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Cited by 92Protein Degradation and InhibitorsCRISPR and Genetic EngineeringChemical Synthesis and Analysis

Advances in targeted degradation of endogenous proteins

Open access
Röth, Sascha, Fulcher, Luke J., Sapkota, Gopal P.·2019·Cellular and Molecular Life Sciences
DOI 10.1007/s00018-019-03112-6PMC PMC6588652
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Abstract

Protein silencing is often employed as a means to aid investigations in protein function and is increasingly desired as a therapeutic approach. Several types of protein silencing methodologies have been developed, including targeting the encoding genes, transcripts, the process of translation or the protein directly. Despite these advances, most silencing systems suffer from limitations. Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible. Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Targeted proteolysis has the potential to overcome some of these limitations. The field of targeted proteolysis has witnessed the emergence of many methodologies aimed at targeting specific proteins for degradation in a spatio-temporal manner. In this review, we provide an appraisal of the different targeted proteolytic systems and discuss their applications in understanding protein function, as well as their potential in therapeutics.

AI snapshot

Written from the paper’s full text.

TL;DR

This review appraises targeted proteolysis methods for degrading endogenous proteins, contrasting them with CRISPR/Cas9 knockout and RNAi. It surveys degron-, nanobody-, and small-molecule-based systems and discusses their research and therapeutic potential.

Key findings

  • Targeted proteolysis directly removes proteins via the ubiquitin–proteasome system while leaving transcription and translation unaffected; approaches include AiD, FKBP12/UnaG degrons, SMASh-tag, TRIM-away, AdPROM, deGradFP/ZIF1, hydrophobic tagging, PROTACs, and SNIPERs.
  • Several systems achieve rapid degradation: TRIM-away depleted GFP with an apparent half-life of roughly 16 min and degraded Rec8 in mouse oocytes within minutes; CM11 depleted VHL30 within 4 h at 10 nM; A1874 degraded BRD4 almost completely in HCT116 cells at 100 nM within 24 h.
  • Some systems have dose or kinetic constraints: RNAi is usually effective only after 48–72 h; SMASh-tag stability is tuneable with asunaprevir from 0.15 nM to 1.5 µM; HyT13 degraded GFP-HALO 50% in 8 h at 1 µM; peptide 1 degraded overexpressed GFP-Tau at 20 µM after 6–12 h.
  • The review notes that only a handful of E3 ubiquitin ligases have been used despite over 600 encoded in the human genome, and that developing PROTACs, SNIPERs, and IMiDs remains costly and time-consuming.

Methods

  • Narrative review; no primary experimental data are reported. It appraises published targeted proteolytic systems and their mechanisms, benefits, and limitations.
  • Discusses CRISPR/Cas9 genome editing, RNAi, and multiple targeted degradation strategies, citing examples from cell lines, primary cells, and model organisms.
  • Includes a table compiling reported high-affinity polypeptide binders—nanobodies, monobodies, DARPins, and Affimer/Affibody—for intracellular proteins.

Limitations

  • Stated: AiD requires inserting the IAA degron at the POI locus and is challenging in metazoan cells; TIR1 overexpression off-target effects need delineation; auxin/IAA can be toxic at high doses.
  • Stated: many systems require POI tagging (e.g., GFP, HALO, dTAG) or selective binders; tagging can compromise POI structure, localization, or activity; PROTAC/SNIPER/IMiD development is costly and time-consuming; only a handful of E3 ligases are used; AdPROM has not yet been applied in whole organisms.
  • Evident: as a narrative review, it does not provide systematic search criteria, quantitative synthesis, or primary validation, and its coverage is selective rather than exhaustive.

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

Integrity signals

Registry

Crossref · 10.1007/s00018-019-03112-6

Cites

167 of 167 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 144 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    Exchanging the HALO reactive linker for the Tau-interacting peptide (C-terminal region of tubulin) induced efficient degradation of Tau.

    “In an attempt to make the system more applicable and remove the need to create HALO-fusions, the HALO reactive linker was exchanged for the Alzheimer’s disease-related protein Tau-interacting peptide (the C-terminal region of tubulin). This induced efficient degradation of Tau [60].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [60] Gao N, Chu T, Li Q et al (2017) Hydrophobic tagging-mediated degradation of Alzheimer’ s disease related Tau. RSC Adv 7:40362–40366. https://doi.org/10.1039/C7RA05347A source

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  • Findingcitation

    The ZIF1 system was adapted to create a deGradFP system.

    “More recently, the ZIF1 system was also adapted to create a deGradFP system [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Caussinus E, Kanca O, Affolter M (2013) Protein knockouts in living eukaryotes using deGradFP and green fluorescent protein fusion targets. In: Current protocols in protein science. Wiley, Hoboken, pp 30.2.1–30.2.13

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  • Findingcitation

    deGradFP, a fusion of an anti-GFP nanobody with the F-box domain from Drosophila melanogaster Slmb, efficiently degrades GFP-tagged proteins in D. melanogaster.

