7 research outputs found
Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors
Novel
engineered NanoLuc (Nluc) luciferase being smaller, brighter,
and superior to traditional firefly (Fluc) or <i>Renilla</i> (Rluc) provides a great opportunity for the development of numerous
biological, biomedical, clinical, and food and environmental safety
applications. This new platform created an urgent need for Nluc inhibitors
that could allow selective bioluminescent suppression and multiplexing
compatibility with existing luminescence or fluorescence assays. Starting
from thienopyrrole carboxylate <b>1</b>, a hit from a 42āÆ000
PubChem compound library with a low micromolar IC<sub>50</sub> against
Nluc, we derivatized four different structural fragments to discover
a family of potent, single digit nanomolar, cell permeable inhibitors.
Further elaboration revealed a channel that allowed access to the
external Nluc surface, resulting in a series of highly potent cell
impermeable Nluc inhibitors with negatively charged groups likely
extending to the protein surface. The permeability was evaluated by
comparing EC<sub>50</sub> shifts calculated from both live and lysed
cells expressing Nluc cytosolically. Luminescence imaging further
confirmed that cell permeable compounds inhibit both intracellular
and extracellular Nluc, whereas less permeable compounds differentially
inhibit extracellular Nluc and Nluc on the cell surface. The compounds
displayed little to no toxicity to cells and high luciferase specificity,
showing no activity against firefly luciferase or even the closely
related NanoBit system. Looking forward, the structural motifs used
to gain access to the Nluc surface can also be appended with other
functional groups, and therefore interesting opportunities for developing
assays based on relief-of-inhibition can be envisioned
NanoBRETīøA Novel BRET Platform for the Analysis of ProteināProtein Interactions
Dynamic
interactions between proteins comprise a key mechanism
for temporal control of cellular function and thus hold promise for
development of novel drug therapies. It remains technically challenging,
however, to quantitatively characterize these interactions within
the biologically relevant context of living cells. Although, bioluminescence
resonance energy transfer (BRET) has often been used for this purpose,
its general applicability has been hindered by limited sensitivity
and dynamic range. We have addressed this by combining an extremely
bright luciferase (Nanoluc) with a means for tagging intracellular
proteins with a long-wavelength fluorophore (HaloTag). The small size
(19 kDa), high emission intensity, and relatively narrow spectrum
(460 nm peak intensity) make Nanoluc luciferase well suited as an
energy donor. By selecting an efficient red-emitting fluorophore (635
nm peak intensity) for attachment onto the HaloTag, an overall spectral
separation exceeding 175 nm was achieved. This combination of greater
light intensity with improved spectral resolution results in substantially
increased detection sensitivity and dynamic range over current BRET
technologies. Enhanced performance is demonstrated using several established
model systems, as well as the ability to image BRET in individual
cells. The capabilities are further exhibited in a novel assay developed
for analyzing the interactions of bromodomain proteins with chromatin
in living cells
Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag
Phenotypic screening of compound
libraries is a significant trend in drug discovery, yet success can
be hindered by difficulties in identifying the underlying cellular
targets. Current approaches rely on tethering bioactive compounds
to a capture tag or surface to allow selective enrichment of interacting
proteins for subsequent identification by mass spectrometry. Such
methods are often constrained by ineffective capture of low affinity
and low abundance targets. In addition, these methods are often not
compatible with living cells and therefore cannot be used to verify
the pharmacological activity of the tethered compounds. We have developed
a novel chloroalkane capture tag that minimally affects compound potency
in cultured cells, allowing binding interactions with the targets
to occur under conditions relevant to the desired cellular phenotype.
Subsequent isolation of the interacting targets is achieved through
rapid lysis and capture onto immobilized HaloTag protein. Exchanging
the chloroalkane tag for a fluorophore, the putative targets identified
by mass spectrometry can be verified for direct binding to the compound
through resonance energy transfer. Using the interaction between histone
deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model
system, we were able to identify and verify all the known HDAC targets
of SAHA as well as two previously undescribed targets, ADO and CPPED1.
The discovery of ADO as a target may provide mechanistic insight into
a reported connection between SAHA and Huntingtonās disease
Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag
Phenotypic screening of compound
libraries is a significant trend in drug discovery, yet success can
be hindered by difficulties in identifying the underlying cellular
targets. Current approaches rely on tethering bioactive compounds
to a capture tag or surface to allow selective enrichment of interacting
proteins for subsequent identification by mass spectrometry. Such
methods are often constrained by ineffective capture of low affinity
and low abundance targets. In addition, these methods are often not
compatible with living cells and therefore cannot be used to verify
the pharmacological activity of the tethered compounds. We have developed
a novel chloroalkane capture tag that minimally affects compound potency
in cultured cells, allowing binding interactions with the targets
to occur under conditions relevant to the desired cellular phenotype.
Subsequent isolation of the interacting targets is achieved through
rapid lysis and capture onto immobilized HaloTag protein. Exchanging
the chloroalkane tag for a fluorophore, the putative targets identified
by mass spectrometry can be verified for direct binding to the compound
through resonance energy transfer. Using the interaction between histone
deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model
system, we were able to identify and verify all the known HDAC targets
of SAHA as well as two previously undescribed targets, ADO and CPPED1.
The discovery of ADO as a target may provide mechanistic insight into
a reported connection between SAHA and Huntingtonās disease
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ā¼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ā¼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 Ā°C or in culture medium for
>15 h at 37 Ā°C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ā¼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ā¼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 Ā°C or in culture medium for
>15 h at 37 Ā°C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ā¼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ā¼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 Ā°C or in culture medium for
>15 h at 37 Ā°C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes