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Cited by 823Click Chemistry and ApplicationsProtein Degradation and InhibitorsComputational Drug Discovery Methods

Advances in covalent drug discovery

Open access
Boike, Lydia, Henning, Nathaniel J., Nomura, Daniel K.·2022·Nature Reviews Drug Discovery
DOI 10.1038/s41573-022-00542-zPMC PMC9403961
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Abstract

Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field. The rational discovery of covalent drugs depends on an expanding toolset of techniques. Here, Daniel Nomura and colleagues highlight covalent drugs that have achieved success over the past decade and discuss the tools and strategies that facilitate their discovery, describing two complementary approaches, namely, ligand-first and electrophile-first strategies.

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Written from the paper’s full text.

TL;DR

This review traces covalent drug discovery from serendipitous historical agents to rational ligand-first and electrophile-first programmes, highlighting approved inhibitors for EGFR, BTK, KRAS(G12C), SARS-CoV-2 Mpro and other targets. It argues that chemoproteomics, covalent screening and structure-guided design now form an integrated toolbox for tackling previously undruggable proteins.

Key findings

  • Covalent drugs can gain potency and selectivity by adding mildly reactive electrophiles to reversible ligands or by screening electrophilic libraries; examples include osimertinib and ibrutinib, with 2020 sales of US$8.43 billion and US$4.33 billion respectively, and sotorasib, approved in 2021 for KRASG12C-mutated NSCLC.
  • Electrophile-first discovery was validated by KRAS(G12C) inhibitors: an initial tethering screen of 480 disulfide fragments and later a 3,300-acrylamide screen contributed to sotorasib; KRAS is mutated in about 25% of cancers, and KRASG12C accounts for 12–14% of KRAS mutations in NSCLC.
  • Covalent protease inhibitors include nirmatrelvir, which forms a reversible covalent nitrile adduct with SARS-CoV-2 Mpro Cys145 and was authorized as part of Paxlovid in December 2021; HCV NS3/4a ketoamide inhibitors boceprevir and telaprevir were approved in 2011 but later discontinued or withdrawn.
  • Chemoproteomic platforms such as isoTOP-ABPP and SLC-ABPP enable profiling of more than 30,000 reactive cysteines, over 8,000 cysteine sites in 18 min per compound, and show that 97% of E3 ligases possess reactive cysteines, supporting covalent E3 recruiters for targeted protein degradation.

Methods

  • Narrative review, not primary research; it synthesizes published literature, drug approval histories, structural biology and chemoproteomic datasets across covalent drug discovery.
  • Evidence is organized by discovery approach: ligand-first (EGFR, BTK and other kinases), electrophile-first (KRAS(G12C), SARS-CoV-2 Mpro, HCV protease, proteasome, boron-containing drugs, voxelotor) and enabling tools (screening platforms, chemoproteomics, covalent docking).
  • Includes tables comparing key covalent drugs and screening methods, plus figures on development timelines, inhibitor–target structures and isoTOP-ABPP workflow; no systematic search strategy or meta-analysis is described.

Limitations

  • The paper states no explicit limitations. Evident: it is a selective narrative review without systematic search criteria or quantitative synthesis, so examples are chosen to illustrate milestones rather than exhaustively cover the field.
  • Evident: many claims depend on cited primary studies and clinical or preclinical data that are not independently reanalysed, and the fast-moving nature of the field may date some coverage.
  • Evident: the review emphasizes cysteine-targeting electrophiles and oncology or infectious disease examples, with comparatively less attention to non-cysteine nucleophilic residues and other covalent strategies.

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Integrity signals

Registry

Crossref · 10.1038/s41573-022-00542-z

Cites

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Statistics

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Claims the paper makes

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  • Premisecitation

    Chemoproteomic platforms enable rapid target identification and selectivity profiling of covalent ligands.

    “This ‘electrophile-first’ approach is partly facilitated by the development of chemoproteomic platforms that enable rapid target identification and selectivity profiling of covalent ligands14–18.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
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  • Premisecitation

    Afatinib was FDA-approved in 2013 as first-line treatment for metastatic NSCLC with activating EGFR mutations.

    “Afatinib (Boehringer Ingelheim) (Figs. 1 and 2b) was approved by the FDA in 2013 as a first-line treatment for patients with metastatic NSCLC with activating mutations in EGFR37,38.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Soria, J.-C. et al. Afatinib versus erlotinib as second-line treatment of patients with advanced squamous cell carcinoma of the lung (LUX-Lung 8): an open-label randomised controlled phase 3 trial. Lancet Oncol. 16, 897–907 (2015). source

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    Prolonged target engagement of covalent drugs can give distinct pharmacodynamic profiles and exceptional potency.

    “The prolonged target engagement of covalent drugs can provide distinct pharmacodynamic profiles and exceptional potency6.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Zhang, T., Hatcher, J. M., Teng, M., Gray, N. S. & Kostic, M. Recent advances in selective and irreversible covalent ligand development and validation. Cell Chem. Biol. 26, 1486–1500 (2019). source

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  • Premisecitation

    Covalent ligand screening from electrophilic compound libraries is becoming more common.

    “Covalent ligand screening is another ligand discovery approach that is becoming more common, whereby various methods are used to discover covalent ligands from libraries of electrophilic compounds7,12,13.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [7] Lonsdale, R. & Ward, R. A. Structure-based design of targeted covalent inhibitors. Chem. Soc. Rev. 47, 3816–3830 (2018). source

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    • cites [13] Lu, W. et al. Fragment-based covalent ligand discovery. RSC Chem. Biol. 2, 354–367 (2021). source

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  • Premisecitation

    The T790M gatekeeper mutation caused disease progression in 60% of cases.

    “in 60% of cases this was due to the acquisition of the T790M ‘gatekeeper mutation’34,35.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] Yu, H. A. et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR -mutant lung cancers. Clin. Cancer Res. 19, 2240–2247 (2013). source

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    Premisecitation

    Osimertinib received accelerated FDA approval in 2015 as second-line NSCLC treatment and was approved first-line for metastatic NSCLC in 2018.

