Site-directed ligand discovery
Open accessAbstract
We report a strategy (called “tethering”) to discover low molecular weight ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (≈1,200 compounds) at concentrations typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand ( K i ≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
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In 2013, Shokat and co-workers discovered a strategy using covalent inhibitors to bind to the cysteine of KRASG12C mutation and screened out lead compounds 6H05 and 2E07 using tethering.
“In 2013, Shokat and co-workers discovered a new strategy where they used covalent inhibitors to bind to the cysteine of KRASG12C mutation, and screened out two lead compounds, 6H05 (17) and 2E07 (18), by utilizing “tethering” technique.47,85,86”
From Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials (2023) · cites this paper as [85]
Tethering uses libraries of compounds linked to disulfides to identify fragments that bind cysteine-adjacent pockets.
“Originally, MS-based compound screening grew out of ‘tethering’, which is a technique employed since 2000 that uses libraries of compounds linked to disulfides to identify fragments that bind cysteine-adjacent pockets90,147.”
From Advances in covalent drug discovery (2022) · cites this paper as [147]
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- Authors
- Erlanson, D. A., Braisted, A. C., Raphael, D. R., Randal, M., Stroud, R. M., Gordon, E. M., & Wells, J. A.
- Affiliations
- University of California, San Francisco (US)
- Corresponding author
- Erlanson, Daniel A.
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2000-08-15
- Volume
- 97
- Issue
- 17
- Pages
- 9367-9372
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC16870
- OpenAlex
- W2150996942
- Cited by
- 466
- References
- 32
- Citations by year
- 2026: 13 · 2025: 14 · 2024: 20 · 2023: 20 · 2022: 12 · 2021: 15 · 2020: 26 · 2019: 17 · 2018: 19 · 2017: 8 · 2016: 24 · 2015: 14 · 2014: 23 · 2013: 24 · 2012: 20
- FWCI
- 13.32
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Small Business Innovation Research
- National Institutes of Health (1r43ca085141-01)
- National Cancer Institute (1 R43 CA85141-01)
- Topics
- Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
- Keywords
- low molecular weight ligands, thymidylate synthase, enzyme inhibitors, pyrimidine metabolism, affinity enhancement
- MeSH terms
- Binding Sites, Cysteine, Disulfides, Escherichia coli, Ligands, Models, Molecular, Molecular Weight, Proline, Protein Conformation, Thermodynamics, Thymidylate Synthase, Drug Design, Protein Engineering, Crystallography, X-Ray, Glutamic Acid, Peptide Library, Amino Acid Substitution
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