20 research outputs found
A Hexylchloride-Based Catch-and-Release System for Chemical Proteomic Applications
Bioorthogonal ligation methods that
allow the selective conjugation
of fluorophores or biotin to proteins and small molecule probes that
contain inert chemical handles are an important component of many
chemical proteomic strategies. Here, we present a new catch-and-release
enrichment strategy that utilizes a hexylchloride group as a bioorthogonal
chemical handle. Proteins and small molecules that contain a hexylchloride
tag can be efficiently captured by an immobilized version of the self-labeling
protein HaloTag. Furthermore, by using a HaloTag fusion protein that
contains a protease cleavage site, captured proteins can be selectively
eluted under mild conditions. We demonstrate the utility of the hexylchloride-based
catch-and-release strategy by enriching protein kinases that are covalently
and noncovalently bound to ATP-binding site-directed probes from mammalian
cell lysates. Our catch-and-release system creates new possibilities
for profiling enzyme families and for the identification of the cellular
targets of bioactive small molecules
A Hexylchloride-Based Catch-and-Release System for Chemical Proteomic Applications
Bioorthogonal ligation methods that
allow the selective conjugation
of fluorophores or biotin to proteins and small molecule probes that
contain inert chemical handles are an important component of many
chemical proteomic strategies. Here, we present a new catch-and-release
enrichment strategy that utilizes a hexylchloride group as a bioorthogonal
chemical handle. Proteins and small molecules that contain a hexylchloride
tag can be efficiently captured by an immobilized version of the self-labeling
protein HaloTag. Furthermore, by using a HaloTag fusion protein that
contains a protease cleavage site, captured proteins can be selectively
eluted under mild conditions. We demonstrate the utility of the hexylchloride-based
catch-and-release strategy by enriching protein kinases that are covalently
and noncovalently bound to ATP-binding site-directed probes from mammalian
cell lysates. Our catch-and-release system creates new possibilities
for profiling enzyme families and for the identification of the cellular
targets of bioactive small molecules
Targeting Diverse Signaling Interaction Sites Allows the Rapid Generation of Bivalent Kinase Inhibitors
The identification of potent and selective modulators
of protein
kinase function remains a challenge, and new strategies are needed
for generating these useful ligands. Here, we describe the generation
of bivalent inhibitors of three unrelated protein kinases: the CAMK
family kinase Pim1, the mitogen-activated protein kinase (MAPK) p38α,
and the receptor tyrosine kinase (RTK) epidermal growth factor receptor
(EGFR). These bivalent inhibitors consist of an ATP-competitive inhibitor
that is covalently tethered to an engineered form of the self-labeling
protein <i>O</i><sup>6</sup>-alkylguanine-DNA alkyltransferase
(SNAP-tag). In each example, SNAP-tag is fused to a peptide ligand
that binds to a signaling interaction site of the kinase being targeted.
These interactions increase the overall selectivity and potency of
the bivalent inhibitors that were generated. The ability to exploit
disparate binding sites in diverse kinases points to the generality
of the method described. Finally, we demonstrate that ATP-competitive
inhibitors that are conjugated to the bio-orthogonal tag <i>O</i><sup>4</sup>-benzyl-2-chloro-6-aminopyrimidine (CLP) are cell-permeable.
The selective labeling of SNAP-tag with CLP conjugates allows the
rapid assembly of bivalent inhibitors in living cells
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Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics
Cyclin-dependent kinases (CDKs) are key mediators of cell proliferation and have been a subject of oncology drug discovery efforts for over two decades. Several CDK and activator cyclin family members have been implicated in regulating the cell division cycle. While it is thought that there are canonical CDK-cyclin pairing preferences, the extent of selectivity is unclear, and increasing evidence suggests that the cell-cycle CDKs can be activated by a pool of available cyclins. The molecular details of CDK-cyclin specificity are not completely understood despite their importance for understanding cancer cell cycles and for pharmacological inhibition of cancer proliferation. We report here a biolayer interferometry assay that allows for facile quantification of CDK binding interactions with their cyclin activators. We applied this assay to measure the impact of Cdk2 inhibitors on Cyclin A (CycA) association and dissociation kinetics. We found that Type I inhibitors increase the affinity between Cdk2 and CycA by virtue of a slowed cyclin dissociation rate. In contrast, Type II inhibitors and other small-molecule Cdk2 binders have distinct effects on the CycA association and dissociation processes to decrease affinity. We propose that the differential impact of small molecules on the cyclin binding kinetics arises from the plasticity of the Cdk2 active site as the kinase transitions between active, intermediate, and inactive states
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Biolayer Interferometry Assay for Cyclin-Dependent Kinase-Cyclin Association Reveals Diverse Effects of Cdk2 Inhibitors on Cyclin Binding Kinetics
Cyclin-dependent kinases (CDKs) are key mediators of
cell proliferation
and have been a subject of oncology drug discovery efforts for over
two decades. Several CDK and activator cyclin family members have
been implicated in regulating the cell division cycle. While it is
thought that there are canonical CDK-cyclin pairing preferences, the
extent of selectivity is unclear, and increasing evidence suggests
that the cell-cycle CDKs can be activated by a pool of available cyclins.
The molecular details of CDK-cyclin specificity are not completely
understood despite their importance for understanding cancer cell
cycles and for pharmacological inhibition of cancer proliferation.
We report here a biolayer interferometry assay that allows for facile
quantification of CDK binding interactions with their cyclin activators.
We applied this assay to measure the impact of Cdk2 inhibitors on
Cyclin A (CycA) association and dissociation kinetics. We found that
Type I inhibitors increase the affinity between Cdk2 and CycA by virtue
of a slowed cyclin dissociation rate. In contrast, Type II inhibitors
and other small-molecule Cdk2 binders have distinct effects on the
CycA association and dissociation processes to decrease affinity.
We propose that the differential impact of small molecules on the
cyclin binding kinetics arises from the plasticity of the Cdk2 active
site as the kinase transitions between active, intermediate, and inactive
states
Affinity-Based Probes Based on Type II Kinase Inhibitors
Protein kinases are key components of most mammalian
signal transduction
networks and are therapeutically relevant drug targets. Efforts to
study protein kinase function would benefit from new technologies
that are able to profile kinases in complex proteomes. Here, we describe
active site-directed probes for profiling kinases in whole cell extracts
and live cells. These probes contain general ligands that stabilize
a specific inactive conformation of the ATP-binding sites of protein
kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties
that allow covalent modification and enrichment of kinases, respectively.
A diverse group of serine/threonine and tyrosine kinases were identified
as specific targets of these probes in whole cell extracts. In addition,
a number of kinase targets were selectively labeled in live cells.
Our chemical proteomics approach should be valuable for interrogating
protein kinase active sites in physiologically relevant environments
ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR.
The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector