Promega @cademy webinar:
"Bioluminescent assays to monitor protease activity"
Information
Proteases are important targets for drug
discovery because of their involvement in myriad
cell signaling pathways. Recent approval for
protease inhibitors of DPPIV, the proteasome,
and renin as drugs for the diseases, type II
diabetes, multiple myeloma, and hypertension,
respectively, attest to their importance. Rapid
and sensitive assays of proteolytic activity are
necessary for the general characterization of
proteases and high-throughput screening for
protease inhibitors. Fluorescence has been
widely used for monitoring protease activity,
but fluorogenic substrates can exhibit high
background and many compound libraries have a
significant percentage of interfering
fluorescent compounds. We have developed an
alternative bioluminescent platform for protease
assays. The bioluminescent chemistry based on
firefly luciferase is particularly versatile. We
have used the luciferin component in the
reaction as a scaffold to create luminogenic
substrates for a wide variety of proteases. By
combining peptide-conjugated aminoluciferin
substrates and a stabilized luciferase in a
coupled-enzyme format, we have developed
homogeneous protease assays that have low
background, improved assay sensitivity,
increased dynamic range, and greater flexibility
in read time. The luminogenic substrates in this
coupled-enzyme format generate a stable signal
that is ideal for high throughput screening.
Examples comparing luminescent protease assays
to fluorescent protease assays will be shown and
the differences in kinetics will be explained.
Both cell-based assays and biochemical assays
using purified proteases have been developed;
the specific requirements and utility for each
will be discussed. We have optimized luminescent
assays for several protease targets, including
caspases, DPPIV, calpain, the proteasome, and
deubiquitinating enzymes, but have capabilities
for a much wider array of targets. The improved
sensitivity achieved with the aminoluciferin-based
protease assays has led to applications that
demonstrate this luminescent technology is
enabling.
In addition to using luciferin as a scaffold, we
have engineered luciferase to be a flexible
vector that can be used to create a substrate
for a protease of interest. This novel
technology complements the aminoluciferin-based
technology and allows rapid generation of
protease substrates through molecular cloning
and coupled transcription/translation cell-free
expression, thus enabling the facile evaluation
of protease function. Our bioluminescent
platform for protease assays utilizes both
luciferin and engineered luciferase as scaffolds
for generating substrates; the combined
technologies can provide solutions for a wide
variety of protease assay needs.
This webinarwill be
presented by Martha O'Brien Ph.D., Sr. Scientist at Research - Biochemical &
Cellular Technologies, Promega Corporation.
