer, 23 cell lines; skin cancer, 35 cell lines; soft tissue cancers, 4 cell lines; upper aeodigestive cancers, 5 cell lines. b,c Dose-response curves for % growth of 19 breast cancer cell lines tested (b) and GI50 values (c) for asukamycin treatment, separated by relative sensitivity to asukamycin. Data from (a-c) were calculated in each cell line from n = 2 biologically independent samples/group.

Extended Data Fig. 2 TP53 levels in 100-140 kDa range in SDS/PAGE gel from FLAG-UBR7 pulldown.

LC-MS/MS proteomic analysis of 100-140 kDa range tryptic digests of SDS/PAGE gels from FLAG-UBR7 pulldown from 231MFP cells treated with DMSO vehicle or asukamycin (50 μM) for 3 h. Data shown as individual replicate spectral count values and average ± sem and are n = 3 biologically independent samples/group. Significance was calculated by a Student’s two-tailed t-test and is shown as *p < 0.05 in asukamycin-treated pulldown samples compared to vehicle-treated controls. Source data can be found in Source Data Tables for Extended Fig. 2.

Extended Data Fig. 3 Understanding the composition of the asukamycin-mediated higher molecular weight band.

a, Quantification of TP53 levels normalized to GAPDH levels in Western blot shown in Fig. 2d. b, Anti-FLAG and anti-TP53 blot in shControl and shTP53 231MFP breast cancer cells expressing FLAG-UBR7 treated with vehicle DMSO or asukamycin (50 μM) for 3 h. * notes the higher molecular FLAG-UBR7 band. c, Quantification of higher molecular weight FLAG-UBR7 band noted with * in (b). Data shown in (a, c) as individual replicate values and average ± sem and are n = 3 biologically independent samples/group. Gel shown in (b) is a representative gel of n = 3 biologically independent samples/group. Statistical significance was calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to shControl cells treated with asukamycin in (a, c). Uncropped blots can be found in Source Data for Extended Fig. 3. Source data for bar graphs can be found in Source Data Tables for Extended Fig. 3.

Source data

Extended Data Fig. 4 TP53 pulldown in wild-type or C374A mutant UBR7-expressing cells.

TP53 and UBR7 levels in input and pulled down eluate from FLAG-UBR7 pulldown in 231MFP cells stably expressing wild-type or C374A mutant FLAG-UBR7 in cells treated with DMSO vehicle or asukamycin (50 μM) treatment for 3 h, assessed by Western blotting. The ‘*’ indicates the higher molecular weight (MW) TP53-asukamycin-UBR7 ternary complex. The gel is representative of n = 3 biologically independent samples/group. Bar graphs below show quantification of parent and higher molecular weight TP53 levels in asukamycin-treated FLAG-pulldown samples from wild-type and C374A-FLAG-UBR7 expressing cells. Statistical significance was calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to WT asukamycin-treated groups. Uncropped blots can be found in Source Data for Extended Fig. 4. Source data for bar graphs can be found in Source Data Tables for Extended Fig. 4.

Source data

Extended Data Fig. 5 Thermal stability of TP53 in 231MFP cell lysate.

a, UBR7 and loading control GAPDH expression in siControl and siUBR7 231MFP cells. b, Thermal stability of TP53 in DMSO vehicle or asukamycin (50 μM, 1 h) treated cell lysate from 231MFP siControl or siUBR7 cells. Gels are representative of n = 3 biologically independent samples/group. c, Quantification of thermal shift assay from (b). Data in (c) shown as individual replicate values and average ± sem and are n = 3 biologically independent samples/group. Statistical significance in (c) was calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to vehicle-treated siControl groups. Data was not significant (p > 0.05) for all asukamycin versus DMSO comparisons in siUBR7 groups. Uncropped blots can be found in Source Data for Extended Fig. 5. Source data for plots can be found in Source Data Tables for Extended Fig. 5.

Source data

Extended Data Fig. 6 TP53 thermal stability.

