8 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) p38Ī±,
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
Conformation-Selective Inhibitors Reveal Differences in the Activation and Phosphate-Binding Loops of the Tyrosine Kinases Abl and Src
Over the past decade, an increasingly
diverse array of potent and
selective inhibitors that target the ATP-binding sites of protein
kinases have been developed. Many of these inhibitors, like the clinically
approved drug imatinib (Gleevec), stabilize a specific catalytically
inactive ATP-binding site conformation of their kinases targets. Imatinib
is notable in that it is highly selective for its kinase target, Abl,
over other closely related tyrosine kinases, such as Src. In addition,
imatinib is highly sensitive to the phosphorylation state of Ablās
activation loop, which is believed to be a general characteristic
of all inhibitors that stabilize a similar inactive ATP-binding site
conformation. In this report, we perform a systematic analysis of
a diverse series of ATP-competitive inhibitors that stabilize a similar
inactive ATP-binding site conformation as imatinib with the tyrosine
kinases Src and Abl. In contrast to imatinib, many of these inhibitors
have very similar potencies against Src and Abl. Furthermore, only
a subset of this class of inhibitors is sensitive to the phosphorylation
state of the activation loop of these kinases. In attempting to explain
this observation, we have uncovered an unexpected correlation between
Ablās activation loop and another flexible active site feature,
called the phosphate-binding loop (p-loop). These studies shed light
on how imatinib is able to obtain its high target selectivity and
reveal how the conformational preference of flexible active site regions
can vary between closely related kinases
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
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
Development of <i>Toxoplasma gondii</i> Calcium-Dependent Protein Kinase 1 (<i>Tg</i>CDPK1) Inhibitors with Potent Anti-<i>Toxoplasma</i> Activity
Toxoplasmosis is a disease of prominent health concern
that is
caused by the protozoan parasite <i>Toxoplasma gondii</i>. Proliferation of <i>T. gondii</i> is dependent on its
ability to invade host cells, which is mediated in part by calcium-dependent
protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors
of <i>Tg</i>CDPK1 that block invasion of parasites into
host cells, preventing their proliferation. The presence of a unique
glycine gatekeeper residue in <i>Tg</i>CDPK1 permits selective
inhibition of the parasite enzyme over human kinases. These potent <i>Tg</i>CDPK1 inhibitors do not inhibit the growth of human cell
lines and represent promising candidates as toxoplasmosis therapeutics
Multiple Determinants for Selective Inhibition of Apicomplexan Calcium-Dependent Protein Kinase CDPK1
Diseases caused by the apicomplexan protozoans Toxoplasma
gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is
regulated by a family of calcium-dependent protein kinases (CDPKs)
that have no direct homologues in the human host. Fortuitously, CDPK1
from both parasites contains a rare glycine gatekeeper residue adjacent
to the ATP-binding pocket. This has allowed creation of a series of
C3-substituted pyrazolopyrimidine compounds that are potent inhibitors
selective for CDPK1 over a panel of human kinases. Here we demonstrate
that selectivity is further enhanced by modification of the scaffold
at the C1 position. The explanation for this unexpected result is
provided by crystal structures of the inhibitors bound to CDPK1 and
the human kinase c-SRC. Furthermore, the insight gained from these
studies was applied to transform an alternative ATP-competitive scaffold
lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor
of this enzyme with no activity against SRC