    “One such approach, named deGradFP, utilized a fusion of an anti-GFP nanobody with the F-box domain from the Drosophila melanogaster Slmb protein, which is a substrate receptor component of the SCF E3 ligase complex, and demonstrated efficient degradation of GFP-tagged proteins in D. melanogaster (Fig. 3b) [50, 51].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [50] Wang S, Tang NH, Lara-Gonzalez P et al (2017) A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans. Development 144:2694–2701. https://doi.org/10.1242/dev.150094 source

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    • cites [51] Caussinus E, Kanca O, Affolter M (2012) Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nat Struct Mol Biol 19:117–121. https://doi.org/10.1038/nsmb.2180 source

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  • Findingcitation

    HALO-tagged fusions of transmembrane proteins with differing amounts of membrane spanning helices could be degraded by the HyT system.

    “Remarkably, HALO-tagged fusions of transmembrane proteins with differing amounts of membrane spanning helices could be degraded by this system as well [56].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [56] Neklesa TK, Tae HS, Schneekloth AR et al (2011) Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat Chem Biol 7:538–543. https://doi.org/10.1038/nchembio.597 source

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  • Findingcitation

    ZIF1 itself is regulated in different stages of development.

    “ZIF1 itself is regulated in different stages of development [53]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] DeRenzo C, Reese KJ, Seydoux G (2003) Exclusion of germ plasm proteins from somatic lineages by cullin-dependent degradation. Nature 424:685–689. https://doi.org/10.1038/nature01887 source

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  • May combine two claims. 45
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    Findingcitation

    Treatment of cells expressing GFP-HALO with HyT13 resulted in 50% degradation in 8 h at 1 μM, without notable toxicity even at concentrations up to 20 μM.

    “Treatment of cells expressing GFP-HALO with this compound (HyT13) resulted in efficient degradation (50% in 8 h at 1 µM), without notable toxicity even at concentrations up to 20 µM [56].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [56] Neklesa TK, Tae HS, Schneekloth AR et al (2011) Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat Chem Biol 7:538–543. https://doi.org/10.1038/nchembio.597 source

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    Findingcitation

    The first PROTAC efficiently degraded MetAP-2 in Xenopus oocyte extracts, but it was not cell permeable.

    “This molecule efficiently degraded MetAP-2 in Xenopus oocyte extracts, however it was not cell permeable [62].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [62] Sakamoto KM, Kim KB, Kumagai A et al (2001) Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc Natl Acad Sci 98:8554–8559. https://doi.org/10.1073/pnas.141230798 source

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    Findingcitation

    Target-specific docking sites can be presented by thalidomide derivatives, as with CC-885 and GSPT1, and pomalidomide and IKZF1 or ZNF692.

    “However, target-specific docking sites can be presented by the thalidomide derivative, as is the case with CC-885 and GSPT1 [77], as well as pomalidomide and the zinc finger proteins IKZF1 or ZNF692 [78].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [77] Matyskiela ME, Lu G, Ito T et al (2016) A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535:252–257. https://doi.org/10.1038/nature18611 source

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    • cites [78] Sievers QL, Petzold G, Bunker RD et al (2018) Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362:eaat0572. https://doi.org/10.1126/science.aat0572 source

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    Findingcitation

    c-Myc levels were strongly depleted, and this depletion lasted longer after washout of the PROTAC than after inhibitor treatment.

    “Concomitantly, c-Myc levels were strongly depleted, and this depletion lasted longer after washout of the PROTAC than after inhibitor treatment [84].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [84] Lu J, Qian Y, Altieri M et al (2015) Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem Biol 22:755–763. https://doi.org/10.1016/j.chembiol.2015.05.009 source

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    Findingcitation

    A1874 treatment led to stabilization of p53 and had an antiproliferative effect that was synergistic compared to treatments with either inhibitor alone.

    “Additionally, PROTAC treatment led to the stabilization of p53 and had an antiproliferative effect that was synergistic when compared to treatments with either inhibitor alone [68].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [68] Hines J, Lartigue S, Dong H et al (2018) MDM2-recruiting PROTAC offers superior, synergistic anti-proliferative activity via simultaneous degradation of BRD4 and stabilization of p53. Cancer Res. https://doi.org/10.1158/0008-5472.can-18-2918 source

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    Findingcitation

    Both ALK PROTACs retained inherent off-target binding of Aurora kinase A, as it too was degraded.

    “However, both PROTACs retained their inherent off-target binding of Aurora kinase A, as it too was degraded [79].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [79] Powell CE, Gao Y, Tan L et al (2018) Chemically induced degradation of anaplastic lymphoma kinase (ALK). J Med Chem 61:4249–4255. https://doi.org/10.1021/acs.jmedchem.7b01655 source

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    Findingcitation

    Switching the HALO reactive linker with a high-affinity agonist for the androgen receptor, RU59063, or with an inhibitor against HER3, TX1-85-1, achieved similar degradation results.