(a) FLAG-UBR7, UBR7, and loading control GAPDH levels from 231MFP cells expressing FLAG-UBR7 after mock depletion or FLAG-UBR7 depletion as assessed by Western blotting. (b) TP53 thermal stability in cell lysate from 231MFP FLAG-UBR7-expressing cells treated with DMSO vehicle or asukamycin (50 μM, 1 h) after mock depletion or FLAG-UBR7 depletion. (c) Quantification of experiments described in (b). Gels in (a) are representative of n = 3 biologically independent samples/group. Gels in (b) are representative of n = 3 biologically independent samples/group which are quantified and shown as individual replicate values in (c). Statistical significance in (c) was calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 comparing UBR7-depleted asukamycin-treated groups to mock-depleted asukamycin-treated groups. Uncropped blots can be found in Source Data for Extended Fig. 6. Source data for plots can be found in Source Data Tables for Extended Fig. 6.

Source data

Extended Data Fig. 7 TP53 binding to DNA consensus sequence.

TP53 DNA binding to p53 DNA consensus sequence in vitro with TP53 spiked into 231MFP shControl or shUBR7 breast cancer cell lysate treated with DMSO vehicle or asukamycin (50 μM). Data shown are individual replicate values and average ± sem from n = 3 biologically independent samples/group. Statistical significance is calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to shControl vehicle-treated groups and #p < 0.05 compared to shControl asukamycin-treated groups. Source data for bar graph can be found in Source Data Tables for Extended Fig. 7.

Source data

Extended Data Fig. 8 TP53 reporter activity.

TP53 reporter activity in HEK293T siControl and siUBR7 cells treated with DMSO vehicle or asukamcyin for 3 h. Data shown are individual replicate values and average ± sem from n = 4 biologically independent samples/group. Statistical significance is calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to corresponding siControl treatment groups. Source data for bar graph can be found in Source Data Tables for Extended Fig. 8.

Source data

Extended Data Fig. 9 Quantitative proteomic analysis of asukamycin treatment in 231MFP cells.

231MFP cells were treated with DMSO vehicle or asukamycin (50 mM, 12 h), and protein expression changes were assessed by quantitative TMT-based proteomics. Data shown are from n = 3 biologically independent samples/group. Highlighted in red are proteins in which their expression was heightened by >2-fold with adjusted p-value<0.05. The full dataset can be found in Source Data Tables for Extended Data Fig. 9. Highlighted are representative known p53 transcriptional targets. The lower table shows pathways that were significantly enriched in a functional enrichment analysis of the proteins that were upregulated by >2-fold with adjusted p-value<0.05, which includes TP53 signaling. Differential abundance significance from TMT-based proteomic data estimated using a background-based ANOVA with Benjamini-Hochberg correction to determine adjusted p-values. Statistical analysis for Pathway Enrichment Analysis was performed using the g:SCS algorithm for computing multiple testing correction for p-values gained from GO and pathway enrichment analysis. It corresponds to an experiment-wide threshold of a = 0.05. Given a fixed input query size, g:SCS analytically approximates a threshold t corresponding to the 5% upper quantile of randomly generated queries of that size. All actual p-values resulting from the query are transformed to corrected p-values by multiplying these to the ratio of the approximate threshold t and the initial experiment-wide threshold a = 0.05.

Source data

Extended Data Fig. 10 CDKN1A level changes with asukamycin treatment.

Tumor suppressor CDKN1A (p21), UBR7, and loading control GAPDH levels in 231MFP siControl and siUBR7 cells treated with DMSO vehicle or asukamycin (50 μM, 12 h), assessed by Western blotting, and quantified by densitometry and normalized to GAPDH levels. Gel on the left is representative of n = 3 biologically independent samples/group. Bar graph on the right shows individual replicate values and average ± sem from n = 3 biologically independent samples/group. Statistical significance is calculated with two-tailed unpaired Student’s t-tests and are shown as *p < 0.05 compared to shControl vehicle-treated groups and #p < 0.05 compared to shControl asukamycin-treated groups. Uncropped blots can be found in Source Data for Extended Fig. 10. Source data for plots can be found in Source Data Tables for Extended Fig. 10.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Synthetic procedures.

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Isobe, Y., Okumura, M., McGregor, L.M. et al. Manumycin polyketides act as molecular glues between UBR7 and P53. Nat Chem Biol 16, 1189–1198 (2020). https://doi.org/10.1038/s41589-020-0557-2

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