20 research outputs found
Effect of Astringent Stimuli on Salivary Protein Interactions Elucidated by Complementary Proteomics Approaches
The
interaction of astringent substances with salivary proteins,
which results in protein precipitation, is considered a key event
in the molecular mechanism underlying the oral sensation of puckering
astringency. As the chemical nature of orally active astringents is
diverse and the knowledge of their interactions with salivary proteins
rather fragmentary, human whole saliva samples were incubated with
suprathreshold and isointensity solutions of the astringent polyphenol
(ā)-epigallocatechin gallate, the multivalent metal salt ironĀ(III)
sulfate, the amino-functionalized polysaccharide chitosan, and the
basic protein lysozyme. After separation of the precipitated proteins,
the proteins affected by the astringents were identified and relatively
quantified for the first time by complementary bottom-up and top-down
mass spectrometry-based proteomics approaches. Major salivary target
proteins, which may be involved in astringency perception, are reported
here for each astringent stimulus
New Affinity Probe Targeting VEGF Receptors for Kinase Inhibitor Selectivity Profiling by Chemical Proteomics
Solid
tumors are dependent for growth on nutrients and the supply
of oxygen, which they often acquire via neoangiogenesis. Vascular
endothelial growth factors and the corresponding receptors (VEGFRs)
play central roles in this process, and consequently, the blockade
of this pathway is one therapeutic strategy for cancer treatment.
A number of small molecules inhibiting VEGFR inhibitors have been
developed for clinical use, and a comprehensive view of target selectivity
is important to assess the therapeutic as well as risk potential of
a drug molecule. Recent advances in mass spectrometry-based chemical
proteomics allow analyses of drugātarget interactions under
close-to-physiological conditions, and in this study, we report on
the design, synthesis, and application of a small molecule affinity
probe as a tool for the selectivity profiling of VEGFR and other kinase
inhibitors. The probe is capable of binding >132 protein kinases,
including angiokinases such as VEGFRs, PDGFRs, and c-KIT from lysates
of cancer cell lines or human placenta tissue. Combining the new probe
with Kinobeads in competitive binding assays, we were able to identify
nanomolar off-targets of the VEGFR/PDGFR inhibitors pazopanib and
axitinib. Because of its broad binding spectrum, the developed chemical
tool can be generically used for the discovery of kinase inhibitor
targets, which may contribute to a more comprehensive understanding
of the mechanisms of action of such drugs
Evaluation of Kinase Activity Profiling Using Chemical Proteomics
Protein
kinases are important mediators of intracellular signaling
and are reversibly activated by phosphorylation. Immobilized kinase
inhibitors can be used to enrich these often low-abundance proteins,
to identify targets of kinase inhibitors, or to probe their selectivity.
It has been suggested that the binding of kinases to affinity beads
reflects a kinaseās activation status, a concept that is under
considerable debate. To assess the merits of the idea, we performed
a series of experiments including quantitative phosphoproteomics and
purification of kinases by single or mixed affinity matrices from
signaling activated or resting cancer cells. The data show that mixed
affinity beads largely bind kinases independent of their activation
status, and experiments using individual immobilized kinase inhibitors
show mixed results in terms of preference for binding the active or
inactive conformation. Taken together, activity- or conformation-dependent
binding to such affinity resins depends (i) on the kinase, (ii) on
the affinity probe, and (iii) on the activation status of the lysate
or cell. As a result, great caution should be exercised when inferring
kinase activity from such binding data. The results also suggest that
assaying kinase activity using binding data is restricted to a limited
number of well-chosen cases
Characterization of a Chemical Affinity Probe Targeting Akt Kinases
Protein kinases are key regulators
of cellular processes, and aberrant
function is often associated with human disease. Consequently, kinases
represent an important class of therapeutic targets and about 20 kinase
inhibitors (KIs) are in clinical use today. Detailed knowledge about
the selectivity of KIs is important for the correct interpretation
of their pharmacological and systems biological effects. Chemical
proteomic approaches for systematic kinase inhibitor selectivity profiling
have emerged as important molecular tools in this regard, but the
coverage of the human kinome is still incomplete. Here, we describe
a new affinity probe targeting Akt and many other members of the AGC
kinase family that considerably extends the scope of KI profiling
by chemical proteomics. In combination with the previously published
kinobeads, the synthesized probe was applied to selectivity profiling
of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells.