    “Osimertinib was granted accelerated approval by the FDA in 2015 as a second-line treatment for NSCLC, and was approved as a first-line treatment for metastatic NSCLC in 2018 (ref.49).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
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    Premisecitation

    Many early covalent drugs were discovered by chance and bind active sites to inhibit enzymatic activity.

    “Many early covalent drugs were discovered serendipitously and bind active sites to inhibit enzymatic activity4.”
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    • cites [4] De Cesco, S., Kurian, J., Dufresne, C., Mittermaier, A. K. & Moitessier, N. Covalent inhibitors design and discovery. Eur. J. Med. Chem. 138, 96–114 (2017). source

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    Premisecitation

    C797X mutations account for 15% of resistance to second-line osimertinib because osimertinib requires Cys797.

    “However, osimertinib depends on Cys797 for covalent binding, and C797X mutations account for 15% of cases of resistance to second-line osimertinib50–52.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
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    Premisecitation

    Aspirin's anti-inflammatory effect is via acetylation of Ser529 in COX-1, preventing arachidonic acid conversion to prostaglandins.

    “Aspirin’s mechanism of action was unknown until 1971, when it was discovered to exert its anti-inflammatory effects by acetylating Ser529 in the substrate-binding channel of cyclooxygenase 1, preventing conversion of the substrate arachidonic acid into prostaglandins20.”
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    Premisecitation

    β-Lactam antibiotics such as penicillin bind PBPs and acylate active-site serines, inhibiting cell wall synthesis and causing membrane rupture.

    “β-Lactam antibiotics such as penicillin (Fig. 1), produced by Penicillium fungi, bind to penicillin-binding proteins (PBPs), which are involved in bacterial cell wall synthesis21.”
    • Also stated in: § History of covalent drugs · plain
      “All PBPs contain active-site serine residues that can be acylated by penicillin, inhibiting PBP activity and leading to cell membrane rupture21.”
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    • cites [21] Nicola, G., Tomberg, J., Pratt, R. F., Nicholas, R. A. & Davies, C. Crystal structures of covalent complexes of β-lactam antibiotics with Escherichia coli penicillin-binding protein 5: toward an understanding of antibiotic specificity. Biochemistry 49, 8094–8104 (2010). source

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    Premisecitation

    Covalent drugs contain a mildly reactive group that forms a covalent bond with protein targets, adding affinity beyond non-covalent binding.

    “Covalent drugs incorporate a mildly reactive functional group that forms a covalent bond with protein targets to confer additional affinity beyond the non-covalent interactions involved in drug binding1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Tuley, A. & Fast, W. The taxonomy of covalent inhibitors. Biochemistry 57, 3326–3337 (2018). This review summarizes the mechanisms of covalent inhibition of enzymes and comprehensively covers examples for each mechanism. source

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    Bortezomib covalently inhibits a catalytic threonine of the 26S proteasome and was FDA-approved in 2003 for multiple myeloma.

    “Bortezomib (Fig. 1), a dipeptide boronic acid that covalently binds to and inhibits a catalytic threonine residue of the 26S proteasome, was approved by the FDA in 2003 to treat patients with multiple myeloma30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
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    Premisecitation

    Over the past 30 years, rational design of covalent drugs has increased and targeting non-conserved amino acids for selectivity has become common.

    “Over the past 30 years, the rational design of covalent drugs has garnered increased interest, and covalently targeting non-conserved amino acids to increase selectivity has become commonplace2,5.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011). This review illustrates the key features of covalent drug discovery with emphasis on targeted covalent inhibitors. source

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    Premisecitation

    Covalent modification has enabled targeting of traditionally undruggable proteins, exemplified by sotorasib approval for KRAS(G12C).

    “Moreover, potent inhibition through covalent modification has enabled targeting of traditionally ‘undruggable’ proteins, exemplified by the approval of sotorasib (Amgen), which is an inhibitor of mutant KRAS(G12C), a GTPase that resisted decades of drug discovery efforts9,10”
    Quote in the paperSubstantial claim0 of 2 sources checked
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    Premisecitation

    Covalent second-generation EGFR inhibitors with acrylamide react with Cys797 and partially restore activity against T790M.

    “Cys797 is located adjacent to the ATP-binding site, and irreversible binding of EGFR ligands to EGFR partially restores activity against the T790M gatekeeper mutant33.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] Ou, S.-H. I. Second-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs): a better mousetrap? A review of the clinical evidence. Crit. Rev. Oncol. Hematol. 83, 407–421 (2012). source

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    Premisecitation

    Third-generation EGFR inhibitors selectively target T790M over wild-type EGFR, including osimertinib.

    “A third generation of EGFR inhibitors followed afatinib; these covalent inhibitors selectively target the T790M mutant over wild-type EGFR, and include WZ4002 (Dana–Farber Cancer Institute)43, osimertinib44,45 and rociletinib (Clovis Oncology; CO-1686)46 (Fig. 2c).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [43] Zhou, W. et al. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 462, 1070–1074 (2009). This paper reports the discovery of covalent EGFR inhibitors selective for the T790M gatekeeper mutation. source

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    Premisecitation

    EGFR overactivity drives NSCLC progression, making EGFR a key oncology target.

    “Overactivity of the receptor tyrosine kinase EGFR drives the progression of non-small-cell lung cancer (NSCLC), making EGFR a key drug target in oncology31.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    Premisecitation

    Fosfomycin reacts with MurA catalytic cysteine, disrupting peptidoglycan synthesis and causing membrane rupture.

    “Another covalent antibiotic is the epoxide-containing fosfomycin (Fig. 1), which is produced by some Streptomyces bacteria and acts by reacting with the catalytic cysteine of UDP-N-acetylglucosamine-enolpyruvyl transferase (MurA) to disrupt peptidoglycan synthesis and induce membrane rupture22–24.”
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    Premisecitation

    Nirmatrelvir inhibits SARS-CoV-2 main protease, showing covalent protease active-site targeting yields valuable drugs.

    “more traditional covalent targeting of protease active sites has continued to yield valuable drugs, such as nirmatrelvir (Pfizer), which inhibits the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro)11”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    Premisecitation

    Ibrutinib and osimertinib had 2020 sales of US$8.43 billion and $4.33 billion, respectively.

