32 research outputs found
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Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize KāRas
Mutationally
activated Ras is one of the most common oncogenic
drivers found across all malignancies, and its selective inhibition
has long been a goal in both pharma and academia. One of the oldest
and most validated methods to inhibit overactive Ras signaling is
by interfering with its post-translational processing and subsequent
cellular localization. Previous attempts to target Ras processing
led to the development of farnesyltransferase inhibitors, which can
inhibit H-Ras localization but not K-Ras due to its ability to bypass
farnesyltransterase inhibition through alternative prenylation by
geranylgeranyltransferase. Here, we present the creation of a neo-substrate
for farnesyltransferase that prevents the alternative prenlation by
geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells
Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize KāRas
Mutationally
activated Ras is one of the most common oncogenic
drivers found across all malignancies, and its selective inhibition
has long been a goal in both pharma and academia. One of the oldest
and most validated methods to inhibit overactive Ras signaling is
by interfering with its post-translational processing and subsequent
cellular localization. Previous attempts to target Ras processing
led to the development of farnesyltransferase inhibitors, which can
inhibit H-Ras localization but not K-Ras due to its ability to bypass
farnesyltransterase inhibition through alternative prenylation by
geranylgeranyltransferase. Here, we present the creation of a neo-substrate
for farnesyltransferase that prevents the alternative prenlation by
geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells
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Expanding the Scope of Electrophiles Capable of Targeting KāRas Oncogenes
There is growing interest in reversible
and irreversible covalent
inhibitors that target noncatalytic amino acids in target proteins.
With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by
expanding the types of amino acids that can be targeted by covalent
inhibitors, we survey a set of electrophiles for their ability to
label carboxylates. We functionalized an optimized ligand for the
K-Ras switch II pocket with a set of electrophiles previously reported
to react with carboxylates and characterized the ability of these
compounds to react with model nucleophiles and oncogenic K-Ras proteins.
Here, we report that aziridines and stabilized diazo groups preferentially
react with free carboxylates over thiols. Although we did not identify
a warhead that potently labels K-Ras G12D, we were able to study the
interactions of many electrophiles with K-Ras, as most of the electrophiles
rapidly label K-Ras G12C. We characterized the resulting complexes
by crystallography, hydrogen/deuterium exchange, and differential
scanning fluorimetry. Our results both demonstrate the ability of
a noncatalytic cysteine to react with a diverse set of electrophiles
and emphasize the importance of proper spatial arrangements between
a covalent inhibitor and its intended nucleophile. We hope that these
results can expand the range of electrophiles and nucleophiles of
use in covalent protein modulation
Staurosporine-Derived Inhibitors Broaden the Scope of Analog-Sensitive Kinase Technology
Analog-sensitive (AS) kinase technology
is a powerful approach
for studying phospho-signaling pathways in diverse organisms and physiological
processes. The key feature of this technique is that a kinase-of-interest
can be mutated to sensitize it to inhibitor analogs that do not target
wild-type (WT) kinases. In theory, this enables specific inhibition
of any kinase in cells and in mouse models of human disease. Typically,
these inhibitors are identified from a small library of molecules
based on the pyrazolopyrimidine (PP) scaffold. However, we recently
identified a subset of native human kinases, including the Ephrin
A kinase family, that are sensitive to commonly used PP inhibitors.
In an effort to develop a bioorthogonal AS-kinase inhibitor and to
extend this technique to PP-sensitive kinases, we sought an alternative
inhibitor scaffold. Here we report the structure-based design of synthetically
tractable, potent, and extremely selective AS-kinase inhibitors based
on the natural product staurosporine. We demonstrate that these molecules,
termed staralogs, potently target AS kinases in cells, and we employ
X-ray crystallography to elucidate their mechanism of efficacy. Finally,
we demonstrate that staralogs target AS mutants of PP-sensitive kinases
at concentrations where there is little to no inhibition of native
human kinases. Thus, staralogs represent a new class of AS-kinase
inhibitors and a core component of the chemical genetic tool kit for
probing kinase-signaling pathways
Staurosporine-Derived Inhibitors Broaden the Scope of Analog-Sensitive Kinase Technology
Analog-sensitive (AS) kinase technology
is a powerful approach
for studying phospho-signaling pathways in diverse organisms and physiological
processes. The key feature of this technique is that a kinase-of-interest
can be mutated to sensitize it to inhibitor analogs that do not target
wild-type (WT) kinases. In theory, this enables specific inhibition
of any kinase in cells and in mouse models of human disease. Typically,
these inhibitors are identified from a small library of molecules
based on the pyrazolopyrimidine (PP) scaffold. However, we recently
identified a subset of native human kinases, including the Ephrin
A kinase family, that are sensitive to commonly used PP inhibitors.
In an effort to develop a bioorthogonal AS-kinase inhibitor and to
extend this technique to PP-sensitive kinases, we sought an alternative
inhibitor scaffold. Here we report the structure-based design of synthetically
tractable, potent, and extremely selective AS-kinase inhibitors based
on the natural product staurosporine. We demonstrate that these molecules,
termed staralogs, potently target AS kinases in cells, and we employ
X-ray crystallography to elucidate their mechanism of efficacy. Finally,
we demonstrate that staralogs target AS mutants of PP-sensitive kinases
at concentrations where there is little to no inhibition of native
human kinases. Thus, staralogs represent a new class of AS-kinase
inhibitors and a core component of the chemical genetic tool kit for
probing kinase-signaling pathways
Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases
The
multienzyme catalytic phosphorylation of phosphatidylinositol
(PI) in a supported lipid membrane platform is demonstrated for the
first time. One-step treatment with PI 4-kinase IIIĪ² (PI4KĪ²)
yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade
of PI4KĪ² followed by PIP 5-kinase produced PI-4,5-bisphosphate
(PIĀ(4,5)ĀP<sub>2</sub> or PIP2). By employing quartz crystal microbalance
with dissipation monitoring, we were able to track membrane association
of kinase enzymes for the first time as well as detect PI4P and PIĀ(4,5)ĀP<sub>2</sub> generation based on subsequent antibody binding to the supported
lipid bilayers. Pharmacologic inhibition of PI4KĪ² by a small
molecule inhibitor was also quantitatively assessed, yielding an EC<sub>50</sub> value that agrees well with conventional biochemical readout.
