Biomedical Engineering Reference
In-Depth Information
biophysical SPR validation experiments. The small molecule discovered may serve
as an alternative to protein A and protein G for industrial antibody production.
Peptide-based libraries have frequently been used in microarray technology
because of their excellent bioactivities and availability, due towell-established peptide
chemistry. Peptide arrays can be employed to decipher substrate specificity and the
inhibition patterns of various classes of enzymes. In 2007, a 1400-member peptide
hydroxamate library was synthesized and immobilized onto avidin-functionalized
glass slides byUttamchandani et al. (Figure 13.2d) [23]. The fabricated arraywas used
to acquire the inhibitor fingerprints of four metalloproteases: thermolysin, collage-
nase, carboxypeptidase, and anthrax lethal factor (LF). The design of a peptide-based
library featured a universal hydroxamate warhead that could bind to a zinc ion in the
active-site pocket of the enzymes, and a distinct peptide fragment that determines
the binding specificity between the enzymes and the ligands. With this strategy, the
group was able to obtain unique inhibitor fingerprints with all four enzymes tested.
Several potent inhibitors against anthrax LF were identified and could be used for
further research development.
Carbohydrates represent another rich and important source for small-molecule
libraries. Carbohydrate arrays have proved to be a valuable tool to use to investi-
gate interactions between sugars and other biomolecules [24]. Microarray applica-
tions of carbohydrates could be valuable for disease diagnosis and vaccine develop-
ment. In the past there was immense difficulty in creating libraries with structurally
diverse carbohydrates. This hindrance has been alleviated with recent developments
in oligosaccharide synthesis. It is now even possible to synthesize defined carbo-
hydrates automatically on the solid support [25], and the duration of the synthesis
is reduced from months to days. In 2004, Blixt et al. constructed a carbohydrate
array with 200 synthetic and natural glycan sequences [26]. The team investigated
a number of fluorescently labeled glycan-binding proteins (GBPs), such as C-type
lectins, siglecs and galectins. The glycan array not only confirmed that the GBP
ligand specificity was consistent with previous reports, but also revealed several new
interactions.
The fragment-based approach is another method that has found wide use in design-
ing potent enzyme inhibitors. This strategy has recently been adopted by Wu et al. for
SMM application [27]. The group designed two hybrid libraries based on the original
14-3-3 peptide substrate RFRpSYPP. As depicted in Figure 13.2e, the pS residue was
retained, and the two flanking peptide fragments were replaced by acid and amine
building blocks, respectively. In total, 243-member N-terminal and 50-member C-
terminal hybrid libraries were synthesized and immobilized onto array. By screening
with the 14-3-3 protein, potent binders from the two sublibraries were identified and
reconstituted into new pS-containing small molecules. Experiments showed that one
of the reconstituted compounds exhibited strong binding affinity to 14-3-3 and could
compete for 14-3-3/ligand binding in vitro. Further experiments showed that it could
induce apoptosis and cell cycle arrest in cell-based assays.
Multiple component reactions (MCRs) have also found use in SMM. Lin et al.
constructed an SMM via Ugi four-component reactions on cellulose supports [28].
First, a photolabile linker was introduced onto a solid support for immobilization
