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Cited by 124Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysCancer-related Molecular Pathways

Manumycin polyketides act as molecular glues between UBR7 and P53

Open access
Isobe, Yosuke, Okumura, Mikiko, McGregor, Lynn M., Brittain, Scott M., Jones, Michael D., Liang, Xiaoyou, White, Ross, Forrester, William +5 more·2020·Nature Chemical Biology
DOI 10.1038/s41589-020-0557-2PMC PMC7572527
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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.

AI snapshot

Written from the paper’s full text.

TL;DR

Using chemoproteomic profiling, the authors show that manumycin polyketides asukamycin and manumycin A covalently target C374 of UBR7 and act as molecular glues that recruit TP53, activating p53 transcription and impairing breast cancer cell proliferation. The epoxide-bearing manumycins require UBR7 C374 and TP53 for anti-proliferative effects, while epoxide-reduced manumycin D loses UBR7–TP53 glue activity.

Key findings

  • Asukamycin impaired 231MFP TNBC proliferation and serum-free survival with EC50 values of 13.8 and 4.5 μM, respectively; across 250 cancer cell lines, GI50 values ranged from 0.08 to >30 μM, and among 19 breast cancer lines 7 were sensitive (GI50 5.7–11.2 μM), 9 intermediate (13–16 μM), and 3 refractory (>20 μM).
  • IsoTOP-ABPP identified C374 of UBR7 as a primary asukamycin target; UBR7 knockdown conferred complete resistance to asukamycin anti-proliferative effects, WT but not C374A UBR7 re-sensitized cells, and overexpression of WT but not C374A UBR7 heightened effects with EC50 values of 10.0, 4.7, and 10.0 μM for GFP-, WT UBR7-, and C374A UBR7-expressing 231MFP cells.
  • Anti-FLAG pulldown found 13 proteins with p<0.01 and >15-fold higher pulldown in asukamycin-treated FLAG-UBR7 versus vehicle-treated FLAG-UBR7 cells; 8 were UBR7-dependent with >4-fold enrichment versus asukamycin-treated FLAG-GFP, including TP53 and PRKDC. TP53 knockdown, but not PRKDC knockdown, conferred complete resistance to asukamycin.
  • Asukamycin increased TP53 thermal stability, TP53 binding to its DNA consensus sequence, TP53AIP1 and CDKN1A/p21 expression, and p53 luciferase reporter activity more than doxorubicin; these effects were attenuated by UBR7 knockdown or C374A UBR7. Manumycin A showed similar UBR7 binding, p53 activation, and UBR7–TP53 glue activity, whereas manumycin D bound UBR7 more weakly, bound TP53 less potently, did not induce UBR7–TP53 interactions, and had attenuated anti-proliferative and p53-activating effects.

Methods

  • Cell-based assays used 231MFP, HCC38, HEK293T, and isogenic HCT116 cells; proliferation/survival was measured with Hoechst 33342, and growth inhibition was tested across 250 cancer cell lines with Cell-Titer Glo and GI50 determination.
  • Target mapping used isoTOP-ABPP with IA-alkyne in 231MFP cells treated with DMSO or 10 μM asukamycin for 3 h, gel-based ABPP with IA-rhodamine, anti-FLAG pulldowns, Western blotting, TMT quantitative proteomics, thermal shift assays, p53 DNA-binding assays, p53 luciferase reporter assays, and qPCR.
  • Genetic validation used shRNA knockdown of UBR7, TP53, or PRKDC, siRNA knockdown of UBR7, and expression of WT or C374A mutant UBR7 in knockdown or overexpression backgrounds.

Limitations

  • The paper states that how the UBR7–asukamycin complex activates p53 is unknown; the targeted cysteine on p53 was not identified because LC-MS/MS did not detect a modified peptide, likely due to adduct instability; the ternary complex crystal structure was not solved; in vitro reconstitution caused asukamycin-mediated self-oligomerization of UBR7 and TP53; the endogenous role of UBR7 remains unclear and a normal UBR7 function in asukamycin activity cannot be excluded; and asukamycin/manumycin A likely have additional targets beyond UBR7.
  • The text makes evident that the 250-cell-line GI50 panel used n=2 biologically independent samples/group, and data/code availability is stated as available from the corresponding author or lab GitHub on reasonable request.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41589-020-0557-2

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 47 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    HCC38 and the MDA-MB-231 line from which 231MFP cells were derived showed intermediate sensitivity to asukamycin.