    “A similar result was achieved when the HALO reactive linker was switched with a high-affinity agonist for the androgen receptor, RU59063 [57], or with a newly developed inhibitor against the EGFR tyrosine kinase HER3, TX1-85-1 [61].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [57] Gustafson JL, Neklesa TK, Cox CS et al (2015) Small-molecule-mediated degradation of the androgen receptor through hydrophobic tagging. Angew Chem Int Ed 54:9659–9662. https://doi.org/10.1002/anie.201503720 source

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    • cites [61] Xie T, Lim SM, Westover KD et al (2014) Pharmacological targeting of the pseudokinase Her3. Nat Chem Biol 10:1006–1012. https://doi.org/10.1038/nchembio.1658 source

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    Findingcitation

    Dasatinib-VHL PROTAC degraded c-Abl at 1 μM, but higher concentrations left c-Abl protein levels unaffected.

    “Interestingly, c-Abl could be degraded with a dasatinib–VHL PROTAC at 1 µM, however, higher concentrations left c-Abl protein levels unaffected [86].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [86] Lai AC, Toure M, Hellerschmied D et al (2016) Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew Chem Int Ed 55:807–810. https://doi.org/10.1002/anie.201507634 source

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    Findingcitation

    CC-885 maintains binding and degradation capability for IKZF1 and targets GSPT1 for degradation.

    “While maintaining the binding and degradation capability for IKZF1, CC-885 targeted GSPT1, a translation termination factor, for degradation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [77] Matyskiela ME, Lu G, Ito T et al (2016) A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535:252–257. https://doi.org/10.1038/nature18611 source

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    Findingcitation

    VHL-PROTACs binding BCR-Abl inhibited downstream signalling, but protein levels remained unchanged.

    “While binding of the PROTACs to BCR–Abl still inhibited downstream signalling, protein levels remained unchanged with any of the tested VHL-PROTACs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [86] Lai AC, Toure M, Hellerschmied D et al (2016) Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew Chem Int Ed 55:807–810. https://doi.org/10.1002/anie.201507634 source

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    Findingcitation

    A VHL-recruiting BRD4 PROTAC exhibited a clear preference for BRD4, but BRD2 and 3 were also degraded at either high concentrations or prolonged treatments.

    “While the PROTAC exhibited a clear preference for BRD4, BRD2 and 3 were also degraded at either high concentrations or prolonged treatments [94].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [94] Zengerle M, Chan KH, Ciulli A (2015) Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem Biol 10:1770–1777. https://doi.org/10.1021/acschembio.5b00216 source

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    Findingcitation

    Bosutinib- and dasatinib-pomalidomide PROTACs efficiently degrade both BCR-Abl and c-Abl at around 100 nM concentrations, and the dasatinib-pomalidomide PROTAC reduces cell viability with a 1000-fold higher efficiency over non-BCR-Abl driven cell lines.

    “In contrast, both bosutinib- and dasatinib–pomalidomide PROTACs were able to efficiently degrade both BCR–Abl and c-Abl at around 100 nM concentrations and the dasatinib–pomalidomide PROTAC was efficient at reducing cell viability with a 1000-fold higher efficiency over non-BCR–Abl driven cell lines [86].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [86] Lai AC, Toure M, Hellerschmied D et al (2016) Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew Chem Int Ed 55:807–810. https://doi.org/10.1002/anie.201507634 source

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    Findingcitation

    SNIPERs that target CRABP-2, estrogen receptor alpha (ERα), and transforming acidic coiled-coil-3 (TACC3) have been generated, and in each case, successful degradation of the target protein has been demonstrated.

    “To date, SNIPERs that target CRABP-2 [101], estrogen receptor alpha (ERα) [103], and transforming acidic coiled-coil-3 (TACC3) [104] have been generated, and in each case, successful degradation of the target protein has been demonstrated.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [101] Okuhira K, Ohoka N, Sai K et al (2011) Specific degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein. FEBS Lett 585:1147–1152. https://doi.org/10.1016/j.febslet.2011.03.019 source

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    • cites [103] Ohoka N, Morita Y, Nagai K et al (2018) Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation. J Biol Chem 293:6776–6790. https://doi.org/10.1074/jbc.RA117.001091 source

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    • cites [104] Ohoka N, Nagai K, Hattori T et al (2014) Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin–proteasome pathway. Cell Death Dis 5:e1513. https://doi.org/10.1038/cddis.2014.471 source

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    Findingcitation

    A new PROTAC with MV1 induced efficient degradation of both c-IAP1 and CRABP-2 and blocked proliferation of neuroblastoma cells; however, effects on either c-IAP2 or XIAP were not analysed.

    “Treatment of cells with this new PROTAC also induced efficient degradation of both c-IAP1 and CRABP-2 and blocked proliferation of neuroblastoma cells; however, the effects on either c-IAP2 or XIAP were not analysed [100].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [100] Itoh Y, Ishikawa M, Kitaguchi R et al (2012) Double protein knockdown of cIAP1 and CRABP-II using a hybrid molecule consisting of ATRA and IAPs antagonist. Bioorg Med Chem Lett 22:4453–4457. https://doi.org/10.1016/J.BMCL.2012.04.134 source

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    Findingcitation

    HaloPROTACs bind to the HALO tag on POIs on one side and VHL on the other, and have been successfully employed to rapidly and inducibly degrade HALO-tagged POIs through the VHL/CUL2/RBX1-dependent UPS.