    “include the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib (AbbVie) and the epidermal growth factor receptor (EGFR) inhibitor osimertinib (AstraZeneca), with sales totalling US$8.43 billion and $4.33 billion in 2020, respectively7,8.”
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    The T790M mutation lowers binding affinity of many reversible EGFR inhibitors and increases EGFR's affinity for ATP.

    “This mutation of the gatekeeper residue in the ATP-binding site of EGFR not only decreases the binding affinity of many reversible inhibitors for EGFR but also increases the binding affinity of EGFR for ATP36.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    KRAS is mutated in about 25% of cancers, especially pancreatic, colorectal and lung cancers.

    “KRAS is a GTPase-encoding oncogene that is mutated in about 25% of all cancers, most notably in pancreatic, colorectal and lung cancers86.”
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    Premisecitation

    Nirmatrelvir inhibits all human coronaviruses in a FRET-based assay and shows no inhibition of human cysteine or serine proteases.

    “displayed potent inhibition in a FRET-based assay across all human coronaviruses, while no inhibitory effects were seen against human cysteine or serine proteases.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    ARS-853 is a selective covalent KRAS(G12C) inhibitor with low micromolar cellular efficacy.

    “In 2016, Wellspring Biosciences disclosed ARS-853, which is a selective covalent inhibitor of KRAS(G12C) with in cellulo efficacy in the low micromolar range92.”
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    • cites [92] Patricelli, M. P. et al. Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov. 6, 316–329 (2016). source

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    ARS-853 treatment reduced active KRAS(G12C), inhibited downstream RAS signalling and induced apoptosis in dependent cell lines.

    “ARS-853 treatment in KRAS(G12C)-dependent cell lines decreased the amount of active KRAS(G12C), inhibited downstream RAS signalling and induced apoptosis92.”
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    • cites [92] Patricelli, M. P. et al. Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state. Cancer Discov. 6, 316–329 (2016). source

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    Compound 12 binding to the switch II pocket impaired KRAS signalling by shifting nucleotide affinity toward GDP and accumulating inactive KRAS.

    “Binding of compound 12 to the switch II pocket impaired KRAS signalling by shifting nucleotide affinity from favouring GTP to GDP and led to the accumulation of KRAS in its inactive state91.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    Covalent FGFR4 inhibitors target Cys552 to gain selectivity over FGFR1-3 and overcome resistance mutations in HCC.

    “Several covalent inhibitors of fibroblast growth factor receptor 4 (FGFR4) target the non-conserved Cys552 residue in FGFR4 to confer selectivity over FGFR1, FGFR2 and FGFR3, as well as to overcome mutations that confer resistance to reversible FGFR inhibitors in hepatocellular carcinoma (HCC)83,84.”
    Quote in the paperSubstantial claim0 of 2 sources checked
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    • cites [84] Tan, L. et al. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors. Proc. Natl Acad. Sci. USA 111, E4869–E4877 (2014).

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    Premisecitation

    JAK3 and mutant EGFR(T790M) both have a methionine gatekeeper and a homologous cysteine next to the ATP-binding site.

    “Both JAK3 and mutant EGFR(T790M) contain a methionine gatekeeper, as well as a homologous cysteine adjacent to the ATP-binding site.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    Many KRAS mutations reduce GTPase activity, causing low GTP hydrolysis and elevated RAS signalling that drives tumorigenesis.

    “Wild-type KRAS is carefully regulated between the active GTP-bound state and inactive GDP-bound state, but many KRAS mutations attenuate GTPase activity, leading to low rates of GTP hydrolysis and elevated RAS signalling, driving tumorigenesis87.”
    Quote in the paperSubstantial claim0 of 1 sources checked
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    Paxlovid received FDA Emergency Use Authorization in December 2021 for mild-to-moderate COVID-19, the first approved oral treatment.

    “In December 2021, the FDA issued an Emergency Use Authorization for Pfizer’s Paxlovid (a combination of nirmatrelvir and ritonavir) to treat mild-to-moderate COVID-19 (caused by SARS-CoV-2) in adults and some paediatric patients, marking the first approved oral treatment for the disease99.”
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    KRAS lacks accessible pockets for reversible inhibitors, competitive inhibitors face picomolar GTP/GDP affinity, and wild-type KRAS inhibitors could cause on-target toxicity.

    “KRAS does not have accessible pockets for reversible inhibitors to bind to, competitive inhibitors would need to overcome the picomolar binding affinities of GTP and GDP, and inhibitors active against wild-type KRAS could show on-target toxicity86,88.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [86] Goebel, L., Müller, M. P., Goody, R. S. & Rauh, D. KRasG12C inhibitors in clinical trials: a short historical perspective. RSC Med. Chem. 11, 760–770 (2020). source

      Not checked in the free run.

    • cites [88] Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A. & Shokat, K. M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013). This work showcases the use of tethering as a screening methodology for discovering covalent ligands against KRAS(G12C) and further identifies ligands against this oncogenic protein. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    SARS-CoV-2 main protease has 96% sequence identity with SARS-CoV-1 main protease, and their catalytic sites overlap completely.

    “SARS-CoV-2 Mpro shares 96% sequence identity with SARS-CoV-1 Mpro, and there is 100% sequence overlap of the catalytic sites102.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [102] Hoffman, R. L. et al. Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19. J. Med. Chem. 63, 12725–12747 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Success of ibrutinib shows rationally designed covalent drugs can achieve acceptable safety and blockbuster status.

    “Overcoming historical concerns relating to the potential toxicity of covalent drugs, the success of ibrutinib demonstrates that rationally designed covalent drugs can achieve acceptable safety profiles and blockbuster status.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    PF-00835231 shows activity in antiviral cell-based assays, stability in plasma, and low clearance in vivo.

    “activity in antiviral cell-based assays, stability in plasma and low clearance in vivo103.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Owen, D. R. et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586–1593 (2021). This work describes the discovery of PF-07321332, later named nirmatrelvir, an orally bioavailable SARS-CoV-2 Mpro inhibitor. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Acalabrutinib was FDA-approved in 2019 for CLL and contains a less reactive butyramide electrophile, hypothesized to explain superior BTK selectivity and fewer cardiovascular events.