Taken together, the development of a PI-containing supported membrane
platform coupled with surface-sensitive measurement techniques for
kinase studies opens the door to exploring the rich biochemistry and
pharmacological targeting of membrane-associated phosphoinositides
Thiophosphorylation of substrate proteins by Prkci<sup>I316</sup> in the zebrafish embryo.
<p>(<b>A</b>) Schematic diagram of the <i>in vivo</i> labeling method for the selective labeling of Prkci<sup>I316A</sup> substrates during zebrafish development. (<b>B</b>) <i>In vivo</i> thiophosphorylation in zebrafish embryos injected at the one-cell stage with 200 Ī¼M <i>N<sup>6</sup></i>-benzyl-ATPĪ³S (6-bn-ATPĪ³S) and mRNA encoding either Prkci<sup>WT</sup> or Prkci<sup>I316A</sup> (AS). Western blot analysis with rabbit monoclonal anti-thiophosphoester (Ī±-thioP) C51-8 antibody (Epitomics) of 80% epiboly (6ā8hpf) samples alkylated with 2.5 mM PNBM reveals a selectively labeled protein in the Prkci<sup>I316A</sup> (AS) sample (asterisk).</p
Design of the analog-sensitive Prkci.
<p>(<b>A</b>) A space-creating mutation (āgatekeeper mutationā) is introduced into the kinase ATP binding pocket which allows the analog-sensitive (AS) kinase mutant to accept a bulky ATP analog (A*TP) required for the chemical genetic identification of kinase substrates [modified after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Cravatt1" target="_blank">[36]</a>]. (<b>B</b>) Alignment of the primary sequence of the ATP binding pocket within the kinase domains of Prkci, v-Src and c-Raf. The residues in red correspond to the amino acids mutated in v-Src <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Liu1" target="_blank">[1]</a>, c-Raf-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040000#pone.0040000-Hindley1" target="_blank">[28]</a>, and Prkci to enlarge the ATP binding pocket.</p
Mutant Prkci<sup>I316A</sup> has normal <i>in vivo</i> biological activity.
<p>(<b>A</b>) Reconstruction of confocal Z-stack sections of embryonic hearts at 28ā30 hpf. Transgenic Tg[<i>cmlc2:GFP</i>]<sup>twu34</sup> one-cell stage embryos were injected with <i>prkci</i> MO alone or together with mRNA encoding Prkci<sup>WT</sup> or analog-sensitive mutant forms of Prkci. Whereas the wild-type heart elongates into a heart tube and towards the left during cardiac jogging, heart development arrests at the cone stage and the heart remains at the embryonic midline in <i>prkci</i> morphants. In functional rescue experiments, injection of <i>prkci</i>MO together with mRNA encoding HisMyc-Prkci<sup>WT</sup> or Prkci<sup>I316A</sup> rescues heart tube elongation. In comparison, the analog-sensitive mutant form Prkci<sup>V300A</sup> fails to rescue heart tube formation in <i>prkci</i> morphants. Percentiles indicate the occurrence of the most common phenotype as depicted in the images and numbers show the total of embryos tested. White dotted line indicates the embryonic midline. L, left; R, right. (<b>B</b>) Membrane localization of endogenous Prkci detected with an anti-Prkci antibody and exogenous Prkci<sup>WT</sup> or Prkci<sup>I316A</sup> in zebrafish cardiomyocytes detected with an anti-Myc antibody. Images are confocal reconstructions of single Z-stack sections of embryonic hearts marked by the transgenic reporter Tg[<i>cmlc2:GFP</i>]<i><sup>twu34</sup></i> at 28ā30 hpf. Expression of exogenous HisMyc-Prkci<sup>WT</sup> or HisMyc-Prkci<sup>I316A</sup> in cardiomyocytes reveals that both recombinant proteins localize to the cell membrane.</p
Optimizing Small Molecule Inhibitors of Calcium-Dependent Protein Kinase 1 to Prevent Infection by Toxoplasma gondii
Toxoplasma gondii is sensitive to
bulky pyrazolo [3,4-<i>d</i>] pyrimidine (PP) inhibitors
due to the presence of a Gly gatekeeper in the essential calcium dependent
protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives
of 3-methyl-benzyl-PP (3-MB-PP, or <b>1</b>). The potency of
PP analogues in inhibiting CDPK1 enzyme activity in vitro (low nM
IC<sub>50</sub> values) and blocking parasite growth in host cell
monolayers in vivo (low Ī¼M EC<sub>50</sub> values) were highly
correlated and occurred in a CDPK1-specific manner. Chemical modification
of the PP scaffold to increase half-life in the presence of microsomes
in vitro led to identification of compounds with enhanced stability
while retaining activity. Several of these more potent compounds were
able to prevent lethal infection with T. gondii in the mouse model. Collectively, the strategies outlined here provide
a route for development of more effective compounds for treatment
of toxoplasmosis and perhaps related parasitic diseases