    “HaloPROTACs, which bind to the HALO tag on POIs on one side and VHL on the other, have been successfully employed to rapidly and inducibly degrade HALO-tagged POIs through the VHL/CUL2/RBX1-dependent UPS [109].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [109] Buckley DL, Raina K, Darricarrere N et al (2015) HaloPROTACS: use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins. ACS Chem Biol 10:1831–1837. https://doi.org/10.1021/acschembio.5b00442 source

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    Findingcitation

    dTAG-13 successfully degraded dTAG-BRD4 fusion proteins generated by CRISPR/Cas9 gene editing, and showed selective, rapid degradation of dTAG-BRD4 upon ligand treatment.

    “This approach proved successful in degrading dTAG–BRD4 fusion proteins generated by CRISPR/Cas9 gene editing, and showed selective, rapid degradation of dTAG–BRD4 upon ligand treatment [108].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [108] Nabet B, Roberts JM, Buckley DL et al (2018) The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14:431–441. https://doi.org/10.1038/s41589-018-0021-8 source

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    Findingcitation

    Generation of the full-length peptide decreased Keap1 affinity tenfold and exhibited affinity towards Tau in the sub-micromolar range.

    “Generation of the full-length peptide decreased Keap1 affinity tenfold and exhibited affinity towards Tau in the sub-micromolar range.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [105] Lu M, Liu T, Jiao Q et al (2018) Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway. Eur J Med Chem 146:251–259. https://doi.org/10.1016/j.ejmech.2018.01.063 source

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    Findingcitation

    MDM2 degradation and p53 stabilization could not be achieved with a VHL warhead.

    “Similar to BCR–Abl, this could not be achieved by the use of a VHL warhead [87].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Li Y, Yang J, Aguilar A et al (2019) Discovery of MD-224 as a first-in-class, highly potent, and efficacious proteolysis targeting chimera murine double minute 2 degrader capable of achieving complete and durable tumor regression. J Med Chem 62(2):448–466. https://doi.org/10.1021/acs.jmedchem.8b00909 source

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    Findingcitation

    dTAG-13 is a heterobifunctional PROTAC that recruits dTAG to CRBN, and thereby degrades dTAG-fused POIs.

    “The authors exploited an FKBP12-F36V-directed ligand called AP1867 (Ariad Pharmaceuticals) to generate a heterobifunctional PROTAC dTAG-13 that recruits dTAG to CRBN, and thereby degrades dTAG-fused POIs [108].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [108] Nabet B, Roberts JM, Buckley DL et al (2018) The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14:431–441. https://doi.org/10.1038/s41589-018-0021-8 source

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    Findingcitation

    VHL PROTACs targeting TBK1 and ALK achieved positive outcomes; for ALK, a clear correlation between efficacy in inhibiting cell proliferation and ALK driver status in different cell lines was observed.

    “Similar positive outcomes could be achieved with PROTACs targeting TANK-binding kinase 1 (TBK1) [92], and ALK, where a clear correlation between efficacy in inhibiting cell proliferation and ALK driver status in different cell lines could be observed [93].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [92] Crew AP, Raina K, Dong H et al (2018) Identification and characterization of von Hippel-Lindau-recruiting proteolysis targeting chimeras (PROTACs) of TANK-binding kinase 1. J Med Chem 61:583–598. https://doi.org/10.1021/acs.jmedchem.7b00635 source

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    • cites [93] Kang CH, Lee DH, Lee CO et al (2018) Induced protein degradation of anaplastic lymphoma kinase (ALK) by proteolysis targeting chimera (PROTAC). Biochem Biophys Res Commun 505:542–547. https://doi.org/10.1016/J.BBRC.2018.09.169 source

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    Findingcitation

    MD-222 and MD-224 efficiently degrade MDM2, stabilize p53, and inhibit cell proliferation at low nanomolar doses.

    “These PROTACs are very efficient at degrading MDM2 and, in turn, stabilize p53 and inhibit cell proliferation at low nanomolar doses.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [87] Li Y, Yang J, Aguilar A et al (2019) Discovery of MD-224 as a first-in-class, highly potent, and efficacious proteolysis targeting chimera murine double minute 2 degrader capable of achieving complete and durable tumor regression. J Med Chem 62(2):448–466. https://doi.org/10.1021/acs.jmedchem.8b00909 source

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    Findingcitation

    A peptidic ligand molecule with an IC50 value of 4.1 μM for VHL binding at the HIF1α binding site was developed, and could be improved to sub-micromolar affinity.

    “This hydroxyproline residue was the starting point for a peptidic ligand molecule with an IC50 value of 4.1 µM for VHL binding at the HIF1α binding site [89], which could be improved to sub-micromolar affinity [90].”
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    • cites [89] Buckley DL, Van Molle I, Gareiss PC et al (2012) Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the vHL/HIF-1α interaction. J Am Chem Soc 134:4465–4468. https://doi.org/10.1021/ja209924v source

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    • cites [90] Buckley DL, Gustafson JL, Van Molle I et al (2012) Small-molecule inhibitors of the interaction between the E3 Ligase VHL and HIF1α. Angew Chem Int Ed 51:11463–11467. https://doi.org/10.1002/anie.201206231 source

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    Findingcitation

    dTAG-13 degradation rates vary depending on the POI used, possibly owing to limited accessibility of the dTAG ligand in different subcellular compartments.

