A high-throughput screen for identification of molecular mimics of Smac/DIABLO utilizing a fluorescence polarization assay

Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.     

A fluorescence polarization assay for native protein substrates of kinases

Protein phosphorylation is the mediator of many important cellular processes of signal transduction and cell regulation. Phosphorylation often occurs on multiple sites within a single protein, whereby the results of individual phosphorylations are not well defined. This is partially due to the lack of tools for analyzing specific phosphorylation states in a quantitative manner. We have developed a high-throughput, rapid, and quantitative method for the determination of the phosphorylation status of peptides and, more importantly, native protein substrates of kinases using a competitive fluorescence-based approach. We have applied our method to measuring the phosphorylation activity of the Wee1 and Myt1 kinases. Our technique allows one to monitor the bis-phosphorylation status of the Cdk2 protein using an antibody specific for bis-phosphorylated Cdk2 and a fluorescently labeled bis-phosphorylated Cdk2 peptide. We have used this assay to screen a library of 16 general kinase inhibitors against Wee1 and Myt1 activity. None of the inhibitors inhibited Wee1, but both staurosporine and K-252a inhibited Myt1, with IC(50) values of 9.2+/-3.6 and 4.0+/-1.3 microM, respectively.     

A fluorescence polarization assay for inhibitors of Hsp90

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.     

Development of a fluorescence polarization based assay for histone deacetylase ligand discovery

Histone deacetylases (HDACs) regulate many important physiological processes and the discovery of small molecules that modulate HDAC activity has both academic and clinical relevance. HDAC inhibitors, most notably SAHA, have been pursued as cancer chemotherapeutics but may be useful in treating psychiatric disorders, malaria, and other diseases. Herein, we describe an inexpensive and robust assay, based on fluorescence polarization, for HDAC ligand discovery. The assay is well suited for high-throughput screening and enzyme kinetic studies.     

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

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.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

Medium throughput cage state stability screen of conditions for the generation of gold nanoparticles encapsulated within a mini-ferritin

Methods for the generation of nanoparticles encapsulated within cage proteins, such as ferritins, provide particles with low polydispersities due to size constraint by the cage. The proteins can provide enhanced water solubility to enable biological applications and affinity and identification tags to facilitate delivery or the assembly of advanced materials. Many effective methods have been developed, however, they are often impeded by cage protein instability in the presence of reagents or conditions for formation of the nanoparticles. Although the stability of ferritin cage quaternary structure can be enhanced, application of ferritins to materials science remains limited by unpredictable behaviour. Recently, we reported a medium throughput technique to directly detect the ferritin cage state. Herein, we expand this strategy to screen conditions commonly used for the formation of gold nanoparticles. Not only do we report nanoparticle formation conditions that permit ferritin stability, we establish a general screening strategy based on protein cage stability that could be applied to other protein cages or for the generation of other types of particles.     

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.     

A fluorescence correlation spectroscopy-based assay for fragment screening of slowly inhibiting protein-peptide interaction inhibitors

A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein-peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein-peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.     

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.     

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.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

布鲁氏菌病荧光偏振抗体检测方法的建立

【目的】布鲁氏菌病(Brucellosis)简称布病,是由布鲁氏菌引起的以感染家畜为主的人畜共患传染病,造成严重的公共卫生问题。目前全世界范围内消除该病的主要方法是扑杀与免疫相结合,所以建立快速准确的诊断方法对防治和清除布病非常必要。本文建立布鲁氏菌病荧光偏振(FPA)抗体检测方法,为布鲁氏菌病(布病)的快速高效诊断提供技术手段。【方法】提纯猪种布鲁氏菌S2株脂多糖O链(OPS),经异硫氰酸荧光素(FITC)标记后作为诊断抗原。通过对样品稀释液、抗原稀释度、反应时间等条件的优化,初步建立了布鲁氏菌荧光偏振诊断方法。用该方法对148份布病阳性血清(其中牛血清70份,羊血清78份)和155份布病阴性血清(其中牛血清82份,羊血清73份)进行检测,确定其敏感性和特异性。按确定的技术参数,制备3批布鲁氏菌FPA抗体检测试剂盒,使用质控阴、阳性血清分别评价试剂盒的批内和批间重复性。用400份临床样本比较本研究开发试剂盒与商品化进口FPA试剂盒的符合率。【结果】使用0.5%蔗糖磷酸缓冲液作为血清样品稀释液;标记抗原的使用浓度为90μg/m L;最佳反应时间为3-5 min。本检测方法的判定标准为:δm P值20时为阴性,δm P值≥20时为阳性。按上述条件建立的FPA检测148份布病阳性血清和155份布病阴性血清,结果敏感性为98.6%,特异性为98.7%。对400份临床样本的比对检测显示,研究建立的FPA方法与进口商品化试剂盒的总符合率为94.0%。【结论】研究建立的布鲁氏菌PFA抗体检测方法具有良好的特异性和敏感性,可作为一种重要的布病诊断快速诊断方法。     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

Binding of bovine prion protein to heparin: a fluorescence polarization study

Glycosaminoglycans (GAGs) are believed to be associated with prion disease pathology and also with metabolism of the prion protein. Fluorescence polarization assay (FPA) of binding between bovine recombinant prion protein (brecPrP) and heparin labelled with AlexaFluor488 was used in model experiments to study glycosaminoglycan-prion protein interaction. Heparin binding to brecPrP was a rapid reversible event which occurred under defined conditions. The interaction of brecPrP with fluorophore-labelled heparin was inhibited by the presence of Cu(2+) ions and was sensitive to competition with heparin, heparan sulphate, and dextran. The dissociation constant of the heparin-brecPrP complex was 73.4+/-3.7 nM. Circular dichroism (CD) experiments indicated that the structure of brecPrP was less helical in the presence of heparin.     

Activation of the Escherichia coli cell division protein FtsZ by a low-affinity interaction with monovalent cations

We have investigated the activation of FtsZ by monovalent cations. FtsZ polymerization was dependent on the concentrations of protein and monovalent salts, and was accompanied by the uptake of a single ion per monomer added. The affinity and the specificity for the cation were low. Potassium, ammonium, rubidium or sodium activated FtsZ to different extents. Electron microscopy showed that polymers formed with either rubidium, or potassium, were very similar, as were their nucleotide turnover rates. The GTPase activity was lower with rubidium than with potassium, indicating that nucleotide exchange is independent of nucleotide hydrolysis. Control of polymerization by binding of a low affinity cation might govern the dynamic behavior of the FtsZ polymers.