Single-step kinase inhibitor screening using a peptide-modified gold nanoparticle platform

Two complementary formats for kinase inhibitor screening are presented in which peptide-modified gold nanoparticles are enzymatically phosphorylated and rapidly aggregate on a surface or in solution by action of phosphospecific antibodies. The simple and rapid colourimetric response of the assays makes them an attractive approach for drug-screening applications.     

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally theo-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.     

Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.     

Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

Ring-opening and ring-closing reactions of a shikimic acid-derived substrate leading to diverse small molecules

An epoxide derived from (-)-shikimic acid was attached to a solid support and used to synthesize over 5000 diverse small molecules. Key transformations include a Lewis acid-catalyzed epoxide opening with amines and an intramolecular Heck reaction with aryl iodides. Compounds derived from this pathway were printed onto small-molecule microarrays and screened for binding to proteins. Compounds that bound to Aurora A kinase were characterized using surface plasmon resonance.     

Following protein kinase acivity by electrochemical means and contact angle measurements

The electrochemical analysis of the protein kinase, casein kinase, is accomplished by the voltammetric response of Ag(+) ions associated with the phosphorylated product; the sensing surface is regenerated by the cleavage of the phosphorylated product with alkaline phosphatase, and the phosphorylation/de-phosphorylation processes are monitored by XPS and contact angle measurements.     

Luminescent kinase activity biosensors based on a versatile bimolecular switch

Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.     

Mitochondrial creatine kinase adsorption to biomimetic membranes: a Langmuir monolayer study

Interaction of mitochondrial creatine kinase (mtCK) with either synthetic or natural zwitterionic or acidic phospholipids was monitored by surface pressure measurements. Injection of mtCK beneath a monolayer at very low surface pressure results in a large increase in the apparent area per lipid molecule reflecting the intrinsic surface activity of the protein. This effect is particularly pronounced with anionic phospholipid-containing films. Upon compression to high lateral pressure, the protein is squeezed out of the lipid monolayer. On the contrary, mtCK injected beneath a monolayer compressed at 30 mN/m, does not insert into the monolayer but is concentrated below the surface by anionic phospholipids as evidenced by the immediate and strong increase in the apparent molecular area occurring upon decompression. Below 8 mN/m the protein adsorbs to the interface and remains intercalated until the lateral pressure increases again. The critical pressure of insertion is higher for anionic lipid-containing monolayers than for films containing only zwitterionic phospholipids. In the former case it is markedly diminished by NaCl. The adsorption of mtCK depends on the percentage of negative charges carried by the monolayer and is reduced by increasing NaCl concentrations. However, the residual interaction existing in the absence of a global negative charge on the membrane may indicate that this interaction also involves a hydrophobic component.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using a multifunctional nanoprobe

We presented a novel electrogenerated chemiluminescence (ECL) biosensor for monitoring the activity and inhibition of protein kinases based on signal amplification using enzyme-functionalized Au NPs nanoprobe. In this design, the biotin-DNA labeled glucose oxidase/Au NPs (GOx/Au NPs/DNA-biotin) nanoprobes, prepared by conjugating Au NPs with biotin-DNA and GOx, were bound to the biotinylated anti-phosphoserine labeled phosphorylated peptide modified electrode surface through a biotin−avidin interaction. The GOx assembled on the nanoprobe can catalyze glucose to generate H₂O₂ in the presence of O₂ while the ECL reaction occurred in the luminol ECL biosensor. At a higher concentration of kinase, there are more nanoprobes on the electrode, which gives a higher amount of GOx at the electrode interface and thus higher electrocatalytic efficiency to the luminol ECL reaction. Therefore, the activity of protein kinases can be monitored by ECL with high sensitivity. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the present proof-of-concept strategy. The as-proposed biosensor presents high sensitivity, low detection limit of 0.013 U mL⁻¹, wide linear range (from 0.02 to 40 U mL⁻¹), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC₅₀ of ellagic acid, a typical PKA inhibitor, was estimated, which is in agreement with those obtained using the conventional kinase assay. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

An Adaptor Domain‐Mediated Autocatalytic Interfacial Kinase Reaction

This paper describes a model system for studying the autocatalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self‐assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an autocatalytic process. The kinetic non‐linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.     

Druggable exosites of the human kino-pocketome

Journal of Computer-Aided Molecular Design - Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding...     

Simulations of the active transport of a neutral solute based on a kinase-channel-phosphatase topology

Simulations of coupled interactions involving two opposite enzymatic reactions, solute diffusions, and electrostatic interactions between membrane charges and charged solutes were conducted under a fixed kinase-channel-phosphatase (KCP) topology oriented from the outside to the inside of a porous membrane structure. Depending on the kinase and phosphatase locations, we recently demonstrated that an active transport of a phosphorylated substrate may occur via the opposite topology, that is, a PCK topology. The present analysis demonstrates that, under a KCP membrane topology, which also behaves as a specific ATP-dependent transporter, the active transport of a neutral substrate may occur. This analogous active transport appears to be dependent on the phosphatase location and on the membrane surface potentials. A broad analysis of the role played by the main parameters taken into account in the model was conducted in order to define precisely the physico-chemical conditions and the membrane topology needed for the highest active transports within the shortest time.     

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis.     

Surrogate AutoShim: predocking into a universal ensemble kinase receptor for three dimensional activity prediction, very quickly, without a crystal structure

"Ensemble surrogate AutoShim" is a kinase specific extension of the AutoShim docking method that solves the three traditional limitations of conventional docking: (1) it gives good correlations with affinity, (2) does not require a target protein structure, and (3) for a preprocessed company archive of 1.5 million compounds, is as fast as traditional 2D QSAR. It does require several hundred experimental IC 50 values for each new target. Original AutoShim adds pharmacophore "shims" to a crystal structure binding site. An iterative partial least squares (PLS) procedure selects the best pose, while adjusting the shim weights to reproduce IC 50 data. Surrogate AutoShim adjusts shims in one crystal structure to reproduce IC 50 data for a different kinase target. Ensemble surrogate AutoShim uses 16 structurally diverse kinase crystal structures as a "universal ensemble kinase receptor", suitable for any kinase target. The 1.5 million member Novartis screening collection has been predocked into the shimmed ensemble, so new kinase models can be built, and the entire corporate archive virtually screened, in hours rather than weeks. A kinase-biased set of 10,000 compounds, that samples the entire corporate archive, has been designed for lead discovery by iterative kinase screening.     

DISTRIBUTION OF ENZYMES INVOLVED IN NUCLEOTIDE METABOLISM IN THE DISK AND OTHER ROD MEMBRANES

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'-nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 m M Mg²⁺. The enzymes dissociated from the membrane when Mg²⁺ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'-Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'-nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light-activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.     

Betulin,a Newly Characterized Compound in Acacia auriculiformis Bark,Is a Multi-Target Protein Kinase Inhibitor

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/β (GSK-3 α/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.     

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.

Analytical Ultracentrifugation as a Matrix-Free Probe for the Study of Kinase Related Cellular and Bacterial Membrane Proteins and Glycans

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.     

蛋白质吸附表面活性剂的测定方法研究

一定ｐＨ值和一定浓度下，蛋白质与表面活性剂相互作用形成复合物产生沉淀。作者利用这一原理，研究了肌酸激酶对表面活性剂的吸附关系，并据此发展了滴定法和表面张力法两种简便测定蛋白质对表面活性剂吸附量的方法。