    “However, the authors noted varying rates of POI degradation, depending on the POI used, possibly owing to limited accessibility of the dTAG ligand in different subcellular compartments [108].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [108] Nabet B, Roberts JM, Buckley DL et al (2018) The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14:431–441. https://doi.org/10.1038/s41589-018-0021-8 source

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    Findingcitation

    ARV-110, an androgen receptor degrader PROTAC, has shown pre-clinical efficacy against prostate cancer and is now a candidate for clinical trials.

    “Indeed, some PROTACs, such as the androgen receptor degrader PROTAC, ARV-110, has shown pre-clinical efficacy against prostate cancer [120] and is now a candidate for clinical trials [121].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [120] Neklesa T, Snyder LB, Willard RR, et al (2018) Abstract 5236: ARV-110: an androgen receptor PROTAC degrader for prostate cancer. In: Cancer Research. American Association for Cancer Research, pp 5236–5236 source

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    • cites [121] Mullard A (2019) First targeted protein degrader hits the clinic. In: Nat Rev Drug Discov. http://www.nature.com/articles/d41573-019-00043-6 . Accessed 19 Mar 2019 source

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    Premisecitation

    The AID degron sequence was successfully knocked into the genes encoding cohesin and cytoplasmic dynein in human colorectal cancer HCT116 cells, and IAA-responsive degradation was demonstrated following TIR1 over-expression.

    “With advances in CRISPR/Cas9 gene editing technologies, the AID degron sequence was successfully knocked into the genes encoding the cohesin and cytoplasmic dynein proteins in human colorectal cancer HCT116 cells. Following TIR1 over-expression, IAA-responsive cohesin and dynein degradation were demonstrated [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT (2016) Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep 15:210–218. https://doi.org/10.1016/J.CELREP.2016.03.001 source

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    Premisecitation

    CRISPR mutagenesis can result in exon skipping, generating a different start codon, resulting in truncations of the protein still being translated.

    “However, mutagenesis by CRISPR can result in exon skipping, generating a different start codon, resulting in truncations of the protein still being translated [3].”
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    • cites [3] Sharpe JJ, Cooper TA (2017) Unexpected consequences: exon skipping caused by CRISPR-generated mutations. Genome Biol 18:109. https://doi.org/10.1186/s13059-017-1240-0 source

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    Premisecitation

    UnaG-mediated degradation completely degrades fusion proteins 4 h after removal of BR from the growth medium, and the system exhibits dose-dependent degradation.

    “Studies on the kinetics of UnaG-mediated degradation have shown that fusion proteins were completely degraded 4 h after removal of BR from the growth medium. Additionally, the system’s mechanism exhibited dose-dependent degradation of fusion proteins [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Navarro R, Chen LC, Rakhit R, Wandless TJ (2016) A novel destabilizing domain based on a small-molecule dependent fluorophore. ACS Chem Biol 11:2101–2104. https://doi.org/10.1021/acschembio.6b00234 source

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    Premisecitation

    Targeted proteolysis by AdPROM is dependent on both proteasomal activity and cullin NEDDylation.

    “The targeted proteolysis by AdPROM is dependent on both proteasomal activity and cullin NEDDylation [18, 19].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [18] Fulcher LJ, Macartney T, Bozatzi P et al (2016) An affinity-directed protein missile system for targeted proteolysis. Open Biol 6:160255. https://doi.org/10.1098/rsob.160255 source

      Not checked in the free run.

    • cites [19] Fulcher LJ, Hutchinson LD, Macartney TJ et al (2017) Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open Biol 7:170066. https://doi.org/10.1098/rsob.170066 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Following microinjection of a target-specific antibody, the POI is recognized by TRIM21, ubiquitinated and rapidly degraded.

    “Following microinjection of a target-specific antibody, the POI is recognized by TRIM21, ubiquitinated and rapidly degraded (Fig. 3a) [40].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Clift D, McEwan WA, Labzin LI et al (2017) A method for the acute and rapid degradation of endogenous proteins. Cell 171:1692.e18–1706.e18. https://doi.org/10.1016/j.cell.2017.10.033 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Upon treatment with asunaprevir, the SMASh-tag degron remains attached to the target protein, leading to rapid POI degradation.

    “Upon treatment with specific protease inhibitors, like asunaprevir, which target the NS3 active site, the degron remains attached to the target protein leading to rapid POI degradation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Chung HK, Jacobs CL, Huo Y et al (2015) Tunable and reversible drug control of protein production via a self-excising degron. Nat Chem Biol 11:713–720. https://doi.org/10.1038/nchembio.1869 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    TRIM21 can target all accessible cellular proteins, although for nuclear-localized proteins, a target-specific nanobody has to be fused to Fc fragments.