    “in which HCC38 and the MDA-MB-231 line from which 231MFP cells were derived showed intermediate sensitivity”
    Quote in the paperNot a substantial claim
  • Findingown data

    UBR7 is a direct target of asukamycin, confirmed by gel-based ABPP competition against IA-rhodamine labeling of recombinant human UBR7.

    “We confirmed that UBR7 was a direct target of asukamycin using gel-based ABPP approaches, wherein we show competition of asukamycin against rhodamine-functionalized iodoacetamide (IA-rhodamine) labeling of recombinant human UBR7 protein (Figure 1d).”
    Quote in the paperSubstantial claim
  • Findingown data

    Asukamycin has EC50 values of 13.8 and 4.5 μM for 231MFP proliferation and survival, respectively.

    “with 50 % effective concentration (EC50) values of 13.8 and 4.5 μM for 231MFP proliferation and survival, respectively.”
    Quote in the paperNot a substantial claim
  • Findingown data

    These 8 targets include PRKDC and TP53.

    “These 8 targets include proteins of great importance in cancer, including DNA protein kinase (PRKDC) and the tumor-suppressor p53 (TP53) 28,29 (Figure 3b).”
    Quote in the paperSubstantial claim
  • Findingown data

    Asukamycin directly interacts with TP53 by gel-based ABPP.

    “we further demonstrated that asukamycin directly interacts with TP53 by gel-based ABPP (Figure 3e).”
    Quote in the paperSubstantial claim
  • May combine two claims. 36
  • The quote only partly states this claim. 6
  • Classified as probably not a substantial claim. 33

  • May combine two claims.

    Findingown data

    Asukamycin impairs proliferation and serum-free survival of the TNBC-derived cell lines 231MFP and HCC38.

    “We show that asukamycin impairs the proliferation (with full serum-containing media) and serum-free cell survival of two TNBC-derived cell lines, 231MFP and HCC38 (Figure 1b, Supplementary Figure 1)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among these 13 targets, 8 are UBR7-dependent, with >4-fold enrichment between asukamycin-treated FLAG-UBR7 cells versus asukamycin-treated FLAG-GFP cells.

    “Among these 13 targets, 8 are UBR7-dependent, with >4-fold enrichment between asukamycin-treated FLAG-UBR7 cells versus asukamycin-treated FLAG-GFP cells (Figure 3a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GAPDH did not interact with the asukamycin-UBR7 complex, arguing for specific molecular interactions over non-specific binding.

    “We also showed that abundant proteins such as GAPDH did not interact with the asukamycin-UBR7 complex, arguing for specific molecular interactions over non-specific binding events (Figure 3c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    C374 of UBR7 is a primary target of asukamycin in 231MFP cells, with the highest control-to-treated ratio in isoTOP-ABPP.

    “We identified C374 of UBR7 as a primary target of asukamycin with the highest control to treated (or light to heavy) ratio in 231MFP cells (Figure 1c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of wild-type but not C374A mutant FLAG-UBR7 conferred significantly heightened asukamycin-induced anti-proliferative effects with EC50 values of 10.0, 4.7, and 10.0 μM for GFP-, WT UBR7-, and C374A mutant UBR7-expressing 231MFP cells, respectively.

    “we found that overexpression of WT, but not C374A mutant FLAG-UBR7 conferred significantly heightened asukamycin-induced anti-proliferative effects with EC50 values of 10.0, 4.7, and 10.0 μM for GFP-, WT UBR7, and C374A mutant UBR7-expressing 231MFP cells, respectively (Figure 2d, 2e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among 19 breast cancer cell lines, 7 showed heightened sensitivity with GI50 values of 5.7-11.2 μM, 9 showed intermediate sensitivity with GI50 values of 13-16 μM, and 3 showed refractory effects with GI50 values >20 μM.

    “Among the 19 breast cancer cell lines tested from receptor positive and TNBC cell lines, 7 showed heightened sensitivity with GI50 values of 5.7–11.2 μM, 9 showed intermediate sensitivity with GI50 values of 13–16 μM, and 3 showed refractory effects with GI50 values >20 μM”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Higher molecular weight species in reducing SDS/PAGE corresponded to estimated combined masses of FLAG-UBR7 and TP53, and even higher species corresponded to PRKDC.

