Biomedical Engineering Reference
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(iii) kinetics of synthesis, steady state level, and intracellular half-life
of the proteins in in vitro transcription-translation systems and/
or transiently or permanently expressing eukaryote cells, and, ide-
ally (iv) testing in animal models (see Fig.
1
for a flow chart of
the complete approach (
13
)). Knowledge of the structure of the
targeted protein might accelerate and guide the search of hit
compounds by virtual screening steps (
14, 15
). Further optimi-
zation of the drug will also largely benefit from knowledge of the
structure of the ligand-protein complex.
Here we present the protocols associated with the HTP
screening and in vitro transcription-translation for the selection
of pharmacological chaperones, for both soluble and membrane
proteins. HTP screening of stabilizing ligands of both types
of proteins is performed by the fluorescence thermal shift assay
(
6, 7, 13
). The positive hits from the HTP thermal assay can be
further analyzed by the isothermal denaturation assay, which repre-
sents a medium throughput screening scheme, at least for stability
assays of membrane proteins (
16
). A protocol for determining the
binding affinity of positive hits from HTP screening by measuring
the quenching of intrinsic fluorescence is also included. Finally,
the effect of the hit compounds on the kinetics of protein synthesis
Virtual screening
of compound database on
3D-structure of protein target
HTP screening
of compound library on protein target
(wild type or mutants).
Search for stabilization
Analysis of binding
affinity
If unwanted
inhibition
Biochemical testing of
activity-stability
on protein target
If unwanted
effects
Testing stabilization
and increased steady-state protein
levels in cells
and/or in vitro transcription-translation
systems
Testing on
animal models
of disease
Potential pharmacological chaperones
Fig. 1. Flowchart of translational approach for search and evaluation of small molecule stabilizing compounds. HTP, high
throughput.
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