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Cited by 179Click Chemistry and ApplicationsAdvanced Proteomics Techniques and ApplicationsChemical Synthesis and Analysis

Expedited mapping of the ligandable proteome using fully functionalized enantiomeric probe pairs

Open access
Wang, Yujia, Dix, Melissa M., Bianco, Giulia, Remsberg, Jarrett R., Lee, Hsin-Yu, Kalocsay, Marian, Gygi, Steven P., Forli, Stefano +4 more·2019·Nature Chemistry
DOI 10.1038/s41557-019-0351-5PMC PMC6874898

Published version of a preprint

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Abstract

A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small-molecule/protein interactions in human cells. Excavating clear structure-activity relationships from these 'ligandability' maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or 'enantioprobes', we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.

AI snapshot

Written from the paper’s full text.

TL;DR

Fully functionalized enantiomeric fragment probe pairs (“enantioprobes”) enable proteome-wide discovery of stereoselective small molecule–protein interactions in human cells. Across eight pairs, the authors identify 176 stereoselective protein targets and validate functional-site engagement for representative proteins, while TMT multiplexing increases throughput.

Key findings

  • Eight enantioprobe pairs differing only in absolute stereochemistry identified 176 proteins with stereoselective interactions, including 119 in PBMCs and 108 in HEK293T cells; >80% of these proteins interacted stereoselectively with only one enantioprobe pair.
  • Enantioprobe targets spanned diverse classes, including enzymes, scaffolding/adaptor proteins, and transcriptional regulators, and showed limited overlap with proteins identified by cysteine- or lysine-reactive covalent fragment profiling.
  • Validation for representative targets (e.g., RPS6KA3, SMYD3, DCTPP1, UNC119B, TSPO, PACSIN2, HDGF, TTC38) showed stereoselective labeling of recombinant proteins that mirrored endogenous proteins; known ligands competitively blocked these interactions.
  • A 10-plex TMT workflow compared four enantioprobe pairs plus a methyl control, recapitulated >85% of pairwise enantioprobe targets, mapped 115 additional stereoselective interactions, and enabled concentration-dependent profiling.

Methods

  • The authors synthesized eight enantioprobe pairs containing a variable fragment recognition element differing only in stereochemistry, plus a constant diazirine photocrosslinker and alkyne handle. HEK293T cells and primary human PBMCs were treated with R/S probes (200 μM, 30 min for MS; 20 μM, 30 min for initial gel), UV-irradiated (365 nm, 10 min), conjugated to azide reporters by CuAAC, enriched, and analyzed by MS-based proteomics.
  • Pairwise quantitative proteomics used reductive dimethylation (PBMCs, replicate both isotopic directions) or SILAC (HEK293T cells); stereoselective interactions were operationally defined as >2.5-fold average preferential enrichment by one enantiomer. Multiplexed TMT experiments required ≥3 unique peptides, >2.5-fold R/S differential enrichment, and >5-fold enrichment over the methyl control probe.
  • Follow-up validation used FLAG-tagged recombinant proteins in HEK293T cells, gel-based fluorescence, competition with known ligands, MS mapping of photolabeled peptides, and docking simulations.

Limitations

  • The paper states that the modest set of eight enantioprobe pairs vastly under-samples the human proteome; low-affinity interactions may not enrich sufficiently for MS detection, and multiple binding sites on a protein may mask stereoselective interactions.
  • The authors state that in most cases they do not know whether stereoselective interactions reflect preferential binding versus stereoselective photoreactivity, and that determining how often interactions occur at functional sites is limited by challenging site mapping and availability of protein structures.
  • Evident from the text: detailed methods are provided only in the Supplementary Information, and functional/structural validation is limited to representative targets rather than all 176 stereoselective proteins.

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

Integrity signals

Registry

Crossref · 10.1038/s41557-019-0351-5

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The enantioprobe pairs showed clear increases in protein labeling across a test concentration range of 5-100 μM.

    “The enantioprobe pairs also showed clear increases in protein labeling across a test concentration range of 5–100 μM (Supplementary Fig. 1)”
    Quote in the paperSubstantial claim
  • Findingown data

    Using a set of eight pairs of enantiomeric FFF probes, the authors identify >170 stereochemistry-dependent small molecule-protein interactions in human cells.