    “Most prominent among these is acalabrutinib (AstraZeneca), approved by the FDA in 2019 to treat CLL, which contains a butyramide electrophile instead of an acrylamide69.”
    • Also stated in: § Covalent BTK inhibitors · hedged
      “The butyramide electrophile is less reactive than an acrylamide, which, in addition to other substitutions, is hypothesized to account for the superior selectivity of acalabrutinib compared with ibrutinib for BTK and could be responsible for the reduced number of adverse cardiovascular events70,71.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [69] Barf, T. et al. Acalabrutinib (ACP-196): a covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J. Pharmacol. Exp. Ther. 363, 240–252 (2017). source

      Not checked in the free run.

    • cites [70] Byrd, J. C. et al. Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia. N. Engl. J. Med. 374, 323–332 (2016). source

      Not checked in the free run.

    • cites [71] Byrd, J. C. et al. Acalabrutinib versus ibrutinib in previously treated chronic lymphocytic leukemia: results of the first randomized phase III trial. J. Clin. Oncol. 39, 3441–3452 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Sotorasib was the first selective KRAS(G12C) inhibitor to enter clinical trials in 2018 and was FDA-approved in May 2021 for KRASG12C-mutated NSCLC.

    “Sotorasib (AMG-510) (Table 1) was the first selective KRAS(G12C) inhibitor to enter clinical trials in 2018 and was developed by Amgen, building on discoveries from a partnership with Carmot Therapeutics in which a custom library of small molecules that covalently bind cysteine were screened against KRAS(G12C)93.”
    • Also stated in: § Covalent KRAS(G12C) inhibitors · plain
      “A phase II clinical trial investigating sotorasib was successfully completed in 2020, and was followed by FDA approval for the treatment of adults with KRASG12C-mutated locally advanced or metastatic NSCLC in May 2021 (ref.95).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [93] Canon, J. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019). source

      Not checked in the free run.

    • cites [95] Skoulidis, F. et al. Sotorasib for lung cancers with KRAS p.G12C mutation. N. Engl. J. Med. 384, 2371–2381 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Success of covalent KRAS(G12C) inhibitors validates electrophile-first approaches and covalent fragment screening.

    “The success of covalent KRAS(G12C) inhibitors validates an electrophile-first approach to covalent drug discovery and affirms the importance of covalent fragment screening techniques (discussed below).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Ibrutinib binds Cys481 in BTK and should be selective because few kinases have a homologous cysteine.

    “Similarly to EGFR inhibitors, ibrutinib binds to a cysteine residue (Cys481) adjacent to the ATP-binding site in BTK, and because only a few kinases have a homologous cysteine, ibrutinib should exhibit a degree of selectivity for BTK over other kinases64.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [64] Davids, M. S. & Brown, J. R. Ibrutinib: a first in class covalent inhibitor of Bruton’s tyrosine kinase. Future Oncol. 10, 957–967 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    ARS-1620 was the first KRAS(G12C) inhibitor suitable for in vivo studies and showed efficacy in patient-derived xenografts at 200 mg/kg.

    “Ultimately, ARS-1620 was identified as the first KRAS(G12C) inhibitor suitable for in vivo studies and showed efficacy in KRAS(G12C) patient-derived xenograft models treated at 200 mg kg−1 once per day or twice per day87.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Janes, M. R. et al. Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell 172, 578–589.e17 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The N-terminal amine of α-haemoglobin was found almost 50 years ago to have a low pKa of 6.9, making it primarily unprotonated and more nucleophilic under physiological conditions.

    “Almost 50 years ago, this N-terminal amine was discovered to have a particularly low pKa of 6.9, indicating that it is primarily unprotonated under physiological conditions and, thus, more nucleophilic144.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [144] Garner, M. H., Bogardt, R. A. & Gurd, F. R. Determination of the pK values for the alpha-amino groups of human hemoglobin. J. Biol. Chem. 250, 4398–4404 (1975). source

      Not checked in the free run.

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

    Premisenone given

    Many emerging covalent ligand screening platforms use MS-based detection, enabled by covalent bond formation between the compound and protein.

    “Many emerging covalent ligand screening platforms involve MS-based detection, enabled by covalent bond formation between the compound and protein.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Voxelotor has a red blood cell to plasma ratio of about 150 that likely reduces off-target effects, and it was approved by the FDA in 2019 at a recommended dose of 1.5 g daily, an unusually high dose for a covalent drug.

    “With a remarkable red blood cell to plasma ratio of ~150 that likely reduces off-target effects, voxelotor was approved by the FDA in 2019 with a recommended dose of 1.5 g daily, which is an unusually high dose for a covalent drug142,146.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [142] Vichinsky, E. et al. A phase 3 randomized trial of voxelotor in sickle cell disease. N. Engl. J. Med. 381, 509–519 (2019). source

      Not checked in the free run.

    • cites [146] Blair, H. A. Voxelotor: first approval. Drugs 80, 209–215 (2020). source

      Not checked in the free run.

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

    Premisecitation

    Sickle cell anaemia is caused by a single mutation in the β-haemoglobin gene that induces polymerization of mutant haemoglobin (HbS) under hypoxic conditions.

    “Sickle cell anaemia is caused by a single mutation in the gene encoding the β-haemoglobin chain that induces polymerization of mutant haemoglobin (HbS) under hypoxic conditions142.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [142] Vichinsky, E. et al. A phase 3 randomized trial of voxelotor in sickle cell disease. N. Engl. J. Med. 381, 509–519 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Bortezomib was the first boron-containing drug approved by the FDA and was approved for multiple myeloma in 2003.

    “Bortezomib (Takeda) was the first boron-containing drug to be approved by the FDA, and was approved for treatment of multiple myeloma in 2003 (ref.120).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [120] Bross, P. F. et al. Approval summary for bortezomib for injection in the treatment of multiple myeloma. Clin. Cancer Res. 10, 3954–3964 (2004). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The chloroacetamide sulfopin was selective for Pin1 in a CITe-Id chemoproteomics experiment and regressed neuroblastoma growth in mice.

