Other screening methods

A technique called tethering, in which a library of di-sulfide containing fragments are reacted with active site cysteine-containing mutant target proteins in order to form covalent adducts, has been applied to the extremely challenging autoimmune disease target IL2 [30]. Ten cysteine mutants of the IL-2 protein were screened against 7000 disulfide fragments. Aromatic acids, such as compound 13, were detected to bind to a lipophilic region of the active site and this guided optimisation of a lead series. Fragment tethering has been successfully applied to a number of challenging targets, and the area has been reviewed [4]. Another example of fragments binding covalently to an enzyme has been reported for the protease thrombin [31]. Finally, compound 14 was identified as one of six hits from screening against thrombin of a library of 100,000 fragments covalently attached in a microarray format using surface plasmon resonance (SPR) [32]. Fragment 14 was a strong SPR hit when combined with a second guanidine-containing fragment in the array. This information was indirectly used to help design larger micromolar inhibitors of the enzyme. Applications and developments of SPR have recently been reviewed [33].

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