Biomedical Engineering Reference
In-Depth Information
fluorescent dyes and applied onto SMMs. The fluorescent reading will provide quali-
tative information on the interaction of small molecules and proteins. As an alternative
approach, researchers can use sandwich-based assays, which provide high specificity
and sensitivity. In this process the protein is first incubated with SMM. After multiple
washing cycles, fluorescently labeled antibodies can be applied to identify the hits
on the array. Corresponding negative control should be included in the experiment
design because some antibodies might bind to microarray nonspecifically. If quan-
titative data such as
K
d
are required, concentration-dependent experiments can be
performed on the array directly by applying fluorescent-labeled proteins of various
concentrations. Using quantitative data has several advantages. For example, it gives
information on how strong the small molecule binds to proteins. It also helps to
significantly reduce the probability of obtaining false positives.
In 2004, Wong et al. synthesized 18,000 enatioenriched 1,3-dioxanes and screened
their activity on a microarray [57]. This is the first report that stereochemically diverse
sets of small molecules had been screened on the array. The library was synthesized
by one bead-one stock solution technology. By probing the array with a fluorescently
labeled calmodulin, the workers showed clear evidence that a heptamethyleneimine
moiety is important for calmodulin-binding activity. In a zebrafish phenotypic assay,
the authors further identified an inhibitor of cardiovascular function in an enantiomer-
dependent manner. Later, Bradner et al. extended the strategy to immobilize around
10,000 small molecules from a DOS library and from natural products onto an
isocyanate-functionalized glass slide (Figure 13.6a) [58]. Several bioactive com-
pounds were detected for their ability to bind to their corresponding antibodies on
the array. The authors demonstrated further that with the strategy they were able to
directly screen small molecules with cellular lysates from human cells without prior
purification of the protein. This technique offers a valuable and workable approach
to identifying small-molecule ligands of the proteins that are inaccessible through
standard protein purification process.
In 2005, Reddy and Kodadek synthesized a 7680-member peptoid library
using a submonomer approach, and printed the peptoid library onto a maleimide-
functionalized surface [59]. Unique and reproducible protein fingerprints were
observed after applying three different fluorescently labeled proteins: MBP, GST,
and ubiquitin. This approach can serve as a useful tool for protein identification with-
out knowing the identity of the small molecules on the array. It can also be applied
to other complicated fingerprinting assay, such as the diagnosis of cancers.
13.4.2 Enzyme Substrate/Inhibitor Profiling
To acquire the substrate specificity of enzymes, several groups have developed differ-
ent strategies with SMMs. Enzymes such as kinases, phosphatases, and proteases play
important roles in the regulation of many cellular functions. The characterization of
an enzyme activity pattern could help researchers to gain a more comprehensive view
of the catalytic mechanism and the properties of the enzyme. The Yao group synthe-
sized and immobilized a variety of fluorogenic coumarin derivatives onto an array
and used it to screen against four different classes of enzymes [60]. Three enzymes