    “The resulting chloroacetamide sulfopin was shown to be selective for Pin1 in a covalent inhibitor target-site identification (CITe-Id) chemoproteomics experiment and was effective in regressing neuroblastoma growth in mice157.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [157] Dubiella, C. et al. Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo. Nat. Chem. Biol. 17, 954–963 (2021). source

      Not checked in the free run.

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

    Interpretationreasoning

    Determination of kinact/Ki provides the most complete information to medicinal chemists about the binding affinity and kinetics of covalent target engagement.

    “Regardless, determination of kinact/Ki provides the most complete information to medicinal chemists about the binding affinity and kinetics of covalent target engagement.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Assaying for reactivity is a crucial step, particularly for key molecules and for compound series containing novel electrophiles.

    “Assaying for reactivity is a crucial step, particularly for key molecules and for compound series containing novel electrophiles.”
    Quote in the paperSubstantial claim

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

    Premisenone given

    ABPP approaches have transformed the characterization of electrophilic compounds, aiding selectivity profiling and target identification.

    “Activity-based protein profiling (ABPP) approaches have transformed the characterization of electrophilic compounds, facilitating selectivity profiling and target identification experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Voxelotor was discovered through a structure-guided effort to increase HbS oxygen affinity and was designed to bind the HbS tetramer in a 1:1 stoichiometry, unlike the 2:1 ratio of earlier compounds.

    “Based on earlier aldehydes, voxelotor was discovered through a structure-guided effort to discover compounds that increase the oxygen affinity of HbS, and was designed to bind the HbS tetramer in a 1:1 stoichiometry, unlike the 2:1 ratio of earlier compounds143,145.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [143] Metcalf, B. et al. Discovery of GBT440, an orally bioavailable r-state stabilizer of sickle cell hemoglobin. ACS Med. Chem. Lett. 8, 321–326 (2017). source

      Not checked in the free run.

    • cites [145] Oksenberg, D. et al. GBT 440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease. Br. J. Haematol. 175, 141–153 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CovDock does not consider electrophile reactivity, which can limit virtual study of docking ligands with different electrophilic functional groups.

    “This approach does not consider the reactivity of the electrophile, which can limit the ability to virtually study the differences in docking ligands with different electrophilic functional groups167.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [167] Scarpino, A., Ferenczy, G. G. & Keserű, G. M. Comparative evaluation of covalent docking tools. J. Chem. Inf. Model. 58, 1441–1458 (2018). This article compares the reproducibility of known binding data for covalent complexes across AutoDock4, CovDock, FITTED, GOLD, ICM-Pro and MOE docking programs. source

      Not checked in the free run.

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

    Premisenone given

    Many covalent drug discovery programmes, including those for covalent EGFR and BTK inhibitors, have added reactive functional groups to previously identified ligands.

    “Many covalent drug discovery programmes, including those for covalent EGFR and BTK inhibitors, have involved the addition of reactive functional groups to previously identified ligands.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The discovery of covalent proteasome inhibitors illustrates the utility of boronic acid electrophiles for protease active sites and shows that a project can start with an unoptimized electrophile and later introduce an alternative one.

    “The discovery of these covalent proteasome inhibitors illustrates the utility of boronic acid electrophiles for targeting protease active sites and demonstrates how a covalent drug discovery project can use an unoptimized electrophile as a starting point and subsequently introduce an alternative electrophile.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisereasoning

    Advances in chemoproteomics have facilitated discovery of covalent ligands against undruggable targets and enabled proteome-wide selectivity profiling to identify targets and off-targets.

    “Advances in chemoproteomics have facilitated the discovery of covalent ligands against undruggable disease targets and enabled the selectivity profiling of covalent ligands across the proteome to identify targets and off-targets of these ligands.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    These aldehydes bind reversibly and covalently, forming a Schiff base with the N-terminal valine of the α-haemoglobin chain.

    “These aldehydes bind in a reversible covalent manner, forming a Schiff base with the N-terminal valine of the α-haemoglobin chain143.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [143] Metcalf, B. et al. Discovery of GBT440, an orally bioavailable r-state stabilizer of sickle cell hemoglobin. ACS Med. Chem. Lett. 8, 321–326 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Voxelotor is a lysine-targeting covalent drug used to treat sickle cell anaemia, and its discovery depended on knowledge of heightened lysine side chain reactivity.

    “Voxelotor (Global Blood Therapeutics) is a lysine-targeting covalent drug used to treat sickle cell anaemia, and the discovery of voxelotor (Table 1) was dependent on knowledge of heightened lysine side chain reactivity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    The development of bortezomib validated the proteasome as a cancer target and encouraged discovery of other proteasome inhibitors.

    “The development of bortezomib validated the proteasome as a cancer target, encouraging the discovery of other proteasome inhibitors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    These boron-based drugs target serine hydrolases, and the weak boron-sulfur bond potentially provides selectivity for serine over cysteine hydrolases.

    “These boron-based drugs target serine hydrolases, and the weak boron–sulfur bond potentially provides selectivity for serine over cysteine hydrolases121.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [121] Adams, J. et al. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg. Med. Chem. Lett. 8, 333–338 (1998). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Using highly reactive electrophiles to gain an initial foothold can be beneficial, although targeting especially nucleophilic protease active sites may allow greater flexibility in electrophile choice.

    “Although targeting especially nucleophilic protease active sites might provide greater flexibility in terms of electrophile choice, using highly reactive electrophiles to gain an initial foothold can be beneficial.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    The growth of commercial libraries of electrophilic fragment-like compounds is a key factor contributing to the rise of electrophile-first discovery strategies.

    “The growth of commercial libraries of electrophilic fragment-like compounds is a key factor contributing to the rise of these electrophile-first discovery strategies.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    The electrophile-first approach has been successful for KRAS(G12C) and has facilitated rapid discovery of E3 ligase ligands for targeted protein degradation.

    “For example, this type of approach proved successful in drugging KRAS(G12C) and has facilitated the rapid discovery of E3 ligase ligands for targeted protein degradation applications.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    This carfilzomib mechanism has been proposed to confer selectivity because most proteases lack nucleophilic side chains at their N terminus.

