Biomedical Engineering Reference
In-Depth Information
inhibition profiles of these small molecules. The Yao group made a similar use of
a protease-sensitive surface to obtain the inhibitor fingerprints of metalloproteases
[63]. The plain glass surface was first functionalized with a layer of fluorogenic
substrate. Nanodroplets with 400 inhibitors and enzymes were then applied to this
protease-sensitive glass surface. The inhibitor fingerprinting profiles could be derived
according to the fluorescent readout. The best inhibitor identified in the experiment
showed a
K
i
value of 2.4 nM against thermolysin. The aforementioned approaches
could present a promising tool for the discovery of new and selective inhibitors in a
high-throughput manner.
Another research group from Kyushu University, Han et al., undertook an exten-
sive investigation on the substrate specificity of various kinases by chemoselectively
immobilizing 290 Tyr peptides and 1100 Ser/Thr peptides onto a glass surface (Figure
13.6b) [64]. Many new, potent, and selective peptides were identified for each kinase.
The peptide array not only identified a previously reported consensus motif, but also
disclosed some novel motifs. Waldmann's and Yao's groups, independently, devel-
oped phosphopeptide arrays to decipher substrate specificity against various protein
phosphatases [46,65,66]. Several groups have also generated carbohydrate arrays to
test the activity of glycosyltransfreases. Bryan et al. screened potential inhibitors of
fucosyltransferase using a carbohydrate array [67]. Park and Shin immobilized 20
different carbohydrate probes on the array and investigated the substrate specificity
of galactosyltransferase [68]. These novel strategies are helpful for understanding the
substrate specificity of enzymes and provided invaluable insights for inhibitor design
and discovery.
13.4.3 Other Applications
Apart from the aforementioned applications, SMMs have found many other novel
applications in light of recent technological advancements. Bailey et al. developed an
array-based system to screen the toxicity of small molecules against mammalian cells
[69]. In this design, small molecules were embedded in the biodegradable poly(lactic
acid)-poly(glycolic acid) copolymer (PLGA) and assembled in a microarray. Cells
were then seeded over the array. As the polymer degraded, the compounds could be
released into neighboring cells in a controlled environment. The strategy was demon-
strated by applying toxic compounds against 1549 cells, as well as by synthetic lethal
screening experiments. In 2005, Lee et al. developed a metabolizing enzyme toxicity
assay on the array to investigate the cytotoxicity of these prodrugs after P450 activa-
tion [70]. The authors successfully assembled mixtures of sol-gel-encapsulated P450
and prodrugs in a microarray. A cell monolayer was then applied to this sol-gel array.
After incubation, the cells can be removed and stained with fluorescence reagents for
conventional microarray analysis. This technology may present an inexpensive and
alternative approach for humanmetabolismand toxicology screeningwith liver slices.
During their recent research, Liang, Liao, and co-workers have constructed a
sialosides array to profile the binding pattern against various influenza hemagglutinin
(HA) subtypes (Figure 13.6c) [71,72]. 27 Sialosides were synthesized and screened
with HA receptors and even complete viruses. Results showed that the HA receptor