    “we also observed several higher molecular weight species in a reducing SDS/PAGE gel that corresponded to the estimated combined masses of FLAG-UBR7 and TP53 as well as even higher molecular weight species that corresponded to the estimated molecular weight of PRKDC (Figure 3c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stable UBR7 knockdown in 231MFP cells did not compromise proliferation but conferred complete resistance to asukamycin-mediated anti-proliferative effects.

    “Stable UBR7 knockdown in 231MFP cells did not compromise proliferation but conferred complete resistance to asukamycin-mediated anti-proliferative effects (Figure 2a, 2b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Manumycin A treatment led to molecular glue interactions between UBR7 and TP53 and resulted in distinct higher molecular weight species corresponding to the added molecular weight of TP53 and UBR7.

    “Manumycin A treatment also led to molecular glue interactions between UBR7 and TP53 and resulted in distinct higher molecular weight species which corresponded to the added molecular weight of TP53 and UBR7 (Figure 5d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Asukamycin significantly induced p53 luciferase reporter transcriptional activity in HEK293T cells, more so than doxorubicin.

    “Asukamycin also significantly induced p53 luciferase reporter transcriptional activity in HEK293T cells, even more so than the positive control topoisomerase inhibitor doxorubicin (dox) that is known to induce apoptosis through activating the p53 pathway (Figure 4e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Manumycin D lacks an epoxide, retains binding to UBR7 but shows less potent binding to TP53 and is not able to covalently modify TP53 to the same extent.

    “Furthermore, we show that manumycin D, which lacks an epoxide, still retains binding to UBR7 but shows less potent binding to TP53 and is not able to covalently modify TP53 to the same extent”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Asukamycin conferred anti-proliferative effects in an isogenic HCT116 colorectal cancer cell line background expressing either wild-type or R248W mutant TP53 but showed resistance in TP53 knockout cells.

    “We also showed that asukamycin conferred anti-proliferative effects in an isogenic HCT116 colorectal cancer cell line background expressing either wild-type or R248W mutant TP53 but showed resistance in TP53 knockout cells (Supplementary Figure 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Functional enrichment analysis of significantly upregulated proteins showed significant enrichment in the TP53 signaling pathway, as well as pathways in protein processing, senescence, and MAPK signaling.

    “Functional enrichment analysis of significantly upregulated proteins showed a significant enrichment in the TP53 signaling pathway, as well as pathways in protein processing, senescence, MAPK signaling (Extended Data Figure 9).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    In 231MFP FLAG-UBR7-expressing cell lysate, asukamycin-induced TP53 thermal stability is significantly lower after FLAG-UBR7 depletion than after mock depletion.

    “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.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Asukamycin treatment (50 μM, 1 h) significantly increases TP53 thermal stability in 231MFP siControl cell lysate compared to vehicle.

    “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.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

    Findingown data

    In 231MFP siUBR7 cells, asukamycin-induced CDKN1A (p21) levels are significantly lower than in siControl cells.

    “#p<0.05 compared to shControl asukamycin-treated groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Among 19 breast cancer cell lines, asukamycin sensitivity varies, as shown by dose-response curves and GI50 values.

    “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.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    TP53 knockdown significantly reduces the higher molecular weight FLAG-UBR7 band in asukamycin-treated 231MFP cells compared to shControl.

    “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).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

    Findingnone given

    Asukamycin treatment (50 μM, 12 h) significantly increases CDKN1A (p21) levels in 231MFP siControl cells compared to vehicle.

    “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”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    In HEK293T cells, TP53 reporter activity is significantly lower in siUBR7 cells than in siControl cells under corresponding vehicle or asukamycin treatment conditions.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Asukamycin has growth inhibitory effects across 250 cancer cell lines, with GI50 values varying by cancer tissue origin.

    “GI50 values for asukamycin across 250 cancer cell lines by cancer tissue origin.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    In vitro, asukamycin treatment (50 μM) significantly increases TP53 binding to p53 DNA consensus sequence in 231MFP shControl cell lysate compared to vehicle.

    “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”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    In asukamycin-treated FLAG-pulldown samples from 231MFP cells, higher molecular weight TP53 levels are significantly lower in C374A mutant UBR7-expressing cells than in wild-type UBR7-expressing cells.