    “this mechanism has been proposed to confer selectivity because most proteases do not have nucleophilic side chains at their N terminus127.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [127] Groll, M., Kim, K. B., Kairies, N., Huber, R. & Crews, C. M. Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of α‘,β‘-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 122, 1237–1238 (2000). source

      Not checked in the free run.

  • May combine two claims.

    Premisereasoning

    DNA-encoded libraries enable screening of massive small-molecule libraries, but covalency provides no specialized advantage for DEL screening.

    “DNA-encoded libraries (DELs) present an alternative approach that enables the screening of massive libraries of small molecules. Unlike MS or ABPP approaches, there is no specialized advantage of covalency in enabling DEL screening”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    CovDock uses traditional non-covalent docking to dock a ligand to a protein target, then models the covalent bond attachment and refines the complex.

    “CovDock uses traditional non-covalent docking approaches to dock a ligand to a protein target, and then models the covalent bond attachment and refines the complex166.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [166] Sotriffer, C. Docking of covalent ligands: challenges and approaches. Mol. Inf. 37, 1800062 (2018). source

      Not checked in the free run.

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

    Premisecitation

    Fixed-time-point IC50 values can serve as a valuable surrogate for kinact/Ki, as suggested by scientists at Pfizer.

    “Scientists working on a covalent JAK3 inhibitor programme at Pfizer, however, suggested that fixed-time-point IC50 values can serve as a valuable surrogate for kinact/Ki (ref. 218).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [218] Thorarensen, A. et al. The advantages of describing covalent inhibitor in vitro potencies by IC50 at a fixed time point. IC50 determination of covalent inhibitors provides meaningful data to medicinal chemistry for SAR optimization. Bioorg. Med. Chem. 29, 115865 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Boceprevir and telaprevir were effective in treating HCV and were approved by the FDA in 2011 after successful trials.

    “Boceprevir and telaprevir were effective in treating HCV and were approved by the FDA in 2011 after successful trials115,116.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [115] Kwo, P. Y. et al. Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial. Lancet 376, 705–716 (2010). source

      Not checked in the free run.

    • cites [116] Sherman, K. E. et al. Response-guided telaprevir combination treatment for hepatitis C virus infection. N. Engl. J. Med. 365, 1014–1024 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In 2019, isoTOP-ABPP identified RNF114 as the target of the enone-containing natural product nimbolide, which was used to make bifunctional degraders of BRD4 and BCR-ABL.

    “In 2019, isoTOP-ABPP was used to identify RNF114 as the target of the enone-containing natural product nimbolide, which was used to make bifunctional degraders of bromodomain-containing protein 4 (BRD4) and BCR–ABL187.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [187] Spradlin, J. N. et al. Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. Nat. Chem. Biol. 15, 747–755 (2019). source

      Not checked in the free run.

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

    Premisecitation

    ABPP probes contain a warhead that covalently reacts with nucleophilic amino acids such as cysteine and a reporter handle such as a fluorophore, biotin, or alkyne moiety for click chemistry.

    “ABPP probes contain a warhead that covalently reacts with nucleophilic amino acids (such as cysteine) and a reporter handle to monitor probe binding, such as a fluorophore, biotin or alkyne moiety for subsequent click chemistry-enabled applications173 (Fig. 5a).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [173] Cravatt, B. F., Wright, A. T. & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008). This review highlights the tools involved in ABPP and explains their corresponding applications. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A TMT-based SLC-ABPP methodology was designed and used to profile an electrophilic fragment library at a depth of more than 8,000 reactive cysteine sites with 18 min total instrument time per compound.

    “a tandem mass tag (TMT)-based streamlined cysteine activity-based protein profiling (SLC-ABPP) methodology was designed and used to profile an electrophilic fragment library at an impressive depth of more than 8,000 reactive cysteine sites with a total instrument time of 18 min per compound183.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [183] Kuljanin, M. et al. Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. Nat. Biotechnol. 39, 630–641 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    Reactivity profiling produces large-scale information across thousands of proteins and gives a relatively unbiased view of nucleophilic amino acid reactivity, usually cysteine.

    “Overall, reactivity profiling generates large quantities of information across thousands of proteins, ultimately providing a relatively unbiased picture of nucleophilic (usually cysteine) amino acid reactivity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Beyond identifying ABHD6 as crucial for metastatic fitness, the approach enabled screening in a biological context more relevant to disease through adaptation of covalent ligand screening to a multiplexed in vivo phenotypic assay.

    “Beyond identifying the lipase ABHD6 as crucial for metastatic fitness, this approach enabled screening in a biological context more relevant to the disease state through adaptation of covalent ligand screening to a multiplexed in vivo phenotypic assay.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [186] Grüner, B. M. et al. An in vivo multiplexed small-molecule screening platform. Nat. Methods 13, 883–889 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    EN4 targets MYC Cys171, which lies in a predicted intrinsically disordered region, and showed proteome-wide selectivity in profiling more than 1,500 cysteines by competitive isoTOP-ABPP.

    “EN4 targets Cys171, which is located within a predicted intrinsically disordered region of MYC, and showed selectivity on a proteome-wide scale in profiling of more than 1,500 cysteines using competitive isoTOP-ABPP.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [179] Boike, L. et al. Discovery of a functional covalent ligand targeting an intrinsically disordered cysteine within MYC. Cell Chem. Biol. 28, 4–13 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Reactivity profiling information can be used to select protein targets for drug discovery or to identify allosteric sites on proteins previously considered un-ligandable or undruggable.

    “This information can be used to either select for appropriate protein targets in drug discovery programmes or to identify allosteric sites on proteins of interest that may have previously been considered un-ligandable or undruggable.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In that study, more than 700 ligandable cysteines were identified and proteome-wide selectivity information was provided for each covalent fragment in the library.

    “In this study, more than 700 ligandable cysteines were identified, and information was provided about the proteome-wide selectivity of each covalent fragment in the library.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [180] Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016). This work demonstrates how chemoproteomics can be paired with covalent ligand screening to identify the protein targets of covalent ligands in complex biological samples. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    IsoTOP-ABPP identified the lipase ABHD6 as the target of hit compounds from that screen, although ABHD6 was not previously known to have a role in metastasis or cancer progression.

