This chapter has presented an introduction to Tethering with a focus on how mass spectrometry enables the technology to rapidly find inhibitors to drug

Fig. 9.10 Tethering with extenders to identify caspase-1 inhibitors. Two of the hits from tethering are shown, as are inhibitors derived from them.

targets. The technology has also been employed to identify inhibitors to proteinprotein interactions and to identify fragments that bind to allosteric sites [31-34]. Although mass spectrometry has been critical in most applications of Tethering, in some cases functional screens are sufficient, particularly where MS is challenging, such as membrane-bound proteins [35-36].

As a fragment-identification method, Tethering is one of many possible approaches. The technique is unique because it uses a covalent bond, which ensures that fragments bind in a stoichiometric fashion, and also allows rapid identification of low-affinity fragments through mass spectrometry. Tethering can target specific sites and wide-ranging conformations of a protein. Moreover, there is some evidence that by employing mass spectrometry, Tethering can identify fragments that bind more weakly than those identified by other methods. For example, the sulfamoyl salicylic acid fragment identified in caspase-3 showed no detectable inhibition by itself. Although this increases the range of fragments accessible to the medicinal chemist, it does raise the question of whether some fragments may be so weak as to be essentially useless for further development. Nonetheless, given the success observed thus far, we believe there are many untapped opportunities for Tethering.

318 | 9 Tethering: Fragment-based Drug Discovery by Mass Spectrometry Acknowledgements

We thank all of our colleagues at Sunesis Pharmaceuticals for their contributions, without which Tethering would not have been possible, and Monya L. Baker for editorial assistance. Portions of this chapter appeared in a similar form in a previous book in this series [20], and Marcus D. Ballinger and James A. Wells both contributed to the editing of these sections.

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