    “Using a set of eight pairs of enantiomeric FFF probes – or “enantioprobes” – we expeditiously identify >170 stereochemistry-dependent small molecule-protein interactions in human cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments confounded the extraction of clear structure-activity relationships from ligandability maps.
  • Findingown data

    The (R) and (S) members within each enantioprobe pair showed similar overall proteomic labeling, with the exception of select proteins that exhibited stereoselective interactions.

    “Encouragingly, however, the (R) and (S) members within each enantioprobe pair showed similar overall proteomic labeling with the exception of select proteins that exhibited stereochemistry-dependent (“stereoselective”) interactions (Fig. 1b and Supplementary Fig. 1, red asterisks).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The eight enantioprobe pairs differ only in absolute stereochemistry of the fragment recognition element and are otherwise physicochemically matched.
  • Findingown data

    The authors observed substantial differences in protein interactions across the enantioprobe pairs, with one probe pair ((R) and (S)-6) exhibiting much greater overall protein labeling compared to others.

    “As expected, we observed substantial differences in protein interactions across the enantioprobe pairs, with one probe pair ((R) and (S)-6) exhibiting much greater overall protein labeling compared to others (Fig. 1b).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Enantioprobe targets observed in PBMCs but not HEK293T cells tended to correspond to immune-enriched proteins.

    “Enantioprobe targets that were observed in PBMCs, but not HEK293T cells, tended to correspond to immune-enriched proteins (e.g., IRAK324, PARP10 25) (Supplementary Dataset 1).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 24
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Findingown data

    The enantioprobe targets span diverse structural and functional classes and include proteins that lack chemical probes.

    “The enantioprobe targets span diverse structural and functional classes and include proteins that lack chemical probes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The authors describe a quantitative, multiplexed workflow capable of performing up to five enantioprobe pair comparisons in a single experiment, thereby greatly increasing the throughput and dimensionality of fragment-based ligand discovery in cells.

    “Finally, we describe a quantitative, multiplexed workflow capable of performing up to five enantioprobe pair comparisons in a single experiment, thereby greatly increasing the throughput and dimensionality of fragment-based ligand discovery in cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The authors validate enantioprobe interactions for several recombinantly expressed proteins and show that the interactions occur at functionally relevant sites on these proteins.

    “We validate enantioprobe interactions for several recombinantly expressed proteins and show that the interactions occur at functionally relevant sites on these proteins.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most stereoselective interactions could be detected with 5-10 μM of the preferred enantioprobe and were preserved across the entire enantioprobe concentration range.

    “Most of these stereoselective interactions could be detected with 5–10 μM of the preferred enantioprobe and were preserved across the entire enantioprobe concentration range (Fig. 3c–f and Supplementary Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The enantioprobe pairs displayed considerable differences in their total number of stereoselective interactions with the human proteome, and these profiles were unrelated to the extent of overall protein labeling displayed by the probes.

    “The enantioprobe pairs displayed considerable differences in their total number of stereoselective interactions with the human proteome (Fig. 2e), and, notably, these profiles were unrelated to the extent of overall protein labeling displayed by the probes (Fig. 1b).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The eight enantioprobe pairs differ only in absolute stereochemistry of the fragment recognition element and are otherwise physicochemically matched.

  • May combine two claims.

    Findingown data

    In total, 176 proteins showed stereoselective interactions with one or more enantioprobe pairs, including 119 proteins in PBMCs and 108 proteins in HEK293T cells.

    “In total, 176 proteins showed stereoselective interactions with one or more enantioprobe pairs, which included 119 proteins identified in PBMCs (Fig. 2b, Supplementary Dataset 1, and Supplementary Dataset 2) and 108 proteins identified in HEK293T cells (Supplementary Fig. 2, Supplementary Dataset 1, and Supplementary Dataset 2).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The eight enantioprobe pairs differ only in absolute stereochemistry of the fragment recognition element and are otherwise physicochemically matched.

  • May combine two claims.

    Findingown data

    While many enantioprobe targets were also enriched by members of the original FFF probe set, these previous profiles often did not provide useful SAR information.

    “Moreover, while many of the enantioprobe targets were also enriched by members of the original FFF probe set13, these previous profiles often did not provide useful SAR information, either reflecting substantial enrichment by all of the FFF probes or mirroring the respective global protein interaction footprints of these probes (Supplementary Fig. 2).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541 e529 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    EPZ031686 blocked (R)-1 interactions with SLC35F2 and PRCP, while PK 11195 decreased (R,R)-7 interactions with ABHD5.

