7 research outputs found
Differential Kinobeads Profiling for Target Identification of Irreversible Kinase Inhibitors
Chemoproteomics
profiling of kinase inhibitors with kinobeads enables
the assessment of inhibitor potency and selectivity for endogenously
expressed protein kinases in cell lines and tissues. Using a small
panel of targeted covalent inhibitors, we demonstrate the importance
of measuring covalent target binding in live cells. We present a differential
kinobeads profiling strategy for covalent kinase inhibitors where
a compound is added either to live cells or to a cell extract that
enables the comprehensive assessment of inhibitor selectivity for
covalent and noncovalent targets. We found that Acalabrutinib, CC-292,
and Ibrutinib potently and covalently bind TEC family kinases, but
only Ibrutinib also potently binds to BLK. ZAK was identified as a
submicromolar affinity Ibrutinib off-target due to covalent modification
of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150
next to the DFG loop, demonstrating an alternative route to covalent
inactivation of this kinase, e.g., to inhibit canonical TGF-ÎČ
dependent processes
A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level
Late
stage failures of candidate drug molecules are frequently
caused by off-target effects or inefficient target engagement <i>in vivo</i>. In order to address these fundamental challenges
in drug discovery, we developed a modular probe strategy based on
bioorthogonal chemistry that enables the attachment of multiple reporters
to the same probe in cell extracts and live cells. In a systematic
evaluation, we identified the inverse electron demand DielsâAlder
reaction between <i>trans</i>-cyclooctene labeled probe
molecules and tetrazine-tagged reporters to be the most efficient
bioorthogonal reaction for this strategy. Bioorthogonal biotinylation
of the probe allows the identification of drug targets in a chemoproteomics
competition binding assay using quantitative mass spectrometry. Attachment
of a fluorescent reporter enables monitoring of spatial localization
of probes as well as drug-target colocalization studies. Finally,
direct target occupancy of unlabeled drugs can be determined at single
cell resolution by competitive binding with fluorescently labeled
probe molecules. The feasibility of the modular probe strategy is
demonstrated with noncovalent PARP inhibitors
A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level
Late
stage failures of candidate drug molecules are frequently
caused by off-target effects or inefficient target engagement <i>in vivo</i>. In order to address these fundamental challenges
in drug discovery, we developed a modular probe strategy based on
bioorthogonal chemistry that enables the attachment of multiple reporters
to the same probe in cell extracts and live cells. In a systematic
evaluation, we identified the inverse electron demand DielsâAlder
reaction between <i>trans</i>-cyclooctene labeled probe
molecules and tetrazine-tagged reporters to be the most efficient
bioorthogonal reaction for this strategy. Bioorthogonal biotinylation
of the probe allows the identification of drug targets in a chemoproteomics
competition binding assay using quantitative mass spectrometry. Attachment
of a fluorescent reporter enables monitoring of spatial localization
of probes as well as drug-target colocalization studies. Finally,
direct target occupancy of unlabeled drugs can be determined at single
cell resolution by competitive binding with fluorescently labeled
probe molecules. The feasibility of the modular probe strategy is
demonstrated with noncovalent PARP inhibitors
A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level
Late
stage failures of candidate drug molecules are frequently
caused by off-target effects or inefficient target engagement <i>in vivo</i>. In order to address these fundamental challenges
in drug discovery, we developed a modular probe strategy based on
bioorthogonal chemistry that enables the attachment of multiple reporters
to the same probe in cell extracts and live cells. In a systematic
evaluation, we identified the inverse electron demand DielsâAlder
reaction between <i>trans</i>-cyclooctene labeled probe
molecules and tetrazine-tagged reporters to be the most efficient
bioorthogonal reaction for this strategy. Bioorthogonal biotinylation
of the probe allows the identification of drug targets in a chemoproteomics
competition binding assay using quantitative mass spectrometry. Attachment
of a fluorescent reporter enables monitoring of spatial localization
of probes as well as drug-target colocalization studies. Finally,
direct target occupancy of unlabeled drugs can be determined at single
cell resolution by competitive binding with fluorescently labeled
probe molecules. The feasibility of the modular probe strategy is
demonstrated with noncovalent PARP inhibitors
Discovery of a Highly Selective Tankyrase Inhibitor Displaying Growth Inhibition Effects against a Diverse Range of Tumor Derived Cell Lines
The
availability of high quality probes for specific protein targets
is fundamental to the investigation of their function and their validation
as therapeutic targets. We report the utilization of a dedicated chemoproteomic
assay platform combining affinity enrichment technology with high-resolution
protein mass spectrometry to the discovery of a novel nicotinamide
isoster, the tetrazoloquinoxaline <b>41</b>, a highly potent
and selective tankyrase inhibitor. We also describe the use of <b>41</b> to investigate the biology of tankyrase, revealing the
compound induced growth inhibition of a number of tumor derived cell
lines, demonstrating the potential of tankyrase inhibitors in oncology
Interrogating the Druggability of the 2âOxoglutarate-Dependent Dioxygenase Target Class by Chemical Proteomics
The 2-oxoglutarate-dependent dioxygenase
target class comprises
around 60 enzymes including several subfamilies with relevance to
human disease, such as the prolyl hydroxylases and the Jumonji-type
lysine demethylases. Current drug discovery approaches are largely
based on small molecule inhibitors targeting the iron/2-oxoglutarate
cofactor binding site. We have devised a chemoproteomics approach
based on a combination of unselective active-site ligands tethered
to beads, enabling affinity capturing of around 40 different dioxygenase
enzymes from human cells. Mass-spectrometry-based quantification of
bead-bound enzymes using a free-ligand competition-binding format
enabled the comprehensive determination of affinities for the cosubstrate
2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate.
We also profiled a set of representative drug-like inhibitor compounds.
The results indicate that intracellular competition by endogenous
cofactors and high active site similarity present substantial challenges
for drug discovery for this target class
Cell Penetrant Inhibitors of the KDM4 and KDM5 Families of Histone Lysine Demethylases. 1. 3âAmino-4-pyridine Carboxylate Derivatives
Optimization
of KDM6B (JMJD3) HTS hit <b>12</b> led to the
identification of 3-((furan-2-ylmethyl)Âamino)Âpyridine-4-carboxylic
acid <b>34</b> and 3-(((3-methylthiophen-2-yl)Âmethyl)Âamino)Âpyridine-4-carboxylic
acid <b>39</b> that are inhibitors of the KDM4 (JMJD2) family
of histone lysine demethylases. Compounds <b>34</b> and <b>39</b> possess activity, IC<sub>50</sub> †100 nM, in KDM4
family biochemical (RFMS) assays with â„50-fold selectivity
against KDM6B and activity in a mechanistic KDM4C cell imaging assay
(IC<sub>50</sub> = 6â8 ÎŒM). Compounds <b>34</b> and <b>39</b> are also potent inhibitors of KDM5C (JARID1C)
(RFMS IC<sub>50</sub> = 100â125 nM)