    “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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Complexes between UBR7-asukamycin and binding partners such as TP53 or PRKDC initially form through reversible interactions which eventually in part lead to multi-covalent interactions.

    “These results show enrichment of both the parent molecular weight protein interaction partner and also a higher molecular weight species, suggesting that the complexes between UBR7-asukamycin and binding partners such as TP53 or PRKDC initially form through reversible interactions which eventually in-part lead to multi-covalent interactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Asukamycin targets C374 of UBR7 and this interaction is responsible for the anti-proliferative effects of asukamycin in breast cancer cells.

    “Taken together, these results demonstrate that asukamycin targets C374 of UBR7 and that this interaction is responsible for the anti-proliferative effects of asukamycin in breast cancer cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Asukamycin promotes formation of a gain-of-function complex between UBR7 and TP53 that results in anti-proliferative effects in TNBC cell lines.

    “Taken together, these results suggest that asukamycin has the ability to promote the formation of a gain-of-function complex between UBR7 and TP53 that results in anti-proliferative effects in TNBC cell lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    This class of molecules targets C374 of UBR7 through a hetero-Michael addition, and then engages TP53 likely initially through reversible binding and then through a covalent bond involving the reactive epoxide.

    “consistent with this class of molecules targeting C374 of UBR7 through a hetero-Michael addition, and then engaging TP53 likely initially through reversible binding and then through a covalent bond involving the reactive epoxide.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    These complexes could also contain additional molecular glue partners, such as those identified in the enrichment proteomic studies.

    “These complexes could also contain additional molecular glue partners, such as those identified in the enrichment proteomic studies.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Manumycin A has been shown to interfere with IKK activity by directly binding to IKKβ and forming stable high molecular mass complexes.

    “Manumycin A has also been shown to interfere with IKK activity by directly binding to IKKβ and forming stable high molecular mass complexes”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Tasaki, T. et al. The substrate recognition domains of the N-end rule pathway. J. Biol. Chem. 284, 1884–1895 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The epoxide contributed to interactions with TP53 and was necessary to engage in multi-covalent interactions between UBR7 and TP53 to exert anti-proliferative effects.

    “These data collectively suggested that the epoxide contributed to interactions with TP53 and was necessary to engage in multi-covalent interactions between UBR7 and TP53 to exert anti-proliferative effects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Asukamycin directly targeted TP53, stabilized TP53 folding, and activated p53 transcriptional activity in a UBR7 C374-dependent manner.

    “Collectively, these results indicated that asukamycin directly targeted TP53, stabilized TP53 folding, and activated p53 transcriptional activity in a UBR7 C374-dependent manner.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Asukamycin activates TP53 transcriptional activity in a UBR7 C374-dependent manner and the anti-proliferative effects observed in breast cancer cells depend on UBR7 and TP53.

    “We demonstrate that asukamycin activates TP53 transcriptional activity in a UBR7 C374-dependent manner and that the anti-proliferative effects observed in breast cancer cells depends on UBR7 and TP53.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Previously reported targets including farnesyltransferase and sphingomyelinase may also play a role in mediating anti-cancer effects or other biological activities.

    “Previously reported targets including farnesyltransferase and sphingomyelinase may also play a role in mediating anti-cancer effects or other biological activities 22,23.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22] Hara, M. et al. Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc. Natl Acad. Sci. USA 90, 2281–2285 (1993). source

      Not checked in the free run.

    • cites [23] Bianchini, G., Balko, J. M., Mayer, I. A., Sanders, M. E. & Gianni, L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat. Rev. Clin. Oncol. 13, 674–690 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    There may be additional higher molecular weight species corresponding to multiple multi-covalent interactions with the three potential reactive sites on asukamycin and manumycin A.

    “While we seem to observe one primary higher molecular weight UBR7-TP53 band, there are also several additional higher molecular weight species observed, which may correspond to multiple multi-covalent interactions with the three potential reactive sites on asukamycin and manumycin A.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Asukamycin reacts with C374 of UBR7 to impair breast cancer cell proliferation through engaging in molecular glue interactions with multiple neo-substrates, including TP53.

    “In this study, we show that asukamycin, a manumycin polyketide bearing multiple electrophilic sites, reacts with C374 of the E3 ligase UBR7 to impair breast cancer cell proliferation through engaging in molecular glue interactions with multiple neo-substrates, including TP53.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Manumycin A has been shown to inhibit farnesyl transferase activity and sphingomyelinase activity.

