Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase,phosphatase, and protease reactions

We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides kinases, such as phosphatases and proteases. By adjustment of the ionic strength of the buffer it is possible to use this method in cases where both the substrate and the enzymatic product are highly negatively charged. All of these enzymatic transformations can be followed in real time, by performing the reactions in the presence of polyarginine and continuously measuring the fluorescence polarization signal. Polyarginine was found to have no effect on the rate of enzymatic conversion of the protease studied (cathepsin G), but its presence decreased the observed rate of phosphorylation by protein kinase A, presumably by decreasing the concentration of free ATP in the reaction solution. Leukocyte antigen related phosphatase catalyzed dephosphorylation reactions were faster in the presence of polyarginine. For all three enzymes, the reaction rates in the presence of polyarginine were found to be sensitive to the presence of known enzyme inhibitors, but the IC(50) values of the kinase inhibitors H-89 and PKI were higher in the presence than in the absence of polyarginine.     

Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3

The serine/threonine kinases Plk1, Plk2, and Plk3 harbor a protein–protein interaction domain dubbed polo-box domain (PBD). Recently, the inhibition of the PBD of the cancer target Plk1 has been successfully explored as an alternative to the inhibition of the kinase by ATP-competitive ligands. However, because the PBDs of Plk1, Plk2, and Plk3 have very similar optimal binding motifs, absolute specificity for the PBD of Plk1 over the PBDs of Plk2 and Plk3 may also represent a big challenge for a small molecule. To aid in the activity profiling of Plk PBD inhibitors, and to identify selective small molecules that will reveal the cellular consequences of inhibiting the PBDs of Plk2 and Plk3, we have developed high-throughput assays based on fluorescence polarization against the PBDs of Plk2 and Plk3. The assays are stable with regard to time and 10% dimethyl sulfoxide and have Z′ values 0.7, making them well-suited for high-throughput screening. Moreover, our data provide insights into the binding preferences of the PBDs of Plk2 and Plk3.     

Evaluation of human microtubule affinity-regulating kinase 4 inhibitors: fluorescence binding studies,enzyme, and cell assays

Human microtubule affinity-regulating kinase 4 (MARK4) is considered as an encouraging drug target for the design and development of inhibitors to cure several life-threatening diseases such as Alzheimer disease, cancer, obesity, and type-II diabetes. Recently, we have reported four ligands namely, BX-912, BX-795, PKR-inhibitor, and OTSSP167 (hydrochloride) which bind preferentially to the two different constructs of human MARK4 containing kinase domain. To ensure the role of ubiquitin-associated (UBA) domain in the ligand binding, we made a newer construct of MARK4 which contains both kinase and UBA domains, named as MARK4-F3. We observed that OTSSP167 (hydrochloride) binds to the MARK4-F3 with a binding constant (K) of 3.16 × 10⁶, M^?1 (±.21). However, UBA-domain of MARK4-F3 doesn’t show any interaction with ligands directly as predicted by the molecular docking. To validate further, ATPase inhibition assays of all three constructs of MARK4 in the presence of mentioned ligands were carried out. An appreciable correlation between the binding experiments and ATPase inhibition assays of MARK4 was observed. In addition, cell-proliferation inhibition activity for all four ligands on the Human embryonic kidney (HEK-293) and breast cancer cell lines (MCF-7) was performed using MTT assay. IC₅₀ values of OTSSP167 for HEK-293 and MCF-7 were found to be 58.88 (±1.5), and 48.2 (±1.6), respectively. OTSSP167 among all four inhibitors, showed very good enzyme inhibition activity against three constructs of MARK4. Moreover, all four inhibitors showed anti-neuroblastoma activity and anticancer properties. In conclusion, OTSSP167 may be considered as a promising scaffold to discover novel inhibitors of MARK4.     

Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative

We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessories which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid analog 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl phosphatidylcholine (I) with Naja-naja venom phospholipase A2 (PLA2) (E.C.3.1.1.4). Upon addition of aliquots of PLA2 containing up to 85 ng protein to a cuvette containing 1-2 microM I, the total measured polarization decreased linearly with time for at least 1000 s. Concomitant analyses of equivalent incubation mixtures and analyses of the contents of the cuvette after incubation and collection of fluorescence data revealed time-dependent formation of N-4-nitrobenzo-2-oxa-1,3-diazole-aminododecanoic acid (II). The decrease in total measured polarization was accelerated by Ca2+ and inhibited by EDTA. These data suggest that PLA2 activity in Naja-naja venom can be measured rapidly at low concentrations of both enzyme (0.01 microgram protein) and substrate (1 microM). Since this technique can be used to collect polarization data over time intervals as short as 4 s, it should be possible to measure the early changes in polarization during the interaction of fluorescent probes with proteins or membranes.     

The fluorescein-mediated interaction of bovine serum albumin with fluorescent derivatives of prolactin and other polypeptides in polarization of fluorescence based assays

During the course of studies relating to the interaction of bovine prolactin with its receptor, it was observed that the fluorescence polarization of prolactin labeled with fluorescein isothiocyanate (fluorescein prolactin) increased from 0.10 to 0.15 upon the addition of bovine serum albumin. Dilution titration measurements show an apparent K_dissociation for the BSA-fluorescein-prolactin complex of 1.1 × 10^?7 M. The stoichiometry of the complex was shown to be approximately 2 mol of fluorescein-prolactin per mole of BSA. The fluorescence emission spectra of the fluorescein moiety in the fluorescein-prolactin is slightly red shifted and increased in intensity in the presence of BSA. The interaction between prolactin and BSA is dependent on the fluorescein attached to the prolactin since [¹²⁵I]prolactin does not form a complex with BSA under identical conditions. The fluorescence polarization of fluorescein-labeled growth hormone and α-lactalbumin also increased in the presence of BSA, suggesting that BSA may interact generally with fluorescein-labeled proteins to form complexes bridged through the fluorescein moiety.     

Evaluation of the fluorescence polarization assay and comparison to other serological assays for detection of brucellosis in cervids

The complement fixation test (CFT), competitive enzyme immunoassay (CELISA), indirect enzyme immunoassay (IELISA) and fluorescence polarization assay (FPA) were evaluated for the detection of antibodies to Brucella abortus and Brucella suis biotype 4 in caribou (Rangifer tarandus caribou), elk (Cervus elapus), red deer (Cervus elapus), and reindeer (Rangifer tarandus tarandus). When combining the data the FPA and the CELISA were determined to be the most suitable tests for serodiagnosis of Cervidae. The overall actual sensitivity of the CFT and the IELISA was 100%. The overall actual sensitivity for the CELISA and FPA was 99%. The overall relative specificity of the CFT (including treatment of anti-complementary data as positive or negative for analysis), the CELISA, the IELISA and the FPA were 65%, 93%, 99%, 99%, and 99%, respectively. The specificities of the buffered plate agglutination test (BPAT), the CFT, the CELISA, the FPA and the IELISA for 55 elk vaccinated with B. abortus strain 19 and tested 4 mo post vaccination were 14%, 31%, 51%, 84%, and 2%, respectively. The FPA is the diagnostic test of choice because it has sensitivity and specificity values comparable to the CELISA; it has the capability to distinguish vaccinal antibody and antibody resulting from exposure to cross-reacting organisms such as Yersinia enterocolitica 0:9 from antibody to Brucella spp. in most cases; it is technically simple to do; it is adaptable to field use and it is relatively inexpensive.     

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.     

An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.     

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization

The proteolytic activity of live Uronema marinum was analyzed by a fluorescence polarization (FP) technique. Protease activity was measured as a decrease in the FP value using fluorescein isothiocynate (FITC)-casein as a protein substrate. A time-dependent decrease in FP occurred in plate wells containing live U. marinum. Supplementation with the cysteine protease inhibitor E-64 had no significant inhibitory effect on the decrease in FP at any of the concentrations used. In contrast, supplementation with 1,10-phenanthroline resulted in complete inhibition of proteolysis for 30 min at 1 mM and for 1 h at 2 and 5 mM. Effective inhibition of the proteolytic activity of live U. marinum by 1,10-phenanthroline indicated that metalloproteases are the main proteases excreted by U. marinum. As U. marinum has a high potential for systemically invading and destroying fish tissues, the metalloproteases excreted by live U. marinum are likely to be involved in the invasion of host tissues and the pathogenicity of the parasite.     

Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays

We have studied the interaction of several phosphopeptides with cationic polyamino acids such as polyarginine and polylysine by fluorescence polarization. The phosphopeptides used were labeled with fluorescein, and their net charges at the experimental pH of 7. 5 were 0, -1, -2, and -3. These phosphopeptides represent the products of enzymatic phosphorylation reactions of the corresponding nonphosphorylated precursors by the protein kinase A, Akt1 (protein kinase Balpha), and protein kinase C. We found that these phosphopeptides bind more strongly to the cationic polyamino acids studied than their nonphosphorylated analogs. This preferential binding of the phosphorylated peptides could be conveniently detected by an increase in the fluorescence polarization signal of the attached fluorescein residue. We have exploited this observation to develop a new approach for the detection of kinase activity that does not require radioactivity or separation of substrate from product. We have successfully used this method to perform K(m) determinations of the kinase enzymes for their substrates and K(i) determinations of one of their inhibitors. This method for measuring kinase activity might be particularly useful for high-throughput screening applications.     

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.     

Protein-tyrosine phosphatase D1, a potential regulator and effector for Tec family kinases

Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain. The signaling mechanisms regulating Etk kinase activity remain largely unknown. To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen. Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated. The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin. In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction. Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction. Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors. The Etk-PTPD1 interaction also increased Stat3 activation. The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation. In addition, Tec (but not Btk) kinase can also be activated by PTPD1. Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.     

From fluorescence polarization to Quenchbody: Recent progress in fluorescent reagentless biosensors based on antibody and other binding proteins

Recently, antibody-based fluorescent biosensors are receiving considerable attention as a suitable biomolecule for diagnostics, namely, homogeneous immunoassay and also as an imaging probe. To date, several strategies for “reagentless biosensors” based on antibodies and natural and engineered binding proteins have been described. In this review, several approaches are introduced including a recently described fluorescent antibody-based biosensor Quenchbody, which works on the principle of fluorescence quenching of attached dye and its antigen-dependent release. The merits and possible demerits of each approach are discussed. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Mutational and inhibitive analysis of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer-based assays

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays key roles in viral replication and is an attractive target for anti-SARS drug discovery. In this report, a fluorescence resonance energy transfer (FRET)-based method was developed to assess the proteolytic activity of SARS-CoV 3CL(pro). Two internally quenched fluorogenic peptides, 1NC and 2NC, corresponding to the N-terminal and the C-terminal autocleavage sites of SARS-CoV 3CL(pro), respectively, were used as substrates. SARS-CoV 3CL(pro) seemed to work more efficiently on 1NC than on 2NC in trans-cleavage assay. Mutational analysis demonstrated that the His41 residue, the N-terminal 7 amino acids, and the domain III of SARS-CoV 3CL(pro) were important for the enzymatic activity. Antibodies recognizing domain III could significantly inhibit the enzymatic activity of SARS-CoV 3CL(pro). The effects of class-specific protease inhibitors on the trans-cleavage activity revealed that this enzyme worked more like a serine protease rather than the papain protease.     

Discovering severe acute respiratory syndrome coronavirus 3CL protease inhibitors: virtual screening, surface plasmon resonance, and fluorescence resonance energy transfer assays

An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C-like protease (SARS-CoV 3CL(pro)) by virtual screening correlating with surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) technologies-based assays. The authors screened 81,287 small molecular compounds against SPECS database by virtual screening; 256 compounds were subsequently selected for biological evaluation. Through SPR technology-based assay, 52 from these 256 compounds were discovered to show binding to SARS-CoV 3CL(pro). The enzymatic inhibition activities of these 52 SARS-CoV 3CL(pro) binders were further applied to FRET-based assay, and IC(50) values were determined. Based on this integrated assay platform, 8 new SARS-CoV 3CL(pro) inhibitors were discovered. The fact that the obtained IC(50) values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants (K(D)s) assayed by SPR implied the reliability of this platform. Our current work is hoped to supply a powerful approach in the discovery of potent SARS-CoV 3CL(pro) inhibitors, and the determined inhibitors could be used as possible lead compounds for further research.     

A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases

Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery.