Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 222

high-throughput screening (HTS) and does not require cell lysis. However,
it does not allow for real-time studies, as the nonphosphorylated substrates
are cleaved and degraded. Moreover, besides their disruptive character, these
approaches merely provide a snapshot of a dynamic process and do not
provide real insights into the dynamics and enzymatic kinetics of the target
kinase in its native environment.
Faced with a real need for tools that enable qualitative and quantitative
assessment of enzymatic activities in their native environment, biologists and
chemists have designed a new generation of tools designated as “fluorescence-
based reporters” or “fluorescent biosensors,” which enable probing the
biochemical function, activity, and dynamics of specific enzymes through
sensitive changes in their fluorescent properties. Fluorescence detection is
currently one of the most widely used approaches in biomolecular imaging
because of its high intrinsic sensitivity and selectivity. Indeed, fluorescence
lends itself to nondestructive imaging, thereby preserving the sample and
the molecules of interest within. Moreover, fluorescence imaging provides
a high degree of temporal and spatial resolution and allows for real-time track-
ing of a molecule in motion in a complex solution or environment.
As such, when fluorescent biosensors are designed to recognize a
target with high specificity and to report on this event with high sensitivity,
they constitute extremely useful tools for the detection of biomolecules, both
in vitro and in vivo , and for monitoring dynamic molecular events, such as
enzymatic activities, conformational changes, or protein/protein interac-
tions. 31 High-resolution imaging and real-time measurements of fluorescent
biosensors provide precious information on the spatial and temporal localiza-
tion of a wide variety of intracellular targets and have proved a successful
means of visualizing dynamic processes in living cells. Fluorescent biosensors
that specifically probe protein kinases allow imaging the behavior of kinases in
a dynamic and quantitative fashion, in space and in time, in cells, tissues, and
whole organisms, thereby allowing tackling questions that could not be
addressed previously. In addition, fluorescent reporters and biosensors can
be exploited to develop high-throughput and high content screens for drug
discovery purposes and for further assessing the efficacy and pharmacokinetics
of lead compounds of potential therapeutic utility. Moreover, fluorescent bio-
sensors offer promising perspectives for diagnostic strategies, for early detec-
tion of protein kinase dysregulation associated with the onset of disease, and
for monitoring disease progression and response to therapeutics.
Two large classes of kinase biosensors have been developed: genetically
encoded autofluorescent protein (AFP) biosensors and nongenetic fluorescent