    “Manumycin A has also been show to inhibit farnesyl transferase activity and sphingomyelinase activity, but asukamycin is not known to inhibit these targets and its anti-cancer mechanism of action is unclear 19–23.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [19] Hu, Y. & Floss, H. G. New type II manumycins produced by Streptomyces nodosus ssp. asukaensis and their biosynthesis. J. Antibiot. 54, 340–348 (2001). source

      Not checked in the free run.

    • cites [20] Shipley, P. R., Donnelly, C. C. A., Le, C. H., Bernauer, A. D. & Klegeris, A. Antitumor activity of asukamycin, a secondary metabolite from the actinomycete bacterium Streptomyces nodosus subspecies asukaensis. Int. J. Mol. Med. 24, 711–715 (2009).

      Not checked in the free run.

    • cites [21] Arenz, C. et al. Manumycin A and its analogues are irreversible inhibitors of neutral sphingomyelinase. Chembiochem. Eur. J. Chem. Biol. 2, 141–143 (2001). source

      Not checked in the free run.

    • cites [22] Hara, M. et al. Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc. Natl Acad. Sci. USA 90, 2281–2285 (1993). source

      Not checked in the free run.

    • cites [23] Bianchini, G., Balko, J. M., Mayer, I. A., Sanders, M. E. & Gianni, L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat. Rev. Clin. Oncol. 13, 674–690 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Thalidomide binds to the E3 ligase cereblon to form a new protein interface that recruits neo-substrates such as SALL4 or Ikaros transcription factors for ubiquitination and degradation.

    “Thalidomide binds to the E3 ligase cereblon to form a new protein interface that recruits neo-substrates such as SALL4 or Ikaros transcription factors for ubiquitination and degradation 9–12.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [9] Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014). source

      Not checked in the free run.

    • cites [10] Chamberlain, P. P. et al. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 21, 803–809 (2014). source

      Not checked in the free run.

    • cites [11] Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14, 981–987 (2018). source

      Not checked in the free run.

    • cites [12] Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray Syndrome. eLife 7, e38430 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The UBR7-asukamycin complex acts like a chaperone to improve TP53 thermal stability and folding, which likely improved its ability to bind to DNA and activate TP53 transcriptional activity.

    “Our data suggest that the UBR7-asukamycin complex acts like a chaperone to improve TP53 thermal stability and folding, which likely improved its ability to bind to DNA and activate TP53 transcriptional activity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Molecular glue-based degraders have potentially lower molecular weight than linker-based bifunctional molecules such as PROTACs, a possible advantage for increased bioavailability and improved pharmacokinetic profiles.

    “Molecular glue-based degraders, as exemplified by the IMiD-family of immunomodulatory drugs including thalidomide, are notable for their potentially lower molecular weight as compared to linker-based bifunctional molecules (e.g. PROTACS), a possible advantage for increased bioavailability and improved pharmacokinetic profiles 4.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Hughes, S. J. & Ciulli, A. Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem 61, 505–516 (2017). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Asukamycin is not known to inhibit farnesyl transferase or sphingomyelinase and its anti-cancer mechanism of action is unclear.

    “Manumycin A has also been show to inhibit farnesyl transferase activity and sphingomyelinase activity, but asukamycin is not known to inhibit these targets and its anti-cancer mechanism of action is unclear 19–23.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [19] Hu, Y. & Floss, H. G. New type II manumycins produced by Streptomyces nodosus ssp. asukaensis and their biosynthesis. J. Antibiot. 54, 340–348 (2001). source

      Not checked in the free run.

    • cites [20] Shipley, P. R., Donnelly, C. C. A., Le, C. H., Bernauer, A. D. & Klegeris, A. Antitumor activity of asukamycin, a secondary metabolite from the actinomycete bacterium Streptomyces nodosus subspecies asukaensis. Int. J. Mol. Med. 24, 711–715 (2009).

      Not checked in the free run.

    • cites [21] Arenz, C. et al. Manumycin A and its analogues are irreversible inhibitors of neutral sphingomyelinase. Chembiochem. Eur. J. Chem. Biol. 2, 141–143 (2001). source

      Not checked in the free run.

