Advances in covalent drug discovery

In the paper

“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).”

What the sources say

• Partly supported

  The introductory passage following Abstract > Conclusions says the FDA approved osimertinib under the Breakthrough Therapy Designation Program, and reference 24 dates the cited FDA announcement to November 13, 2015. The same passage places treatment after first-line EGFR-TKI therapy, but it does not call the approval accelerated. Our copy does not address a 2018 first-line approval for metastatic NSCLC (searched: 2018 first-line approval). Page numbers were not supplied. (LLM, conf=0.88)

  cites [49] Mok, T. S. et al. Osimertinib or platinum–pemetrexed in EGFR T790M–positive lung cancer. N. Engl. J. Med. 376, 629–640 (2016). sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Supported

  The Introduction (page not provided) says that a KRAS codon 12 mutation attenuates GTPase activity, causing activated GTP-bound KRAS to accumulate and downstream signalling pathways to activate. The Discussion (page not provided) describes mutant KRAS as a driver of tumorigenesis. Together, these passages support the attributed mechanism, although the paper discusses codon 12 mutations specifically. (LLM, conf=0.88)

  cites [87] Janes, M. R. et al. Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell 172, 578–589.e17 (2018). source

In the paper

“Many early covalent drugs were discovered serendipitously and bind active sites to inhibit enzymatic activity4.”

What the sources say

• Not checked yetcites [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

In the paper

“However, osimertinib depends on Cys797 for covalent binding, and C797X mutations account for 15% of cases of resistance to second-line osimertinib50–52.”

What the sources say

• Not checked yetcites [50] Passaro, A., Jänne, P. A., Mok, T. & Peters, S. Overcoming therapy resistance in EGFR-mutant lung cancer. Nat. Cancer 2, 377–391 (2021). source
• Not checked yetcites [51] Leonetti, A. et al. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br. J. Cancer 121, 725–737 (2019). source
• Not checked yetcites [52] Thress, K. S. et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non–small cell lung cancer harboring EGFR T790M. Nat. Med. 21, 560–562 (2015). source

In the paper

“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.”

What the sources say

• Not checked yetcites [20] Vane, J. R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 231, 232–235 (1971).

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [30] Curran, M. P. & McKeage, K. Bortezomib: a review of its use in patients with multiple myeloma. Drugs 69, 859–888 (2009). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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
• Not checked yetcites [5] Sutanto, F., Konstantinidou, M. & Dömling, A. Covalent inhibitors: a rational approach to drug discovery. RSC Med. Chem. 11, 876–884 (2020). source

In the paper

“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”

What the sources say

• Not checked yetcites [9] Moore, A. R., Rosenberg, S. C., McCormick, F. & Malek, S. RAS-targeted therapies: is the undruggable drugged? Nat. Rev. Drug Discov. 19, 533–552 (2020). source
• Not checked yetcites [10] Huang, L., Guo, Z., Wang, F. & Fu, L. KRAS mutation: from undruggable to druggable in cancer. Signal Transduct. Target. Ther. 6, 386 (2021). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [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
• Not checked yetcites [44] Cross, D. A. E. et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 4, 1046–1061 (2014). source
• Not checked yetcites [45] Finlay, M. R. V. et al. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J. Med. Chem. 57, 8249–8267 (2014). source
• Not checked yetcites [46] Walter, A. O. et al. Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 3, 1404–1415 (2013). source

In the paper

“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.”

What the sources say

• Not checked yetcites [31] Lynch, T. J., Okimoto, R. A., Supko, J. G. & Settleman, J. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non–small-cell lung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139 (2004). source

In the paper

“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.”

What the sources say

• Not checked yetcites [22] Dijkmans, A. C. et al. Fosfomycin: pharmacological, clinical and future perspectives. Antibiotics 6, 24 (2017). source
• Not checked yetcites [23] Hendlin, D. et al. Phosphonomycin, a new antibiotic produced by strains of Streptomyces. Science 166, 122–123 (1969). source
• Not checked yetcites [24] Kahan, F. M., Kahan, J. S., Cassidy, P. J. & Kropp, H. The mechanism of action of fosfomycin (phosphonomycin). Ann. NY Acad. Sci. 235, 364–386 (1974). source

In the paper

“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”

What the sources say

• Not checked yetcites [11] Vandyck, K. & Deval, J. Considerations for the discovery and development of 3-chymotrypsin-like cysteine protease inhibitors targeting SARS-CoV-2 infection. Curr. Opin. Virol. 49, 36–40 (2021). source

In the paper

“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.”

What the sources say

• Not checked yetcites [7] Lonsdale, R. & Ward, R. A. Structure-based design of targeted covalent inhibitors. Chem. Soc. Rev. 47, 3816–3830 (2018). source
• Not checked yetcites [8] Sagonowsky, E. The top 20 drugs by worldwide sales in 2020. Fierce Pharma https://www.fiercepharma.com/special-report/top-20-drugs-by-2020-sales (2021).