    “TRIM21 was able to target all accessible cellular proteins, although for nuclear-localized proteins, a target-specific nanobody has to be fused to Fc fragments due to the full-length antibody being too large to pass through the nuclear pore complex.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Clift D, McEwan WA, Labzin LI et al (2017) A method for the acute and rapid degradation of endogenous proteins. Cell 171:1692.e18–1706.e18. https://doi.org/10.1016/j.cell.2017.10.033 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The C. elegans SOCS-Box protein ZIF1 recruits proteins containing a 36 amino acid ZF1 motif and targets them for degradation.

    “The C. elegans SOCS-Box protein ZIF1 recruits proteins containing a 36 amino acid ZF1 motif and targets them for degradation [52].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Armenti ST, Lohmer LL, Sherwood DR, Nance J (2014) Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins. Development 141:4640–4647. https://doi.org/10.1242/dev.115048 source

      Not checked in the free run.

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

    Premisecitation

    Unfolded proteins are bound by molecular chaperones, which prevent their aggregation and help them regain their original folding.

    “Unfolded proteins are bound by molecular chaperones, which prevent their aggregation and help them regain their original folding [55].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [55] Balchin D, Hayer-Hartl M, Hartl FU (2016) In vivo aspects of protein folding and quality control. Science 353:aac4354. https://doi.org/10.1126/science.aac4354 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Expression of AdPROM does not appear to interfere with hypoxia signalling, as evidenced by no detectable stabilization of HIF1α in AdPROM-expressing cells.

    “Crucially, despite VHL being the substrate adaptor for endogenous hypoxia-inducible factor 1α (HIF1α) [49], expression of AdPROM does not appear to interfere with hypoxia signalling, as evidenced by no detectable stabilization of HIF1α in AdPROM-expressing cells [18, 19].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [18] Fulcher LJ, Macartney T, Bozatzi P et al (2016) An affinity-directed protein missile system for targeted proteolysis. Open Biol 6:160255. https://doi.org/10.1098/rsob.160255 source

      Not checked in the free run.

    • cites [19] Fulcher LJ, Hutchinson LD, Macartney TJ et al (2017) Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open Biol 7:170066. https://doi.org/10.1098/rsob.170066 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Fusion of the UnaG degron to either terminus elicited degradation of the fusion proteins by the 26S proteasome, and this degradation could be halted by BR treatment.

    “Notably, fusion of the UnaG degron to either terminus elicited degradation of the fusion proteins by the 26S proteasome, and this degradation could be halted by BR treatment [22].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [22] Navarro R, Chen LC, Rakhit R, Wandless TJ (2016) A novel destabilizing domain based on a small-molecule dependent fluorophore. ACS Chem Biol 11:2101–2104. https://doi.org/10.1021/acschembio.6b00234 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Getting large amounts of pure and highly selective IgG antibodies into target cells is challenging and costly, making TRIM-away less feasible than other proteolytic methods for large-scale applications.

    “However, getting large amounts of pure and highly selective IgG antibodies into target cells is not only challenging, but extremely costly and therefore makes TRIM-away not as feasible as other proteolytic methods, especially when considering large-scale applications.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    MDM2 is one of the most upregulated E3 ligases in cancer, leading to constant removal of p53.

    “However, MDM2 is one of the most upregulated E3 ligases in cancer, leading to constant removal of p53 [63–65].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [63] Niazi S, Purohit M, Niazi JH (2018) Role of p53 circuitry in tumorigenesis: a brief review. Eur J Med Chem 158:7–24. https://doi.org/10.1016/J.EJMECH.2018.08.099 source

      Not checked in the free run.

    • cites [64] Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387:296–299. https://doi.org/10.1038/387296a0 source

      Not checked in the free run.

    • cites [65] Honda R, Tanaka H, Yasuda H (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 420:25–27. https://doi.org/10.1016/S0014-5793(97)01480-4 source

      Not checked in the free run.

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

    Premisecitation

    Thalidomide and its derivatives, e.g. lenalidomide and pomalidomide, are used as immunomodulatory imide drugs (IMiDs) in the treatment of several diseases, including ENL, multiple myeloma, MDS, systemic lupus erythematosus and inflammatory bowel disease.

    “Nevertheless, thalidomide and its derivatives, e.g. lenalidomide and pomalidomide, have been used for a newfound purpose in their immunomodulatory imide drug (IMiD) function and are applied in the treatment of several diseases, including erythema nodosum leprosum (ENL), multiple myeloma, myelodysplastic syndrome (MDS), systemic lupus erythematosus and inflammatory bowel disease [75, 76].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [75] Millrine D, Kishimoto T (2017) a brighter side to thalidomide: its potential use in immunological disorders. Trends Mol Med 23:348–361. https://doi.org/10.1016/j.molmed.2017.02.006 source

      Not checked in the free run.

    • cites [76] Sheskin J (1965) Thalidomide in the treatment of lepra reactions. Clin Pharmacol Ther 6:303–306. https://doi.org/10.1002/cpt196563303 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Thalidomide displaces endogenous CRBN substrates such as MEIS2, perhaps explaining its teratogenic effects in pregnant women.