    “For instance, EPZ031686 blocked (R)-1 interactions with the solute carrier SLC35F2 and the peptidase PRCP (Fig. 4a), while PK 11195 decreased (R,R)-7 interactions with the lipid-binding protein ABHD5 (Supplementary Fig. 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Photolabeling of the SMYD3 aa 255-265 peptide by (R)-1 was blocked by co-incubation with EPZ031686 and was not observed with the inactive enantioprobe (S)-1.

    “Quantitative MS-based proteomics further demonstrated that photolabeling of the aa 255–265 peptide by (R)-1 was blocked by co-incubation with EPZ031686 and was not observed with the inactive enantioprobe (S)-1 (Fig. 4c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The authors mapped the primary sites of enantioprobe labeling for UNC119B and TSPO and confirmed the stereoselectivity of these labeling events and their blockade by treatment with competitive ligands.

    “We also mapped the primary sites of enantioprobe labeling for UNC119B (Fig. 4e) and TSPO (Supplementary Fig. 4) and confirmed the stereoselectivity of these labeling events and their blockade by treatment with competitive ligands (squarunkin A and PK 11195, respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For SMYD3, only EPZ031686, but not SAM or SAH, blocked (R)-1 interactions.

    “For SMYD3, only EPZ031686, but not SAM or SAH, blocked (R)-1 interactions (Fig. 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The non-crosslinkable analogue of (S)-4, but not (R)-4, competitively blocked enantioprobe binding to TTC38.

    “we found that the non-crosslinkable analogue of (S)-4, but not (R)-4, competitively blocked enantioprobe binding to TTC38 (Supplementary Fig. 6)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.

  • May combine two claims.

    Findingown data

    TTC38 has been previously identified as an off-target of the HDAC inhibitor panobinostat and was found herein to display stereoselective interaction with the (S)-4 probe that was blocked by panobinostat.

    “Consider TTC38, for instance, a poorly characterized protein that has been previously identified as an off-target of the HDAC inhibitor panobinostat36 and found herein to display stereoselective interaction with the (S)-4 probe that was blocked by panobinostat.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Becher, I. et al. Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat. Nat. Chem. Biol. 12, 908–910 (2016). source

      Not checked in the free run.

    • Rests on an assumption: The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.

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

    Premisecitation

    Fragment-based ligand discovery (FBLD) has emerged as a versatile approach for the discovery of atom-efficient, small-molecule binders for a wide range of proteins.

    “Fragment-based ligand discovery (FBLD) has emerged as a versatile approach for the discovery of atom-efficient, small-molecule binders for a wide range of proteins10–12.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [10] Bembenek, S. D., Tounge, B. A. & Reynolds, C. H. Ligand efficiency and fragment-based drug discovery. Drug Discov. Today 14, 278–283 (2009). source

      Not checked in the free run.

    • cites [11] Scott, D. E., Coyne, A. G., Hudson, S. A. & Abell, C. Fragment-based approaches in drug discovery and chemical biology. Biochemistry 51, 4990–5003 (2012). source

      Not checked in the free run.

    • cites [12] Erlanson, D. A., Fesik, S. W., Hubbard, R. E., Jahnke, W. & Jhoti, H. Twenty years on: the impact of fragments on drug discovery. Nat. Rev. Drug Discov. 15, 605–619 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premiseown data

    Individual FFF probes in the authors' initial studies showed substantial differences in their overall proteomic interaction profiles, which made for complicated structure-activity relationships requiring careful manual review to identify authentic fragment-protein interactions.

    “As a consequence, we found that individual FFF probes showed substantial differences in their overall proteomic interaction profiles, which made for complicated structure-activity relationships (SARs) requiring careful manual review to identify fragment-protein interactions that reflected authentic recognition events (versus simply correlating with the overall proteomic interaction profiles of the”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541 e529 (2017). source

      Not checked in the free run.

    • Rests on an assumption: The distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments confounded the extraction of clear structure-activity relationships from ligandability maps.

  • Classified as probably not a substantial claim.

    Premisecitation

    The stereoselective engagement of protein targets is a feature of numerous chemical probes and drugs.

    “The stereoselective engagement of protein targets is a feature of numerous chemical probes and drugs15–19”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [15] Agranat, I., Caner, H. & Caldwell, J. Putting chirality to work: the strategy of chiral switches. Nat. Rev. Drug Discov. 1, 753–768 (2002). source

      Not checked in the free run.