In the paper

“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.”

What the sources say

• Not checked yetcites [36] Yun, C.-H. et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl Acad. Sci. USA 105, 2070–2075 (2008). source

In the paper

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

What the sources say

• Not checked yetcites [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

In the paper

“displayed potent inhibition in a FRET-based assay across all human coronaviruses, while no inhibitory effects were seen against human cysteine or serine proteases.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“ARS-853 treatment in KRAS(G12C)-dependent cell lines decreased the amount of active KRAS(G12C), inhibited downstream RAS signalling and induced apoptosis92.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [91] Lito, P., Solomon, M., Li, L.-S., Hansen, R. & Rosen, N. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science 351, 604–608 (2016). source

In the paper

“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.”

What the sources say

• Not checked yetcites [83] Kim, R. D. et al. First-in-human phase I study of fisogatinib (BLU-554) validates aberrant FGF19 signaling as a driver event in hepatocellular carcinoma. Cancer Discov. 9, 1696–1707 (2019). source
• Not checked yetcites [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).

In the paper

“Both JAK3 and mutant EGFR(T790M) contain a methionine gatekeeper, as well as a homologous cysteine adjacent to the ATP-binding site.”

What the sources say

• Not checked yetcites [79] Tan, L. et al. Development of selective covalent Janus kinase 3 inhibitors. J. Med. Chem. 58, 6589–6606 (2015). source

In the paper

“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.”

What the sources say

• Not checked yetcites [99] US FDA. Coronavirus (COVID-19) Update: FDA Authorizes First Oral Antiviral for Treatment of COVID-19. FDA https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-first-oral-antiviral-treatment-covid-19 (2021).

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

“SARS-CoV-2 Mpro shares 96% sequence identity with SARS-CoV-1 Mpro, and there is 100% sequence overlap of the catalytic sites102.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“activity in antiviral cell-based assays, stability in plasma and low clearance in vivo103.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [93] Canon, J. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019). source
• Not checked yetcites [95] Skoulidis, F. et al. Sotorasib for lung cancers with KRAS p.G12C mutation. N. Engl. J. Med. 384, 2371–2381 (2021). source

In the paper

“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).”

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [87] Janes, M. R. et al. Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor. Cell 172, 578–589.e17 (2018). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

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

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [146] Blair, H. A. Voxelotor: first approval. Drugs 80, 209–215 (2020). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

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

In the paper

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

In the paper

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

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

In the paper

“These aldehydes bind in a reversible covalent manner, forming a Schiff base with the N-terminal valine of the α-haemoglobin chain143.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“The development of bortezomib validated the proteasome as a cancer target, encouraging the discovery of other proteasome inhibitors.”

In the paper

“These boron-based drugs target serine hydrolases, and the weak boron–sulfur bond potentially provides selectivity for serine over cysteine hydrolases121.”

What the sources say

• Not checked yetcites [121] Adams, J. et al. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg. Med. Chem. Lett. 8, 333–338 (1998). source

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“this mechanism has been proposed to confer selectivity because most proteases do not have nucleophilic side chains at their N terminus127.”

What the sources say

• Not checked yetcites [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

In the paper

“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”

In the paper

“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.”

What the sources say

• Not checked yetcites [166] Sotriffer, C. Docking of covalent ligands: challenges and approaches. Mol. Inf. 37, 1800062 (2018). source

In the paper

“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).”

What the sources say

• Not checked yetcites [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

In the paper

“Boceprevir and telaprevir were effective in treating HCV and were approved by the FDA in 2011 after successful trials115,116.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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).”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

What the sources say

• Not checked yetcites [186] Grüner, B. M. et al. An in vivo multiplexed small-molecule screening platform. Nat. Methods 13, 883–889 (2016). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [186] Grüner, B. M. et al. An in vivo multiplexed small-molecule screening platform. Nat. Methods 13, 883–889 (2016). source

In the paper

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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

What the sources say

• Not checked yetcites [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

In the paper

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

In the paper

“ABPP can also be used to identify off-targets and generally assess the selectivity of covalent molecules.”

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [206] Anscombe, E. et al. Identification and characterization of an irreversible inhibitor of CDK2. Chem. Biol. 22, 1159–1164 (2015). source
• Not checked yetcites [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

In the paper

“However, sulfonyl fluoride-based probes are not completely selective for lysine and have also been used to target tyrosine residues209.”

What the sources say

• Not checked yetcites [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

In the paper

“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.”

In the paper

“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.”

What the sources say

• Not checked yetcites [202] Abbasov, M. E. et al. A proteome-wide atlas of lysine-reactive chemistry. Nat. Chem. 13, 1081–1092 (2021). source

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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

In the paper

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

In the paper

“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.”

What the sources say

• Not checked yetcites [202] Abbasov, M. E. et al. A proteome-wide atlas of lysine-reactive chemistry. Nat. Chem. 13, 1081–1092 (2021). source
• Not checked yetcites [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

In the paper

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

In the paper

“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.”