    “However, thalidomide displaces endogenous CRBN substrates, such as the transcription factor MEIS2 which is involved in embryonic development, perhaps explaining its teratogenic effects in pregnant women [69, 73–75].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [69] Fischer ES, Böhm K, Lydeard JR et al (2014) Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512:49–53. https://doi.org/10.1038/nature13527 source

      Not checked in the free run.

    • cites [73] Cecconi F, Proetzel G, Alvarez-Bolado G et al (1997) Expression of Meis2, a Knotted-related murine homeobox gene, indicates a role in the differentiation of the forebrain and the somitic mesoderm. Dev Dyn 210:184–190. https://doi.org/10.1002/(SICI)1097-0177(199710)210:2<184::AID-AJA10>3.0.CO;2-E source

      Not checked in the free run.

    • cites [74] Ito T, Ando H, Suzuki T et al (2010) Identification of a primary target of thalidomide teratogenicity. Science 327:1345–1350. https://doi.org/10.1126/science.1177319 source

      Not checked in the free run.

    • cites [75] Millrine D, Kishimoto T (2017) a brighter side to thalidomide: its potential use in immunological disorders. Trends Mol Med 23:348–361. https://doi.org/10.1016/j.molmed.2017.02.006 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MDM2 binds the p53 transactivation domain, leading to inhibition of p53 function, nuclear export and degradation under physiological conditions.

    “MDM2 binds the p53 transactivation domain, leading to the inhibition of p53 function, nuclear export and degradation under physiological conditions.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [63] Niazi S, Purohit M, Niazi JH (2018) Role of p53 circuitry in tumorigenesis: a brief review. Eur J Med Chem 158:7–24. https://doi.org/10.1016/J.EJMECH.2018.08.099 source

      Not checked in the free run.

    • cites [64] Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387:296–299. https://doi.org/10.1038/387296a0 source

      Not checked in the free run.

    • cites [65] Honda R, Tanaka H, Yasuda H (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 420:25–27. https://doi.org/10.1016/S0014-5793(97)01480-4 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MDM2 is an oncoprotein with E3 ligase activity that targets the tumour suppressor p53 for ubiquitination.

    “Mouse double minute 2 homolog (MDM2) is an oncoprotein with E3 ligase activity that targets the tumour suppressor p53 for ubiquitination.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [63] Niazi S, Purohit M, Niazi JH (2018) Role of p53 circuitry in tumorigenesis: a brief review. Eur J Med Chem 158:7–24. https://doi.org/10.1016/J.EJMECH.2018.08.099 source

      Not checked in the free run.

    • cites [64] Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387:296–299. https://doi.org/10.1038/387296a0 source

      Not checked in the free run.

    • cites [65] Honda R, Tanaka H, Yasuda H (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 420:25–27. https://doi.org/10.1016/S0014-5793(97)01480-4 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Keap1 functions as a substrate receptor unit for the cullin3-RBX1 complex, targeting Nrf2 for ubiquitination under physiological conditions, while oxidative stress inactivates Keap1, leading to accumulation of active Nrf2.

    “Keap1 functions as a substrate receptor unit for the cullin3–RBX1 complex, targeting nuclear factor erythroid 2 related factor 2 (Nrf2) for ubiquitination under physiological conditions, while oxidative stress inactivates Keap1, leading to accumulation of active Nrf2 [106].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [106] Lu M-C, Ji J-A, Jiang Z-Y, You Q-D (2016) The Keap1-Nrf2-ARE pathway as a potential preventive and therapeutic target: an update. Med Res Rev 36:924–963. https://doi.org/10.1002/med.21396 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    Tagging proteins can compromise the structural integrity, subcellular distribution and biological activity of the POI itself.

    “Moreover, tagging proteins can compromise the structural integrity, subcellular distribution and biological activity of the POI itself.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Tagging proteins can compromise the structural integrity, subcellular distribution and biological activity of the POI itself.

  • May combine two claims.

    Interpretationnone given

    Most targeted proteolytic systems that require tagging of the POI with a polypeptide tag using CRISPR/Cas9 are useful for understanding POI function, but are limited in scope to the cell systems in which CRISPR/Cas9 modifications are feasible.

    “Most of the targeted proteolytic systems that require tagging of the POI with a polypeptide tag, such as GFP or dTAG, using CRISPR/Cas9 are useful for understanding POI function, but are limited in scope to the cell systems in which CRISPR/Cas9 modifications are feasible.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    High-affinity binders are already used for immunoprecipitation and immunofluorescence applications.

    “While high-affinity binders are already used for immunoprecipitation and immunofluorescence applications [117], their use with AdPROM and AdPROM-like technologies will vastly expand the toolkit for researchers.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [117] Rothbauer U, Zolghadr K, Tillib S et al (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3:887–889. https://doi.org/10.1038/nmeth953 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    These small polypeptide scaffolds consist of a stable backbone and variable loop regions, which determine POI-binding specificity.

    “These small polypeptide scaffolds consist of a stable backbone and variable loop regions, which determine POI-binding specificity.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [116] Banta S, Dooley K, Shur O (2013) Replacing antibodies: engineering new binding proteins. Annu Rev Biomed Eng 15:93–113. https://doi.org/10.1146/annurev-bioeng-071812-152412 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    For some proteins, it might transpire that the UPS is not able to cause proteolysis of a POI despite its ubiquitination.