The results confirmed the inhibition of all Akt isoforms and of a
number of known as well as CDC42BPB as a novel putative target for
GSK690693. This work also established, for the first time, the kinase
selectivity profile of the clinical phase I drug GSK2141795 and identified
PRKG1 as a low nanomolar kinase target as well as the ATP-dependent
5ā²-3ā² DNA helicase ERCC2 as a potential new non-kinase
off-target
Evaluation of Kinase Activity Profiling Using Chemical Proteomics
Protein
kinases are important mediators of intracellular signaling
and are reversibly activated by phosphorylation. Immobilized kinase
inhibitors can be used to enrich these often low-abundance proteins,
to identify targets of kinase inhibitors, or to probe their selectivity.
It has been suggested that the binding of kinases to affinity beads
reflects a kinaseās activation status, a concept that is under
considerable debate. To assess the merits of the idea, we performed
a series of experiments including quantitative phosphoproteomics and
purification of kinases by single or mixed affinity matrices from
signaling activated or resting cancer cells. The data show that mixed
affinity beads largely bind kinases independent of their activation
status, and experiments using individual immobilized kinase inhibitors
show mixed results in terms of preference for binding the active or
inactive conformation. Taken together, activity- or conformation-dependent
binding to such affinity resins depends (i) on the kinase, (ii) on
the affinity probe, and (iii) on the activation status of the lysate
or cell. As a result, great caution should be exercised when inferring
kinase activity from such binding data. The results also suggest that
assaying kinase activity using binding data is restricted to a limited
number of well-chosen cases
Characterization of a Chemical Affinity Probe Targeting Akt Kinases
Protein kinases are key regulators
of cellular processes, and aberrant
function is often associated with human disease. Consequently, kinases
represent an important class of therapeutic targets and about 20 kinase
inhibitors (KIs) are in clinical use today. Detailed knowledge about
the selectivity of KIs is important for the correct interpretation
of their pharmacological and systems biological effects. Chemical
proteomic approaches for systematic kinase inhibitor selectivity profiling
have emerged as important molecular tools in this regard, but the
coverage of the human kinome is still incomplete. Here, we describe
a new affinity probe targeting Akt and many other members of the AGC
kinase family that considerably extends the scope of KI profiling
by chemical proteomics. In combination with the previously published
kinobeads, the synthesized probe was applied to selectivity profiling
of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells.
The results confirmed the inhibition of all Akt isoforms and of a
number of known as well as CDC42BPB as a novel putative target for
GSK690693. This work also established, for the first time, the kinase
selectivity profile of the clinical phase I drug GSK2141795 and identified
PRKG1 as a low nanomolar kinase target as well as the ATP-dependent
5ā²-3ā² DNA helicase ERCC2 as a potential new non-kinase
off-target
Chemical Proteomics Uncovers EPHA2 as a Mechanism of Acquired Resistance to Small Molecule EGFR Kinase Inhibition
Tyrosine kinase inhibitors (TKIs)
have become an important therapeutic
option for treating several forms of cancer. Gefitinib, an inhibitor
of the epidermal growth factor receptor (EGFR), is in clinical use
for treating non-small cell lung cancer (NSCLC) harboring activating
EGFR mutations. However, despite high initial response rates, many
patients develop resistance to gefitinib. The molecular mechanisms
of TKI resistance often remain unclear. Here, we describe a chemical
proteomic approach comprising kinase affinity purification (kinobeads)
and quantitative mass spectrometry for the identification of kinase
inhibitor resistance mechanisms in cancer cells. We identified the
previously described amplification of MET and found EPHA2 to be more
than 10-fold overexpressed (<i>p</i> < 0.001) in gefitinib-resistant
HCC827 cells suggesting a potential role in developing resistance.
siRNA-mediated EPHA2 knock-down or treating cells with the multikinase
inhibitor dasatinib restored sensitivity to gefitinib. Of all dasatinib
targets, EPHA2 exhibited the most drastic effect (<i>p</i> < 0.001). In addition, EPHA2 knockdown or ephrin-A1 treatment
of resistant cells decreased FAK phosphorylation and cell migration.