    • cites [22] Hara, M. et al. Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc. Natl Acad. Sci. USA 90, 2281–2285 (1993). source

      Not checked in the free run.

    • cites [23] Bianchini, G., Balko, J. M., Mayer, I. A., Sanders, M. E. & Gianni, L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat. Rev. Clin. Oncol. 13, 674–690 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Premisecitation

    Electrophilic natural products represent a vastly underexplored subset of natural products from which molecular glues can originate, and such species could have unique and positive attributes owing to their potential for covalent bond formation.

    “Electrophilic natural products represent a vastly underexplored subset of natural products from which molecular glues can originate, and such species could have unique and positive attributes owing to their potential for covalent bond formation16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies suggested UBR7 functions as a tumor suppressor in triple-negative breast cancer cells through reducing monoubiquitination of histone H2B and reducing CDH4 levels.

    “Previous studies have suggested that UBR7 functions as a tumor suppressor in triple-negative breast cancer cells 27, through reducing monoubiquitination of histone H2B and reducing CDH4 levels”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Goodwin, J. F. & Knudsen, K. E. Beyond DNA repair: DNA-PK function in cancer. Cancer Discov 4, 1126–1139 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cotylenin diterpenes form interfaces between the 14-3-3 protein family and a variety of interacting partners.

    “The cotylenin diterpenes form interfaces between the 14-3-3 protein family and a variety of interacting partners 8.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Ottmann, C. et al. A structural rationale for selective stabilization of anti-tumor interactions of 14-3-3 proteins by cotylenin A. J. Mol. Biol. 386, 913–919 (2009). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Degradation of SALL4 leads to teratogenic effects while degradation of Ikaros is a proven chemotherapeutic strategy.

    “Paradoxically, degradation of the former leads to teratogenic effects while degradation of the latter is a proven chemotherapeutic strategy4,6.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Hughes, S. J. & Ciulli, A. Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem 61, 505–516 (2017). source

      Not checked in the free run.

    • cites [6] Milroy, L.-G., Grossmann, T. N., Hennig, S., Brunsveld, L. & Ottmann, C. Modulators of protein-protein interactions. Chem. Rev. 114, 4695–4748 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    UBR7 belongs to the UBR family of N-recognin E3 ligases, but unlike UBR1-6, contains a plant homeodomain rather than an F-box domain.

    “UBR7 belongs to the UBR family of N-recognin E3 ligases, but unlike UBR1–6, contains a plant homeodomain (PHD) rather than an F-box domain 41.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Kleiner, R. E., Hang, L. E., Molloy, K. R., Chait, B. T. & Kapoor, T. M. A chemical proteomics approach to reveal direct protein–protein interactions in living cells. Cell Chem. Biol. 25, 110–120.e3 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationcitation

    These alternate biological activities may also be mediated through additional molecular glue-type interactions beyond those reported here.

    “indicating that these alternate biological activities may also be mediated through additional molecular glue-type interactions beyond those reported here 40.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Tasaki, T. et al. The substrate recognition domains of the N-end rule pathway. J. Biol. Chem. 284, 1884–1895 (2009). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Rapamycin binds FKBP12, recruits mTORC1, and results in partial inhibition of mTORC1.

    “The natural product rapamycin binds to FKBP12, leading to the recruitment of mTORC1, resulting in partial inhibition of mTORC1 7.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Schreiber, S. L. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251, 283–287 (1991). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    UBR7 can ubiquitinylate histones and has a variety of interacting partners in other contexts.

    “It has been reported that UBR7 can ubiquitinylate histones and has a variety of interacting partners in other contexts 42.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Haritakun, R., Srikitikulchai, P., Khoyaiklang, P. & Isaka, M. Isariotins A–D, alkaloids from the insect pathogenic fungus Isaria tenuipes BCC 7831. J. Nat. Prod. 70, 1478–1480 (2007). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    UBR7 is annotated as an E3 ubiquitin ligase and postulated to be involved in histone ubiquitination.

    “While UBR7 is annotated as an E3 ubiquitin ligase and postulated to be involved in histone ubiquitination 27, its biochemical and physiological functions are poorly understood especially with regard to cancer cell proliferation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Goodwin, J. F. & Knudsen, K. E. Beyond DNA repair: DNA-PK function in cancer. Cancer Discov 4, 1126–1139 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    IsoTOP-ABPP is a well-validated method for covalent ligand target identification.

