Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize K‑Ras

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 K‑Ras

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

Expanding the Scope of Electrophiles Capable of Targeting K‑Ras 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 IIIβ (PI4Kβ) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kβ followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI­(4,5)­P₂ 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 PI­(4,5)­P₂ generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kβ by a small molecule inhibitor was also quantitatively assessed, yielding an EC₅₀ 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^I316 in the zebrafish embryo.

<p>(<b>A</b>) Schematic diagram of the <i>in vivo</i> labeling method for the selective labeling of Prkci^I316A substrates during zebrafish development. (<b>B</b>) <i>In vivo</i> thiophosphorylation in zebrafish embryos injected at the one-cell stage with 200 μM <i>N⁶</i>-benzyl-ATPγS (6-bn-ATPγS) and mRNA encoding either Prkci^WT or Prkci^I316A (AS). Western blot analysis with rabbit monoclonal anti-thiophosphoester (α-thioP) C51-8 antibody (Epitomics) of 80% epiboly (6–8hpf) samples alkylated with 2.5 mM PNBM reveals a selectively labeled protein in the Prkci^I316A (AS) sample (asterisk).</p

Design of the analog-sensitive Prkci.

<p>(<b>A</b>) A space-creating mutation (“gatekeeper mutation”) 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^I316A has normal <i>in vivo</i> biological activity.

<p>(<b>A</b>) Reconstruction of confocal Z-stack sections of embryonic hearts at 28–30 hpf. Transgenic Tg[<i>cmlc2:GFP</i>]^twu34 one-cell stage embryos were injected with <i>prkci</i> MO alone or together with mRNA encoding Prkci^WT 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^WT or Prkci^I316A rescues heart tube elongation. In comparison, the analog-sensitive mutant form Prkci^V300A 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^WT or Prkci^I316A 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>^twu34</i> at 28–30 hpf. Expression of exogenous HisMyc-Prkci^WT or HisMyc-Prkci^I316A 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 IC₅₀ values) and blocking parasite growth in host cell monolayers in vivo (low μM EC₅₀ 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