The reversible covalent bond formation in Tethering has both advantages and disadvantages. On the positive side, ESI/mass spectrometry detects hits rapidly. As a "positive" detection method, it is less prone to false positives than are typical inhibition assays [22]. Since the bond must be within several Angstroms of the cysteine residue, it roughly indicates where fragments bind. Moreoever, the bond facilitates modeling and crystallography: if the fragment is not highly soluble, the non-covalent complex may be difficult to crystallize and the disulfide-bonded complex is more likely to yield a structure. A related advantage is that the stoichi-ometry of the fragment in the complex is exactly one-to-one.

Of course, the very ability to detect real but weak binders is a double-edged sword. Typical affinities for fragments are initially weak (in the millimolar range). They can be improved through chemical optimization, but optimizing a very weakly binding fragment can be challenging. However, a second-generation version of the technology, Tethering with extenders, largely solves this problem (see below).

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