A homogeneous,nonradioactive high-throughput fluorogenic protein kinase assay

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective. Here we present a novel, rapid, robust assay to measure the enzyme activity of low concentrations of several serine/threonine and tyrosine protein kinases. It is based on the use of fluorogenic peptide substrates (Rhodamine 110, bis peptide amide) that are cleaved before phosphorylation to release the free Rhodamine 110; upon phosphorylation, cleavage is hindered, and the compound remains as a nonfluorescent peptide conjugate. The assay can be carried out in single- as well as multiwell plate formats such as 96- and 384-well plates. The signal-to-noise ratio is very high (40), the Z(') is over 0.8, and the signal is stable for at least 4h. Finally, the assay is easily adapted to a robotic system for drug discovery programs targeting protein kinases.     

Phosphatidic acid regulation of phosphatidylinositol 4-phosphate 5-kinases

Phosphatidic acid (PA) production by receptor-stimulated phospholipase D is believed to play an important role in the regulation of cell function. The second messenger function of PA remains to be elucidated. PA can bind and affect the activities of different enzymes and here we summarise the current status of activation of Type I phosphatidylinositol 4-phosphate 5-kinase by PA. Type 1 phosphatidylinositol 4-phosphate 5-kinase is also regulated by ARF proteins as is phospholipase D and we discuss the contributions of ARF and PA towards phosphatidylinositol(4,5)bisphosphate synthesis at the plasma membrane.     

The intracellular localisation and mobility of Type Igamma phosphatidylinositol 4P 5-kinase splice variants

There are three known splice variants of Type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPkin Iγ): PIPkins Iγ87, Iγ90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489–496) PIPkin IγC (here called PIPkin Iγ93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Iγ93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Iγ93 provide insights into the factors that dictate cellular targeting of Type Iγ PIPkins.     

Development of a homogeneous binding assay for histamine receptors

Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts.     

A direct fluorometric assay for tissue transglutaminase

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.     

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence ⁹¹⁶NFNHIHKRIRRVADKYLSG⁹³⁴ comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence ¹⁴¹⁴SKKTNRGSQLHKYYMKRRTL¹⁴³³, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166–1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.     

A fluorescence polarization assay for identifying ligands that bind to vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a homodimeric proangiogenic protein that induces endothelial cell migration and proliferation primarily through interactions with its major receptors, VEGFR-1 and VEGFR-2. Inhibitors of one or both of these VEGF-receptor interactions could be beneficial as therapeutics for diseases caused by dysfunctional angiogenesis (e.g., cancer). Others have reported small peptides that bind to the VEGF dimer at surface regions that are recognized by the receptors. Here we report the development of a fluorescence polarization assay based on the binding to VEGF of a derivative of one of these peptides that has been labeled with BODIPY-tetramethylrhodamine (BODIPY^TMR). This 384-well format assay is tolerant to dimethyl sulfoxide (DMSO, up to 4% [v/v]) and has a Z′ factor of 0.76, making it useful for identifying molecules that associate with the receptor-binding surface of the VEGF dimer.     

A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)₂ with the released halogen ion of the substrate in the presence of Fe³⁺. The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes.     

Homogeneous fluorescent assay for RNA polymerase

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.     

A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors

Phosphorylation of extracellular signal-regulated kinase (ERK) is linked to activation of many cell surface receptors and kinases. However, phosphorylated ERK has not been used as a biochemical marker to monitor pharmacology of these biomolecules, largely because commonly used methods to detect the phosphoprotein are not quantitative and do not have sufficient throughput. In this article, a high-throughput, 384-well, cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines is described. The assay uses infrared-labeled secondary antibodies to detect phospho-ERK, and the signals in the wells of the microtiter plate are quantified by a LI-COR infrared scanner. V(max), EC(50), and functional K(i) values of various D2 and D3 agonists and antagonists determined in this assay are similar to those in the literature. The assay is nonradioactive, is quantitative, and has a good signal-to-noise ratio. In addition, the signal is stable. This assay can be used to monitor the activities of many G protein-coupled receptors and other signaling biomolecules that are linked to phosphorylation of ERK. The methodology can potentially be used to detect the change in level of any cellular protein in which highly selective antibodies are available.     

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.     

A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries

Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group α-(1→3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and α-L-Fucp-(1→2)-β-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-O-R. A closely related α-(1→3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen α-D-Galp-NAc-(1→3)-[α-L-Fuc(1→2)]-β-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the color change of the pH indicator bromothymol blue when a proton is released during the transfer of Gal/GalNAc from UDP-Gal/UDP-GalNAc to the acceptor substrate. Saturation mutagenesis of GTB enzyme at M214, a hot spot adjacent to the ²¹¹DVD²¹³ metal binding motif, was performed and the resulting library was screened for increases in UDP-GalNAc transfer activity. Two novel mutants, M214G and M214S, identified by pH indicator screening, were purified and kinetically characterized. M214S and M214G both exhibited two-fold higher k_cat and specific activity than wild-type GTB for UDP-GalNAc. The results confirm the importance of residue M214 for donor enzyme specificity.     

A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.     

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours.     

Development of a high-throughput AlphaScreen assay measuring full-length LRRK2(G2019S) kinase activity using moesin protein substrate

Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson’s disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75 nM. The estimated apparent K_m value for adenosine triphosphate (6 μM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z′ factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors.     

A colorimetric assay optimization for high-throughput screening of dihydroorotase by detecting ureido groups

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway and is a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40-μl assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)–thiosemicarbazide (TSC) or DAMO–antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives, including dimethyl sulfoxide, reducing agents, detergents, and bovine serum albumin. DAMO–TSC (Z′-factors 0.7–0.8) was determined to be superior to DAMO–antipyrine (Z′-factors 0.5–0.6) with significantly less variability within replicates. An HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO–TSC. This robust method has no heating requirement, which was the main obstacle to applying previous assays to HTS. More important, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large-scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.     

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.     

Phosphorylation of glycosyl-phosphatidylinositol by phosphatidylinositol 3-kinase changes its properties as a substrate for phospholipases

Phosphatidylinositol 3-kinases (PI3K) phosphorylate the 3-position of the inositol ring of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate. It is not clear whether PI3K can phosphorylate the inositol group in other biomolecules. We sought to determine whether PI3K was able to use glycosyl-phosphatidylinositol (GPI) as a substrate. This phospholipid may exist either in free form (GPIfree) or forming a lipid anchor (GPIanchor) for the attachment of extracellular proteins to the plasma membrane. We demonstrate the specific PI3K-mediated phosphorylation of the inositol 3-hydroxyl group within both types of GPI by incubating this phospholipid with immunoprecipitated PI3K. The phosphorylated product behaves in HPLC as a derivative of a PI3K lipid product. To our knowledge, this is the first demonstration that PI3K uses lipid substrates other than phosphoinositides. Further, we show that this has potential functional consequences. When GPIfree is phosphorylated, it becomes a poorer substrate for GPI-specific phospholipase D, but a better substrate for phosphatidylinositol-specific phospholipase C. These phosphorylation events may constitute the basis of a previously undescribed signal transduction mechanism.     

A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.