Biomedical Engineering Reference
In-Depth Information
FIGURE 12.4
Diverse biochemical screening approaches.
It is quite common for an enzyme or protein to have more than one binding site or
partner. For example, p38 MAP kinase has three ligand-binding sites, corresponding
to Mg
2
+
ATP, protein substrate, and MKP1 dual-specificity protein phosphatase, as
well as an allosteric site generated by movement of the enzyme's activation loop.
The center panel of Figure 12.4 illustrates a protein similar to p38. This hypothetical
protein performs catalytic conversion of a substrate and binds to a small-molecule
ligand as well as to a protein partner, thus providing multiple venues for designing
functional and displacement assays (Figure 12.4a to c).
One main commonality between all displacement and enzymatic assays is that
they are sensitive to a binding of small molecules in a specific (“orthosteric”) site and
frequently are insensitive to the binding in allosteric sites. Thus, screening with any
single ligand of choice will result in a narrow set of compounds targeting the specific
ligand-binding site, as illustrated through the outcomes of the “screening funnels”
shown in Figure 12.4a to c. Therefore, compounds against different binding sites of
a single target could be identified but would require a panel of several displacement
assays. This paradigm is not only costly, in general, but clearly impossible for a
protein with no known ligands. Thus, detection of direct compound binding (Figure
12.4d) may be a preferred approach, especially for novel, poorly characterized targets.