    “IsoTOP-ABPP experiments also enabled the identification of the lipase ABHD6 as the target of hit compounds from this screen, even though ABHD6 was not previously known to have a role in metastasis or cancer progression.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [186] Grüner, B. M. et al. An in vivo multiplexed small-molecule screening platform. Nat. Methods 13, 883–889 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Covalent ligands are useful not only as functional inhibitors but also have important roles in emerging induced proximity modalities.

    “Covalent ligands are not only useful as functional inhibitors, but also have important roles in emerging induced proximity modalities.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Cys171 was first identified as a ligandable hotspot on MYC from compiled cysteine-reactive chemoproteomics data, which prompted the search for a selective covalent ligand against it.

    “Cys171 was initially identified as a ligandable hotspot on MYC through analysis of compiled cysteine-reactive chemoproteomics data, and this information spurred the subsequent search for a selective covalent ligand against that cysteine.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [179] Boike, L. et al. Discovery of a functional covalent ligand targeting an intrinsically disordered cysteine within MYC. Cell Chem. Biol. 28, 4–13 (2020). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In a separate study, scout fragments were used to construct bifunctional FKBP12 and BRD4 degraders, and DCAF16 was identified as the target E3 ligase responsible for degradation.

    “In a separate study, scout fragments were used to construct bifunctional FKBP12 and BRD4 degraders, and the authors identified DCAF16 as the target E3 ligase responsible for degradation190.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [190] Zhang, X., Crowley, V. M., Wucherpfennig, T. G., Dix, M. M. & Cravatt, B. F. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat. Chem. Biol. 15, 737–746 (2019). source

      Not checked in the free run.

    • Rests on an assumption: Most bifunctional degraders recruit CRBN or VHL, but there are more than 600 E3 ligases with varying substrate scopes.

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

    Interpretationreasoning

    Target identification experiments using chemoproteomic platforms are crucial for investigating the mechanism of action of electrophilic compounds discovered through phenotypic assays.

    “In general, target identification experiments using chemoproteomic platforms are crucial in investigating the mechanism of action of electrophilic compounds discovered through phenotypic assays.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    ABPP can identify off-targets and assess the selectivity of covalent molecules.

    “ABPP can also be used to identify off-targets and generally assess the selectivity of covalent molecules.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Such ligands include a kinetic transthyretin stabilizer, an isoform-selective PI3Kδ inhibitor, and inhibitors of CDK2 and Hsp90.

    “Such ligands include a kinetic transthyretin stabilizer204, an isoform-selective PI3Kδ inhibitor205 and inhibitors of cyclin-dependent kinase 2 (CDK2)206 and Hsp90 (ref.207).”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [204] Grimster, N. P. et al. Aromatic sulfonyl fluorides covalently kinetically stabilize transthyretin to prevent amyloidogenesis while affording a fluorescent conjugate. J. Am. Chem. Soc. 135, 5656–5668 (2013). source

      Not checked in the free run.

    • cites [205] Dalton, S. E. et al. Selectively targeting the kinome-conserved lysine of PI3Kδ as a general approach to covalent kinase inhibition. J. Am. Chem. Soc. 140, 932–939 (2018). source

      Not checked in the free run.

    • cites [206] Anscombe, E. et al. Identification and characterization of an irreversible inhibitor of CDK2. Chem. Biol. 22, 1159–1164 (2015). source

      Not checked in the free run.

    • cites [207] Cuesta, A., Wan, X., Burlingame, A. L. & Taunton, J. Ligand conformational bias drives enantioselective modification of a surface-exposed lysine on Hsp90. J. Am. Chem. Soc. 142, 3392–3400 (2020). source

      Not checked in the free run.

    • Rests on an assumption: Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.
    • Rests on an assumption: The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

  • May combine two claims.

    Premisecitation

    Sulfonyl fluoride-based probes are not completely selective for lysine and have also been used to target tyrosine residues.

    “However, sulfonyl fluoride-based probes are not completely selective for lysine and have also been used to target tyrosine residues209.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [209] Hett, E. C. et al. Rational targeting of active-site tyrosine residues using sulfonyl fluoride probes. ACS Chem. Biol. 10, 1094–1098 (2015). source

      Not checked in the free run.

    • Rests on an assumption: Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.
    • Rests on an assumption: The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

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

    Premisenone given

    Over the past decade, advances in covalent drug discovery have led to successful drugs including inhibitors of EGFR, BTK, KRAS(G12C) and SARS-CoV-2 Mpro.

    “Over the past decade, advances in covalent drug discovery have led to successful drugs, including inhibitors of EGFR, BTK, KRAS(G12C) and SARS-CoV-2 Mpro.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    A library of approximately 180 electrophiles was assembled and isoTOP-ABPP experiments were performed to assess selectivity of different chemotypes and identify lysines ligandable with small molecules.

    “a library of approximately 180 electrophiles was assembled and isoTOP-ABPP experiments then performed to assess the selectivity of different chemotypes and identify lysines ligandable with small molecules202.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [202] Abbasov, M. E. et al. A proteome-wide atlas of lysine-reactive chemistry. Nat. Chem. 13, 1081–1092 (2021). source

      Not checked in the free run.

    • Rests on an assumption: Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.
    • Rests on an assumption: The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

  • May combine two claims.

    Premisecitation

    Through isoTOP-ABPP experiments, more than 9,000 ligandable lysines were identified, and more elaborated pentafluorophenol-containing or N-hydroxysuccinimide-ester-containing compounds could selectively label specific proteins of interest.

    “Through isoTOP-ABPP experiments, more than 9,000 ligandable lysines were identified and more elaborated pentafluorophenol-containing or N-hydroxysuccinimide-ester-containing compounds could selectively label specific proteins of interest200,201.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [200] Hacker, S. M. et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem. 9, 1181–1190 (2017). source

      Not checked in the free run.

    • cites [201] Ward, C. C., Kleinman, J. I. & Nomura, D. K. NHS-esters as versatile reactivity-based probes for mapping proteome-wide ligandable hotspots. ACS Chem. Biol. 12, 1478–1483 (2017). source

      Not checked in the free run.