    • cites [16] Nguyen, L. A., He, H. & Pham-Huy, C. Chiral drugs: an overview. Int. J. Biomed. Sci. 2, 85–100 (2006). source

      Not checked in the free run.

    • cites [17] Zanos, P. et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481–486 (2016). source

      Not checked in the free run.

    • cites [18] Sui, J. J., Zhang, J. H., Ching, C. B. & Chen, W. N. Expanding proteomics into the analysis of chiral drugs. Mol. Biosyst. 5, 603–608 (2009). source

      Not checked in the free run.

    • cites [19] Sanna, M. G. et al. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat. Chem. Biol. 2, 434–441 (2006). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The fragment binding elements in the authors' initial studies were selected based on their representation in drug-like molecules and were diverse in structure and physicochemical properties.

    “The fragment binding elements in our initial studies were selected based on their representation in drug-like molecules14 and were accordingly diverse in structure and physicochemical properties.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [14] Welsch, M. E., Snyder, S. A. & Stockwell, B. R. Privileged scaffolds for library design and drug discovery. Curr. Opin. Chem. Biol. 14, 347–361 (2010). source

      Not checked in the free run.

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

    Study design and conductown data

    For PBMCs, experiments were performed in replicate in both isotopic directions to furnish four independent experiments for each enantioprobe pair.

    “For PBMCs, these experiments were performed in replicate in both isotopic directions (heavy vs light and light vs heavy) to furnish four independent experiments for each enantioprobe pair”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    EPZ031686 binds in the lysine substrate binding pocket of SMYD3 and noncompetitively with SAM.

    “Structural studies have shown that EPZ031686 binds in the lysine substrate binding pocket of SMYD3 and noncompetitively with SAM29.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Mitchell, L. H. et al. Novel oxindole sulfonamides and sulfamides: EPZ031686, the first orally bioavailable small molecule SMYD3 inhibitor. ACS Med. Chem. Lett. 7, 134–138 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The docking results helped explain the stereoselectivity of the (R)-1-SMYD3 interaction: while (S)-1 reproduced a similar binding mode, the inverted chiral center reduced the quality of the docking match.

    “The docking results also helped to explain the stereoselectivity of the (R)-1-SMYD3 interaction, as, while (S)-1 was able to reproduce a similar binding mode, the inverted chiral center reduced the quality of the docking match by placing the hydrophobic phenyl ring outside the molecular envelope of the inhibitor EPZ030456 and toward hydrophilic side chains T184 and E192 (Fig. 4d, bottom).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    For some protein targets (e.g., UNC119B), the magnitude of observed stereoselectivity reflects a difference in specific interactions with both enantioprobes, while for other proteins (e.g., SMYD3), the degree of stereoselectivity may be even greater than experimentally measured, being suppressed by low-level nonspecific interactions with the non-preferred enantioprobe.

    “These data indicate that, for some protein targets (e.g., UNC119B), the magnitude of observed stereoselectivity reflects a difference in specific interactions with both enantioprobes, while, for other proteins (e.g., SMYD3), the degree of stereoselectivity may be even greater than that experimentally measured, being instead suppressed by low-level nonspecific interactions with the non-preferred”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The eight enantioprobe pairs differ only in absolute stereochemistry of the fragment recognition element and are otherwise physicochemically matched.

  • May combine two claims.

    Premisecitation

    Residues D255-Y257 of SMYD3 are located within 3.6 angstroms of an EPZ031686 analog in the SMYD3 co-crystal structure, and Y257 interacts with the methylated lysine in substrate co-crystal structures.

    “These residues are located within 3.6 angstroms of an EPZ031686 analog in the SMYD3 co-crystal structure (Fig. 4c), and Y257 specifically has been found to interact with the methylated lysine in substrate co-crystal structures39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Fu, W. Q. et al. Structural basis for substrate preference of SMYD3, a SET domain-containing protein lysine methyltransferase. J. Biol. Chem. 291, 9173–9180 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    SLC35F2 and PRCP may specifically bind enantioprobes, but without stereochemical preference.

    “suggesting that these latter proteins may specifically bind enantioprobes, but without stereochemical preference.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The enrichment profiles of proteins enriched by one or more enantioprobe pairs without stereopreference reflect specific probe-protein interactions, with SAR across the enantioprobe set driven by chemotype rather than stereotype.

