8 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
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1Ī±) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1Ī± levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1Ī±) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1Ī± levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
Protein-fragment
complementation assays (PCAs) are widely used
for investigating protein interactions. However, the fragments used
are structurally compromised and have not been optimized nor thoroughly
characterized for accurately assessing these interactions. We took
advantage of the small size and bright luminescence of NanoLuc to
engineer a new complementation reporter (NanoBiT). By design, the
NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly
associate so that their assembly into a luminescent complex is dictated
by the interaction characteristics of the target proteins onto which
they are appended. To ascertain their general suitability for measuring
interaction affinities and kinetics, we determined that their intrinsic
affinity (<i>K</i><sub>D</sub> = 190 Ī¼M) and association
constants (<i>k</i><sub>on</sub> = 500 M<sup>ā1</sup> s<sup>ā1</sup>, <i>k</i><sub>off</sub> = 0.2 s<sup>ā1</sup>) are outside of the ranges typical for protein interactions.
The accuracy of NanoBiT was verified under defined biochemical conditions
using the previously characterized interaction between SME-1 Ī²-lactamase
and a set of inhibitor binding proteins. In cells, NanoBiT fusions
to FRB/FKBP produced luminescence consistent with the linear characteristics
of NanoLuc. Response dynamics, evaluated using both protein kinase
A and Ī²-arrestin-2, were rapid, reversible, and robust to temperature
(21ā37 Ā°C). Finally, NanoBiT provided a means to measure
pharmacology of kinase inhibitors known to induce the interaction
between BRAF and CRAF. Our results demonstrate that the intrinsic
properties of NanoBiT allow accurate representation of protein interactions
and that the reporter responds reliably and dynamically in cells
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