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Disulfide Trapping

Disulfide trapping is a fragment screening method that allows the targeting of specific sites of interest on proteins. In this approach, a protein containing a native or introduced cysteine residue near a site of interest is screened against a library of thiol-containing drug-like fragments under partially reducing conditions that promote thiol-disulfide exchange. Fragments that form both non-covalent and covalent (disulfide bond) binding interactions near a cysteine residue are stabilized and therefore persist, whereas fragments that bind non-specifically (disulfide bond only) are reduced away. The fragment-modified protein is stable and is readily detected by high throughput mass spectrometry, allowing unequivocal identification of the bound fragment. By screening pools of distinct-mass fragments, thousands of fragments can be screened per week. The low hit rate observed (~1 in 1000) speaks to the selectivity of the method and permits the screening of proteins containing multiple thiols without fear of multiple-fragment binding. Crystallographic studies have demonstrated that disulfide-trapped fragments bind in the same mode as the corresponding free (un-tethered) fragment, thus validating this approach for the design of non-covalently bound ligands. The method is particularly well-suited to the identification of novel allosteric sites in proteins, as demonstrated by recent work in the Wells lab on the caspases.