Biomedical Engineering Reference
In-Depth Information
Several detection approaches are available for the study of direct ligand binding.
For example, surface plasmon resonance (SPR) detects binding to an immobilized
analyte and provides information on the kinetics of binding and dissociation. Comple-
mentarily to SPR, microcalorimetry detects ligand binding either through isothermal
heat release (isothermal titration calorimetry, ITC) or changes in the thermodynam-
ics of the protein (differential scanning calorimetry, DSC). Nuclear magnetic reso-
nance could detect binding through changes in spectral data for either labeled protein
residues or a ligand itself. Mass spectrometry analysis could potentially identify bind-
ing of multiple ligands from a complex mixture of compounds; however, the need to
separate bound and unbound ligands through gel-filtration steps makes it insensitive
to weak binders. The mentioned technologies provide a powerful arsenal for studying
protein-ligand binding; unfortunately, none of them have sufficient throughput for
screening tens to hundreds of thousands of compounds against numerous targets.
An example of a high-throughput non-mechanism-based binding screening
approach is the ThermoFluor technology pioneered by 3D Pharmaceuticals [12].
This approach is also known as differential scanning fluorimetry (DSF) and pro-
tein thermal shift assays. Technically, the protein thermal denaturation is performed
on a real-time PCR type of instrument and monitored through the binding of an
environment-sensitive dye such as 1-anilinonaphthalene-8-sulfonate or Sypro Orange
[13]. These dyes bind to hydrophobic patches and surfaces of unfolding protein giving
rise to a significant increase in fluorescence; their fluorescence is high in hydropho-
bic environment while significantly quenched in aqueous milieu (Figure 12.5). The
screening of compounds is based on their effect on thermal stability of proteins.
Compounds binding to the protein through hydrogen bonding and van der Waals
interactions are expected to stabilize the protein molecules and result in the increased
temperature of protein thermal denaturation, as visualized by the shift in transition
temperature
T
m
(Figure 12.5b and c). This approach is normally based on 384-well
generic PCR plates and could provide a throughput of five to seven plates per hour
per instrument.
Another example of the target-based, mechanism-agnostic binding HTS approach
is provided by resonant waveguide grating (RWG) technology. This technology
utilizes evanescent waves generated at the boundary between a waveguide high-
refractive-index layer deposited on the glass plate bottom and the low-refractive-index
solution in the well. These waves extend into the solution above the glass bottom
surface and are sensitive to any change in refractive index in the local region. This
approach can detect direct binding of small-molecule ligands to protein molecules
immobilized at the surface of the well. Understandably, this approach requires utiliza-
tion of special plates with a high-quality grating layer that are much more expensive
than generic screening plates, yet it provides an effective and rapid method for HTS
using direct binding assay in high-density formats.
Noteworthy, a mechanism-agnostic binding approach not only provides an envi-
ronment for unbiased identification of compounds targeting all potential binding
sites on a protein molecule, but importantly, serves as an inherent enrichment
process for finding sites on the macromolecule surface that are most suitable to
binding with a particular set of compounds. This represents a great advantage for