    “For some proteins, it might transpire that the UPS is not able to cause proteolysis of a POI despite its ubiquitination.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationnone given

    Developing small molecule PROTACs, SNIPERs and IMiDs to degrade specific POIs remains extremely challenging, as the generation of high-affinity and selective POI-binding ligands that can be fused with the E3-binding ligands is costly and time-consuming.

    “Developing small molecule PROTACs, SNIPERs and IMiDs to degrade specific POIs remains extremely challenging, as the generation of high-affinity and selective POI-binding ligands that can be fused with the E3-binding ligands is costly and time-consuming.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developing selective high-affinity binders of POIs is both resource- and time-intensive process.

  • May combine two claims.

    Interpretationreasoning

    The large HALO tag has the potential of affecting the function of the POI by obstructing key interactors or substrates from accessing the POI, and in certain cases, the HALO tag itself might become the primary target of ubiquitination and degradation.

    “This has the potential of affecting the function of the POI by obstructing key interactors, or substrates from accessing the POI, and in certain cases, the HALO tag itself might become the primary target of ubiquitination and degradation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Nanobody-based targeted proteolytic systems, such as AdPROM, can rapidly address the druggability of any given target in different disease models.

    “Even in the absence of robust PROTAC molecules to target a wide range of so-called undruggable POIs, nanobody-based targeted proteolytic systems, such as AdPROM, can rapidly address the druggability of any given target in different disease models.”
    Quote in the paperNot a substantial claim
50 of 50

  • The paper relies on this without support.

    The SCF degradation pathway is conserved across eukaryotes.

    “Whilst other eukaryotes lack this auxin-inducible response, the SCF degradation pathway is conserved, thereby raising the tantalizing possibility that the auxin-inducible degradation pathway could be transferred into other eukaryotic cells.”

  • The paper relies on this without support.

    CRISPR/Cas9 genome editing is irreversible.

    “Silencing protein expression through genetic ablation, for example by CRISPR/Cas9 genome editing, is irreversible, time consuming and not always feasible.”

  • The paper relies on this without support.

    RNAi approaches warrant prolonged treatments.

    “Similarly, RNA interference approaches warrant prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects.”

  • The paper relies on this without support.

    For any targeted proteolytic system to work effectively, the POI when recruited to the E3 ligase component has to present lysine (or other) residues for ubiquitination.

    “For any targeted proteolytic system to work effectively, the POI when recruited to the E3 ligase component has to present lysine (or other) residues for ubiquitination.”

  • The paper relies on this without support.

    Tagging proteins can compromise the structural integrity, subcellular distribution and biological activity of the POI itself.

    “Moreover, tagging proteins can compromise the structural integrity, subcellular distribution and biological activity of the POI itself.”

  • The paper relies on this without support.

    Developing selective high-affinity binders of POIs is both resource- and time-intensive process.

    “Developing selective high-affinity binders of POIs is both resource- and time-intensive process.”
6 of 6

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?

  • HaloPROTACs, dTAGs and other degradation proof-of-concept strategies, such as the AID system, now serve as crucial target validation tools when approaching a new therapeutic POI.

    “HaloPROTACs, dTAGs and other degradation proof-of-concept strategies, such as the AID system, have been reviewed extensively elsewhere96,97 and now serve as crucial target validation tools when approaching a new therapeutic POI.”

    From PROTAC targeted protein degraders: the past is prologue (2022) · cites this paper as [96]

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.

  • Slow protein turnover is said to prevent transcript knockdown

    Minor

    The paper says RNAi can degrade a target RNA, but later says slow turnover of the protein prevents knockdown of the target transcript. Protein turnover affects how quickly protein levels fall, not whether RNAi depletes its transcript.

1 of 1

Cites

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Related

Authors
Röth, S., Fulcher, L. J., & Sapkota, G. P.
Affiliations
University of Dundee (GB)
Corresponding author
Sapkota, Gopal P.
ORCID
Venue
Cellular and Molecular Life Sciences
Publisher
Springer Science and Business Media LLC
Published
2019-04-27
Volume
76
Issue
14
Pages
2761-2777
ISSN
  • 1420-682X (ISSN-L)
  • 1420-9071
DOI
10.1007/s00018-019-03112-6
PMC
PMC6588652
OpenAlex
W2942679535
Semantic Scholar
853176d484342afe31afcdcc1ce2b555d1e1ec59
Cited by
92
References
167
Citations by year
2026: 2 · 2025: 15 · 2024: 7 · 2023: 11 · 2022: 18 · 2021: 14 · 2020: 20 · 2019: 5
FWCI
1.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Cellular and Molecular Life Sciences (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
Funders
UK Medical Research Council (MC_UU_12016/3)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
Keywords
targeted proteolysis, CRISPR/Cas9 genome editing, off-target effects
MeSH terms
Gene Editing, Humans, Proteins, Proteasome Endopeptidase Complex, Ubiquitination, Proteolysis

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.

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