    “We interpret these enrichment profiles to also reflect specific probe-protein interactions, where the SAR across the enantioprobe set is driven by chemotype rather than stereotype.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.

  • May combine two claims.

    Premisecitation

    The initial set of fully functionalized fragments uncovered many new small molecule-protein interactions, some of which were advanced to selective and cell-active chemical probes.

    “The initial set of fully functionalized (clickable, photoreactive) fragments (FFFs) uncovered many new small molecule-protein interactions, some of which were advanced to selective and cell-active chemical probes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541 e529 (2017). source

      Not checked in the free run.

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

    Interpretationown data

    Saturated enrichment at lower concentrations of the preferred enantioprobe possibly reflects higher affinity binding events.

    “possibly reflecting higher affinity binding events”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    We previously described a chemical proteomic strategy to perform fragment-based ligand discovery experiments in human cells.

    “We previously described a chemical proteomic strategy to perform fragment-based ligand discovery experiments in human cells13.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541 e529 (2017). source

      Not checked in the free run.

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

    Interpretationown data

    The enantioprobes offer a convenient target engagement assay for assessing competitive binding of elaborated analogues in cells.

    “Toward this end, we note that the enantioprobes offer a convenient target engagement assay for assessing competitive binding of elaborated analogues in cells, and that several of the enantiopure fragment recognition groups deployed herein are poised for direct modification using synthetic methodologies such as C-H bond activation chemistry44–46.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.

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

    Premisecitation

    Several of the enantiopure fragment recognition groups deployed herein are poised for direct modification using synthetic methodologies such as C-H bond activation chemistry.

    “Toward this end, we note that the enantioprobes offer a convenient target engagement assay for assessing competitive binding of elaborated analogues in cells, and that several of the enantiopure fragment recognition groups deployed herein are poised for direct modification using synthetic methodologies such as C-H bond activation chemistry44–46.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [44] Ming, S. et al. Modular, Stereocontrolled Cβ–H/Cα–C Activation of Alkyl Carboxylic Acids. Proc. Natl Acad. Sci. USA 116, 8721–8727 (2019). source

      Not checked in the free run.

    • cites [45] Tran, A. T. & Yu, J. Q. Practical alkoxythiocarbonyl auxiliaries for iridium(I)-catalyzed C–H alkylation of azacycles. Angew. Chem. Int. Ed. Engl. 56, 10530–10534 (2017). source

      Not checked in the free run.

    • cites [46] Jain, P., Verma, P., Xia, G. Q. & Yu, J. Q. Enantioselective amine alpha-functionalization via palladium-catalysed C–H arylation of thioamides. Nat. Chem. 9, 140–144 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premiseown data

    Individual FFFs showed substantially different overall protein interaction profiles in human cells, which complicated the assignment of small molecule-protein interactions displaying authentic SARs versus nonspecific binding to the FFFs.

    “Nonetheless, we also found that individual FFFs showed substantially different overall protein interaction profiles in human cells, which complicated the assignment of small molecule-protein interactions displaying authentic SARs (vs nonspecific binding to the FFFs).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Parker, C. G. et al. Ligand and target discovery by fragment-based screening in human cells. Cell 168, 527–541 e529 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The study's data highlight the value of incorporating TMT-based multiplexing readouts into enantioprobe profiling experiments, which greatly expedited discovery of stereoselective interactions without substantial losses in sensitivity or accuracy.

    “Finally, our data highlight the value of incorporating TMT-based multiplexing readouts into enantioprobe profiling experiments, which greatly expedited the discovery of stereoselective interactions without substantial losses in sensitivity or accuracy.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.
    • Rests on an assumption: The stochastic nature of protein identification events in untargeted MS-based proteomic experiments hinders confident assignment of proteins that selectively interact with one or a subset of enantioprobes across different experiments.
    • Rests on an assumption: The limited throughput of pairwise comparisons prevents deeper exploration of SAR both within and across enantioprobe pairs.
    • Rests on an assumption: The throughput of pairwise comparisons is restrictive when comparing protein interaction profiles of several enantioprobes under various conditions.

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

    Interpretationreasoning

    The third profile might reflect cases where equivalent binding is observed for both enantioprobes, but one probe generates a greater amount of photoadduct with the protein target.

    “This outcome might reflect cases where equivalent binding is observed for both enantioprobes, but one of the probes generates a greater amount of photoadduct with the protein target (see Discussion below).”
    Quote in the paperNot a substantial claim
32 of 32

  • The paper relies on this without support.