    “prior to further processing using a well-validated method for covalent ligand target identification—isotopic tandem orthogonal proteolysis ABPP (isoTOP-ABPP) 2,17,26.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [2] Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016). source

      Not checked in the free run.

    • cites [17] Sattler, I., Thiericke, R. & Zeeck, A. The manumycin-group metabolites. Nat. Prod. Rep. 15, 221–240 (1998). source

      Not checked in the free run.

    • cites [26] Adhikary, S. et al. Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor. Nat. Commun. 10, 1398 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Analogs of rapamycin and thalidomide can maintain binding to FKBP12 and cereblon respectively, but shift their neo-substrate binding profiles to confer new and distinct biological functions.

    “Moreover, analogs of rapamycin and thalidomide can still maintain binding to FKBP12 and cereblon respectively, but shift their neo-substrate binding profiles to confer new and distinct biological functions 13,14.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] Guo, Z. et al. Rapamycin-inspired macrocycles with new target specificity. Nat. Chem. 11, 254 (2019). source

      Not checked in the free run.

    • cites [14] Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, eaat0572 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Multi-covalent small-molecules can potentially act as new molecular glues and these interactions can be quickly deciphered using chemoproteomic approaches.

    “our study showcases the possibility that multi-covalent small-molecules can potentially act as new molecular glues and that these interactions can be quickly deciphered using chemoproteomic approaches.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    For probe-modified peptides with ratios >2, only targets present across all three biological replicates, statistically significant, and with good quality MS1 peak shapes across all biological replicates were interpreted.

    “For those probe-modified peptides that showed ratios >2, we only interpreted those targets that were present across all three biological replicates, were statistically significant, and showed good quality MS1 peak shapes across all biological replicates.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    ProLUCID data was filtered through DTASelect to achieve a peptide false-positive rate below 5%.

    “ProLUCID data was filtered through DTASelect to achieve a peptide false-positive rate below 5%.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Only probe-modified peptides evident across two out of three biological replicates were interpreted for their isotopic light to heavy ratios.

    “Only those probe-modified peptides that were evident across two out of three biological replicates were interpreted for their isotopic light to heavy ratios.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Differential abundance significance was estimated using a background-based ANOVA with Benjamini-Hochberg correction to determine adjusted p-values.

    “Differential abundance significance was estimated using a background-based ANOVA with Benjamini-Hochberg correction to determine adjusted p-values.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    High confidence protein identifications were reported using a Percolator estimated <1% false discovery rate (FDR) cut-off.

    “High confidence protein identifications were reported using a Percolator estimated <1% false discovery rate (FDR) cut-off.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Ashburner, M. et al. Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Statistical analysis for pathway enrichment was performed using the g:SCS algorithm for multiple testing correction of p-values from GO and pathway enrichment analysis, corresponding to an experiment-wide threshold of a=0.05.

    “Statistical 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.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [54] The Gene Ontology Consortium. The Gene Ontology resource: 20 years and still GOing strong. Nucleic Acids Res. 47, D330–D338 (2019). source

      Not checked in the free run.

    • cites [55] Raudvere, U. et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 47, W191–W198 (2019). source

      Not checked in the free run.

    • cites [56]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Data validation of peptide and protein identifications was done at the level of the complete dataset consisting of combined Mascot search results for all individual samples per experiment via the Percolator validation node in Proteome Discoverer.

    “Data validation of peptide and protein identifications was done at the level of the complete dataset consisting of combined Mascot search results for all individual samples per experiment via the Percolator validation node in Proteome Discoverer.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Ashburner, M. et al. Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    P-values were corrected using the Benjamini/Hochberg method.

    “P-values were corrected using the Benjamini/Hochberg method.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductcitation

    Under serum-free survival conditions, the cells do not proliferate, so the assay measures loss of viability under pharmacological treatment.

    “Under serum-free survival conditions, we have previously observed that the cells do not proliferate, and thus we are measuring loss of viability in these cells under serum-free conditions with pharmacological treatment 49.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] Xu, T. et al. ProLuCID: an improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J. Proteomics 129, 16–24 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Only peptide-to-spectrum matches that are unique assignments to a given identified protein within the total dataset are considered for protein quantitation.