These findings confirm EPHA2 as an actionable drug target, provide
a rational basis for drug combination approaches, and indicate that
chemical proteomics is broadly applicable for the discovery of kinase
inhibitor resistance
Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity
To
investigate the role of perireceptor events in inter-individual
variability in salt taste sensitivity, 31 volunteers were monitored
in their detection functions for sodium chloride (NaCl) and classified
into sensitive (0.6ā1.7 mmol/L), medium-sensitive (1.8ā6.9
mmol/L), and nonsensitive (7.0ā11.2 mmol/L) subjects. Chemosensory
intervention of NaCl-sensitive (S<sup>+</sup>) and nonsensitive (S<sup>ā</sup>) panellists with potassium chloride, ammonium chloride,
and sodium gluconate showed the salt taste sensitivity to be specific
for NaCl. As no significant differences were found between S<sup>+</sup> and S<sup>ā</sup> subjects in salivary sodium and protein
content, salivary proteome differences and their stimulus-induced
dynamic changes were analyzed by tryptic digestion, iTRAQ labeling,
and liquid chromatographyātandem mass spectrometry analysis.
Differences in the salivary proteome between S<sup>+</sup> and S<sup>ā</sup> subjects were found primarily in resting saliva and
were largely independent of the dynamic alterations observed upon
salt stimulation. Gene ontology enrichment analysis of key proteins,
i.e., immunoglobulin heavy constant y1, myeloblastin, cathepsin G,
and kallikrein, revealed significantly increased serine-type endopeptidase
activity for the S<sup>+</sup> group, while the S<sup>ā</sup> group exhibited augmented cysteine-type endopeptidase inhibitor
activity by increased abundances in lipocalin-1 and cystatin-D, -S,
and -SN, respectively. As proteases have been suggested to facilitate
transepithelial sodium transport by cleaving the y-subunit of the
epithelial sodium channel (ENaC) and protease inhibitors have been
shown to reduce ENaC-mediated sodium transport, the differentially
modulated proteolytic activity patterns observed <i>in vivo</i> for S<sup>+</sup> and S<sup>ā</sup> subjects show evidence
of them playing a crucial role in affecting human NaCl sensitivity
Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors
Many
protein kinases are valid drug targets in oncology because
they are key components of signal transduction pathways. The number
of clinical kinase inhibitors is on the rise, but these molecules
often exhibit polypharmacology, potentially eliciting desired and
toxic effects. Therefore, a comprehensive assessment of a compoundās
target space is desirable for a better understanding of its biological
effects. The enzyme ferrochelatase (FECH) catalyzes the conversion
of protoporphyrin IX into heme and was recently found to be an off-target
of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity
associated with this drug in melanoma patients. This raises the question
of whether FECH binding is a more general feature of kinase inhibitors.
To address this, we applied a chemical proteomics approach using kinobeads
to evaluate 226 clinical kinase inhibitors for their ability to bind
FECH. Surprisingly, low or submicromolar FECH binding was detected
for 29 of all compounds tested and isothermal dose response measurements
confirmed target engagement in cells. We also show that Vemurafenib,
Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells,
verifying that drug binding leads to a loss of FECH activity. Further
biochemical and docking experiments identified the protoporphyrin
pocket in FECH as one major drug binding site. Since the genetic loss
of FECH activity leads to photosensitivity in humans, our data strongly
suggest that FECH inhibition by kinase inhibitors is the molecular
mechanism triggering photosensitivity in patients. We therefore suggest
that a FECH assay should generally be part of the preclinical molecular
toxicology package for the development of kinase inhibitors
Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs
The
receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays
important roles in oncogenesis, metastasis, and treatment resistance,
yet therapeutic targeting, drug discovery, or investigation of EPHA2
biology is hampered by the lack of appropriate inhibitors and structural
information. Here, we used chemical proteomics to survey 235 clinical
kinase inhibitors for their kinase selectivity and identified 24 drugs
with submicromolar affinities for EPHA2. NMR-based conformational
dynamics together with nine new cocrystal structures delineated drugāEPHA2
interactions in full detail. The combination of selectivity profiling,
structure determination, and kinome wide sequence alignment allowed
the development of a classification system in which amino acids in
the drug binding site of EPHA2 are categorized into key, scaffold,
potency, and selectivity residues. This scheme should be generally
applicable in kinase drug discovery, and we anticipate that the provided
information will greatly facilitate the development of selective EPHA2
inhibitors in particular and the repurposing of clinical kinase inhibitors
in general