    The eight enantioprobe pairs differ only in absolute stereochemistry of the fragment recognition element and are otherwise physicochemically matched.

    “We synthesized a library of eight enantioprobe pairs, where members of each pair differ only in absolute stereochemistry of the fragment recognition element (Fig. 1a).”

  • The paper relies on this without support.

    The distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments confounded the extraction of clear structure-activity relationships from ligandability maps.

    “Excavating clear structure-activity relationships from these “ligandability” maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments.”

  • The paper relies on this without support.

    The methyl control probe is a valid negative control for nonspecific protein enrichment, such that >5-fold enrichment over it indicates specific probe-protein interaction.

    “alongside a previously described methyl control probe13 (in duplicate)”
    “along with > 5-fold enrichment over the methyl control probe.”

  • The paper relies on this without support.

    The stochastic nature of protein identification events in untargeted MS-based proteomic experiments hinders confident assignment of proteins that selectively interact with one or a subset of enantioprobes across different experiments.

    “And, relatedly, the stochastic nature of protein identification events in untargeted MS-based proteomic experiments hindered confident assignment of proteins that selectively interacted with one or a subset of enantioprobes across different experiments.”

  • The paper relies on this without support.

    The limited throughput of pairwise comparisons prevents deeper exploration of SAR both within and across enantioprobe pairs.

    “Prominently, the limited throughput prevented a deeper exploration of SAR both within and across enantioprobe pairs.”

  • The paper relies on this without support.

    The throughput of pairwise comparisons is restrictive when comparing protein interaction profiles of several enantioprobes under various conditions.

    “Finally, the throughput of pairwise comparisons also becomes restrictive when attempting to compare the protein interaction profiles of several enantioprobes under various conditions (e.g., different cell types, probe concentrations, etc.).”
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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?

  • Equipping noncovalent fragments with a photoreactive diazirine moiety enables their proteome-wide target identification without prerequisite of a particular amino acid in or nearby a ligandable site.

    “Equipping noncovalent fragments with a photoreactive diazirine moiety similarly enables their proteome-wide target identification without the prerequisite of a particular amino acid in or nearby a ligandable site.98−100”

    From Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence (2023) · cites this paper as [100]

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.

  • HEK293T cell line is called HEK239T in one passage

    Minor

    The cell type used for the proteomic comparison is named HEK239T here, but the same passage subsequently identifies it as HEK293T.

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References 53 · Cited by 179

Authors
Wang, Y., Dix, M. M., Bianco, G., Remsberg, J. R., Lee, H.-Y., Kalocsay, M., Gygi, S. P., Forli, S., Vite, G., Lawrence, R. M., Parker, C. G., & Cravatt, B. F.
Affiliations
  • Scripps Research Institute (US)
  • Harvard University (US)
  • Bristol-Myers Squibb (United States) (US)
Corresponding author
  • Parker, Christopher G.
  • Cravatt, Benjamin F.
ORCID
Venue
Nature Chemistry
Publisher
Springer Science and Business Media LLC
Published
2019-10-28
Volume
11
Issue
12
Pages
1113-1123
ISSN
  • 1755-4330 (ISSN-L)
  • 1755-4349
DOI
10.1038/s41557-019-0351-5
PMC
PMC6874898
OpenAlex
W2982116122
Cited by
179
References
53
Citations by year
2026: 22 · 2025: 24 · 2024: 40 · 2023: 25 · 2022: 19 · 2021: 22 · 2020: 17 · 2019: 10
FWCI
6.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Chemistry (publishedVersion)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
  • Figshare (submittedVersion, cc-by-nc-nd)
Funders
  • U.S. Department of Health and Human Services (814099)
  • American Cancer Society
  • National Institutes of Health (DK114785, CA231991)
  • National Cancer Institute (R50CA211526, R35CA231991)
  • National Institute of General Medical Sciences (R01GM067945, R01GM069832)
  • National Institute of Diabetes and Digestive and Kidney Diseases (RC2DK114785)
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
Keywords
chemoproteomics, absolute configuration, protein-small molecule interactions, ligandability, structure-activity relationship, human cells
MeSH terms
Humans, Ligands, Proteins, Stereoisomerism, Molecular Probes, Molecular Structure, Proteome, Small Molecule Libraries

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