    “Only peptide-to-spectrum matches that are unique assignments to a given identified protein within the total dataset are considered for protein quantitation.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    Under serum-containing conditions, the cells proliferate normally, so pharmacological effects in this assay indicate a combination of loss of proliferation and viability.

    “Under serum-containing conditions, the cells proliferate normally, and thus pharmacological effects in this assay indicate a combination of loss of proliferation and viability.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Only proteins that showed >2 peptides in at least one sample were subsequently interpreted.

    “Only those proteins that showed >2 peptides in at least one sample was subsequently interpreted.”
    Quote in the paperNot a substantial claim
64 of 64

  • The paper relies on this without support.

    231MFP cells are a representative TNBC line for mechanistic studies of asukamycin.

    “Given our interest in TNBCs, we focused our subsequent mechanistic studies for asukamycin in 231MFP cells, as a representative TNBC line.”

  • The paper relies on this without support.

    The p53 luciferase reporter assay specifically measures p53 transcriptional activity.

    “Asukamycin also significantly induced p53 luciferase reporter transcriptional activity in HEK293T cells, even more so than the positive control topoisomerase inhibitor doxorubicin (dox) that is known to induce apoptosis through activating the p53 pathway (Figure 4e).”

  • The paper relies on this without support.

    TP53AIP1 is a p53 target gene.

    “asukamycin induced the expression of the p53 target gene TP53AIP1 in 231MFP cells.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Streptomyces-derived manumycin polyketides asukamycin and manumycin A function as covalent MG degraders that engage UBR7 and TP53.

    “These natural products seem to function as covalent MG degraders that engage UBR7 (a putative E3) and TP53.101”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [101]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Reporter-assay treatment duration differs between caption and methods

    Major

    Extended Data Fig. 8 describes a three-hour asukamycin treatment for the TP53 reporter assay, whereas the Luciferase Reporter Assay method specifies six hours of compound treatment.

  • Cancer-type counts do not total 250 cell lines

    Minor

    The cancer-type counts in Extended Data Fig. 1 sum to 257 because liver cancer is listed twice. Counting liver cancer only once gives 249, still not the stated 250.

    Reported 250 · recomputed 257

  • Extended Data Fig. 3a cites the wrong main figure

    Minor

    The Extended Data caption says it quantifies a Western blot in Figure 2d, while the results identify Figure 3d as the blot associated with Extended Data Fig. 3a.

3 of 3

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Authors
Isobe, Y., Okumura, M., McGregor, L. M., Brittain, S. M., Jones, M. D., Liang, X., White, R., Forrester, W., McKenna, J. M., Tallarico, J. A., Schirle, M., Maimone, T. J., & Nomura, D. K.
Affiliations
  • University of California, Berkeley (US)
  • Innovative Genomics Institute (US)
Corresponding author
  • Maimone, Thomas J.
  • Nomura, Daniel K.
ORCID
Venue
Nature Chemical Biology
Publisher
Springer Science and Business Media LLC
Published
2020-06-22
Volume
16
Issue
11
Pages
1189-1198
ISSN
  • 1552-4450 (ISSN-L)
  • 1552-4469
DOI
10.1038/s41589-020-0557-2
PMC
PMC7572527
OpenAlex
W3036135201
Cited by
124
References
55
Citations by year
2026: 10 · 2025: 16 · 2024: 24 · 2023: 15 · 2022: 28 · 2021: 28 · 2020: 3
FWCI
6.16
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Chemical Biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • The Novartis Repository (Novartis) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | National Institutes of Health (R01CA240981)
  • Novartis Institutes for BioMedical Research, Mark Foundation for Cancer Research and Chordoma Foundation ASPIRE Award, Japanese Society for the Promotion of Science (JSPS) postdoctoral fellowships
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Keywords
molecular glues, TP53, E3 ubiquitin ligase, p53 transcriptional activation, breast cancer, chemoproteomics, anticancer mechanism, cell death
MeSH terms
Antineoplastic Agents, Breast Neoplasms, Cross-Linking Reagents, Female, Humans, Molecular Conformation, Polyenes, Structure-Activity Relationship, Molecular Structure, Gene Expression Regulation, Neoplastic, Tumor Suppressor Protein p53, Ubiquitin-Protein Ligases, Cell Line, Tumor, Polyunsaturated Alkamides, Static Electricity, Gene Knockdown Techniques, Drug Discovery, Polyketides

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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