Biomedical Engineering Reference
In-Depth Information
FIGURE 18.6
SL209 inhibit heterodimerization of GST-core106/FLAG-core106. (
See
insert for color representation of the figure
.)
antibodies fused to fluorophores were added, an anti-GST-europium cryptate (Eu) and
an anti-FLAG-allophycocyanin (XL-665). Finally, each well was irradiated with 330-
nm light to excite the Eu fluorophore. The interaction between the two fluorophore-
tagged proteins led to transfer of fluorescence energy and emission at 665 nm in the
negative control. Small-molecule inhibitors of heterodimerization diminished FRET,
which resulted in Eu-specific emission at 620 nm.
The TR-FRET assay was used to screen a DOS library of 2240 compounds devel-
oped at the Center for Chemical Methodology for Library Development at Boston
University (CMLD-BU) for inhibitors of core heterodimerization [51b]. Based on an
initial hit, the compound SL209 was identified as an inhibitor of core106 dimerization
that exhibited low toxicity toward uninfected hepatoma cells (Figure 18.6) [51b]. The
dose-dependent inhibitory activity of the compound was confirmed in an amplified
luminescent proximity homogeneous assay (ALPHA).
To validate core as the target of SL209, an SL209-biotin conjugate was prepared
as an affinity probe. The conjugate retained the activity of the untagged compound in
the ALPHA assay. In affinity-isolation experiments with hepatoma cells, the SL209-
biotin conjugate was found to associate directly with the core protein. In addition
to core, several other HCV proteins were pulled down by the conjugate, including
the NS3 viral helicase and the NS5A viral regulatory protein. When the HCV pSGR
subgenomic replicon, which produces NS3 and NS5A but not core, was used in
pull-down experiments, neither NS3 nor NS5A was retained. However, capture of
NS3 and NS5A was rescued upon addition of core106 to the replicon lysate, strongly
suggesting that these proteins associate with core in the cell and that the SL209-biotin
interaction is specific to core. Further studies showed that SL209-biotin co-localizes
with core on the lipid droplets that act as surfaces for core-directed assembly of HCV
viral particles [51a].
The discovery of SL209 as a new dimerization inhibitor of the HCV nucleocapsid
protein core exemplifies the utility of DOS libraries in developing small-molecule