    • Rests on an assumption: Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.
    • Rests on an assumption: The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

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

    Interpretationnone given

    Adoption of electrophile-first discovery strategies represents a notable shift in the field.

    “Adoption of electrophile-first discovery strategies represents a notable shift in the field.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    That study yielded more broadly reactive electrophiles, such as dicarboxaldehydes, for further lysine profiling, and identified less-reactive electrophiles, including N-acyl-N-alkyl sulfonamides, previously used as bioconjugation tools in cells.

    “This study yielded more broadly reactive electrophiles, such as dicarboxaldehydes, that could be used for further lysine profiling experiments, but also identified less-reactive electrophiles, including N-acyl-N-alkyl sulfonamides, which had been previously used as tools for bioconjugation in cells202,203.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [202] Abbasov, M. E. et al. A proteome-wide atlas of lysine-reactive chemistry. Nat. Chem. 13, 1081–1092 (2021). source

      Not checked in the free run.

    • cites [203] Tamura, T. et al. Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide. Nat. Commun. 9, 1870 (2018). source

      Not checked in the free run.

    • Rests on an assumption: Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.
    • Rests on an assumption: The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

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

    Interpretationnone given

    Covalent drug discovery overcomes obstacles in designing ligands against otherwise undruggable protein targets.

    “Covalent drug discovery overcomes obstacles in designing ligands against otherwise undruggable protein targets.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The approvals of these drugs are milestones that show covalent drug discovery has evolved from a serendipitous effort to a field with established roadmaps for success.

    “The approvals of these drugs represent milestones that showcase the evolution of covalent drug discovery from a serendipitous effort to a field with established roadmaps for success.”
    Quote in the paperNot a substantial claim
83 of 83

  • The paper relies on this without support.

    Each covalent drug candidate must balance reactivity, potency, and selectivity.

    “each compound must balance reactivity, potency and selectivity.”

  • The paper relies on this without support.

    Cysteine's low abundance provides selectivity but restricts opportunities to covalently target specific proteins.

    “The low abundance of cysteine enables selectivity but limits opportunities for covalently targeting specific proteins of interest.”

  • The paper relies on this without support.

    The ε-amino group of lysine is not nucleophilic enough under physiological conditions, so efficient lysine-targeting covalent ligands require unusually reactive lysines.

    “Owing to the low nucleophilicity of the ε-amino group of lysine under physiological conditions, the discovery of efficient lysine-targeting covalent ligands requires identification of unusually reactive lysines.”

  • The paper relies on this without support.

    Most protein-reactive covalent drugs are electrophilic so that they can react with nucleophilic amino acids.

    “Most protein-reactive covalent drugs tend to be electrophilic, to enable reactions with nucleophilic amino acids.”

  • The paper relies on this without support.

    Nucleophilic drugs can react with electrophilic cofactors and post-translational modifications.

    “By contrast, nucleophilic drugs can react with electrophilic cofactors and post-translational modifications.”

  • The paper relies on this without support.

    Most bifunctional degraders recruit CRBN or VHL, but there are more than 600 E3 ligases with varying substrate scopes.

    “Although most bifunctional degrader molecules (proteolysis-targeting chimeras (PROTACs)) recruit the E3 ligases cereblon (CRBN) or von Hippel–Lindau (VHL) protein to degrade target proteins, there are more than 600 E3 ligases with varying substrate scopes.”
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?

  • Afatinib forms hydrogen bonds to the main chain of Met793 in the hinge region and interacts with hydrophobic regions; the furanyl group is exposed to the solvent, and the 3-chloro-4-fluorophenyl group is located near the gatekeeper residue.

    “Similar to the first-generation EGFR TKI, afatinib forms hydrogen bonds to the main chain of Met793 in the hinge region and interacts with hydrophobic regions. The furanyl group is exposed to the solvent, and the 3-chloro-4-fluorophenyl group is located near the “gatekeeper” residue.50”

    From Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials (2023) · cites this paper as [50]

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.

  • Voxelotor is assigned two different amino-acid targets

    Minor

    The text calls voxelotor lysine-targeting, but later says its aldehyde-based compounds form a covalent bond with the N-terminal valine of the α-haemoglobin chain. Valine and lysine are different amino acids.

  • Tavaborole does not fit the stated target class

    Minor

    The paragraph groups these boron-based drugs as serine-hydrolase inhibitors, but describes tavaborole as trapping tRNA in leucyl-tRNA synthetase through bonds to tRNA hydroxy groups.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. McWhirter, C. in Annual Reports in Medicinal Chemistry (eds Ward, R. A. & Grimster, N. P.) vol. 56 1–31 (Elsevier, 2021).

Cited by

Sorted by citations

Authors
Boike, L., Henning, N. J., & Nomura, D. K.
Affiliations
Innovative Genomics Institute (US)
Corresponding author
Nomura, Daniel K.
ORCID
Venue
Nature Reviews Drug Discovery
Publisher
Springer Science and Business Media LLC
Published
2022-08-25
Volume
21
Issue
12
Pages
881-898
ISSN
  • 1474-1776 (ISSN-L)
  • 1474-1784
DOI
10.1038/s41573-022-00542-z
PMC
PMC9403961
OpenAlex
W4293084539
Semantic Scholar
b94c42b5629eb6be15dea45d3c538519c52f5ee7
Cited by
823
References
219
Citations by year
2026: 172 · 2025: 256 · 2024: 236 · 2023: 147 · 2022: 12
FWCI
15.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Reviews Drug Discovery (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Science Foundation
  • Mark Foundation For Cancer Research
  • National Institutes of Health
  • National Cancer Institute (F31CA268278, R01CA240981, R35CA263814)
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
Keywords
covalent drug discovery, covalent inhibitors, chemoproteomics, KRAS G12C inhibitors, EGFR inhibitors, BTK inhibitors, reactive functional groups, target identification, selectivity profiling
MeSH terms
Agammaglobulinaemia Tyrosine Kinase, SARS-CoV-2, COVID-19 Drug Treatment, Humans, Ligands, Structure-Activity Relationship, Proto-Oncogene Proteins p21(ras), Protein Kinase Inhibitors, Drug Discovery, ErbB Receptors

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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 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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