Fluoresceinated FKBP12 ligands for a high-throughput fluorescence polarization assay

Several fluoresceinated FKBP12 ligands have been prepared for a high-throughput fluorescence polarization assay. K(i)s for FKBP12 rotamase inhibition by these ligands range from 1.3 microM to 32 nM, and their design is based on X-ray crystal structures of FKBP12 complexed with known immunophilin ligands.     

Characterization of HERG potassium channel inhibition using CoMSiA 3D QSAR and homology modeling approaches

A data set consisting of twenty-two sertindole analogues and ten structurally diverse inhibitors, spanning a wide range in potency, was analyzed using CoMSiA. A homology model of HERG was constructed from the crystal structure of the open MthK potassium channel. A complementary relationship between our CoMSiA and homology models is apparent when the long inhibitor axis is oriented parallel to the longitudinal axis of the pore, with the tail region pointed toward the selectivity filter. The key elements of the pharmacophore, the CoMSiA and the homology model are: (1) The hydrophobic feature optimally consists of an aromatic group that is capable of engaging in pi-stacking with a Phe656 side chain. Optionally, a second aromatic or hydrophobic group present in some inhibitors may contact an additional Phe656 side chain. (2) The basic nitrogen appears to undergo a pi-cation interaction with Tyr652. (3) The pore diameter (12A+), and depth of the selectivity loop relative to the intracellular opening, act as constraints on the conformation-dependent inhibitor dimensions.     

A discriminant model constructed by the support vector machine method for HERG potassium channel inhibitors

HERG attracts attention as a risk factor for arrhythmia, which might trigger torsade de pointes. A highly accurate classifier of chemical compounds for inhibition of the HERG potassium channel is constructed using support vector machine. For two test sets, our discriminant models achieved 90% and 95% accuracy, respectively. The classifier is even applied for the prediction of cardio vascular adverse effects to achieve about 70% accuracy. While modest inhibitors are partly characterized by properties linked to global structure of a molecule including hydrophobicity and diameter, strong inhibitors are exclusively characterized by properties linked to substructures of a molecule.     

A real-time high-throughput fluorescence assay for sphingosine kinases

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC₅₀ values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.     

HERG potassium channel regulation by the N-terminal eag domain

Human ether-á-go-go related gene (hERG, K(v)11.1) potassium channels play a significant role in cardiac excitability. Like other K(v) channels, hERG is activated by membrane voltage; however, distinct from other K(v) channels, hERG channels have unusually slow kinetics of closing (deactivation). The mechanism for slow deactivation involves an N-terminal "eag domain" which comprises a PAS (Per-Arnt-Sim) domain and a short Cap domain. Here we review recent advances in understanding how the eag domain regulates deactivation, including several new Nuclear Magnetic Resonance (NMR) solution structures of the eag domain, and evidence showing that the eag domain makes a direct interaction with the C-terminal C-linker and Cyclic Nucleotide-Binding Homology Domain.     

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.     

Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate

Deactivation of voltage-gated potassium (K(+)) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC(50) = 32 nM) of HERG K(+) channels. This bulky compound (7 x 20 A) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 microM MK-499 to occur with time constants of 10.5 and 52.7 s at -160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K(+) channel.     

A high-throughput fluorescence polarization assay for inhibitors of gyrase B

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.     

A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.     

Inhibition of HERG potassium channel current by the class 1a antiarrhythmic agent disopyramide

The Class 1a antiarrhythmic drug disopyramide (DISO) is associated with 'acquired' prolongation of the QT interval of the electrocardiogram (ECG). This potentially proarrhythmic effect is likely to reflect drug actions on ion channels involved in ventricular action potential repolarisation. In this study, we examined the effects of DISO on potassium channels encoded by HERG, as this K channel type has been implicated in both congenital and acquired long-QT syndromes (LQTS). Chinese hamster ovary cells were transiently transfected with HERG cDNA for subsequent whole cell patch clamp recording. HERG tail currents recorded at -40 mV following test pulses to +30 mV were inhibited in a dose-dependent fashion by DISO concentrations within the clinical range (IC50 = 7.23 +/- 0.72 microM; mean +/- SEM). Experiments with 10 microM DISO indicated that the degree of HERG blockade showed some voltage dependence. Further data obtained using an 'envelope of tails' protocol (pulse potential +40 mV) were consistent with a significant role for open-channel blockade at lower drug concentrations. At higher concentrations it is possible that blockade may have involved drug binding to both resting and open channels. Inhibition of the inactivation-deficient mutant HERG-S631A was comparable to that seen for wild-type HERG. Therefore, channel inactivation was not obligatory for DISO to exert its effect. Native delayed rectifier tail currents from rabbit isolated ventricular myocytes were also inhibited by DISO. We conclude (a) that DISO inhibits HERG encoded potassium channels at clinically relevant concentrations and (b) that this action may constitute the molecular basis for acquired LQTS associated with this drug.     

Coupled K+-water flux through the HERG potassium channel measured by an osmotic pulse method

The streaming potential (V(stream)) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. V(stream) provides a quantitative measure of the ion and water flux (the water-ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure V(stream) with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (<1 s) by an ultrafast solution switching system (the osmotic pulse [quick jump-and-away] method). The reversal potentials were monitored by a series of voltage ramps before, during, and after the osmotic pulse. The shifts of the reversal potentials immediately after the osmotic jump gave V(stream). In symmetrical K+ solutions (10 mM), the V(stream)s measured at different osmolalities showed a linear relationship with a slope of -0.7 mV/DeltaOsm, from which the water-ion coupling ratio (n, the ratio of the flux of water to the flux of cations; Levitt, D.G., S.R. Elias, and J.M. Hautman. 1978. Biochim. Biophys. Acta. 512:436-451) was calculated to be 1.4. In symmetrical 100 mM K+ solutions, the coupling ratio was decreased significantly (n = 0.9), indicating that the permeation process through states with increased ion occupancy became significant. We presented a diagrammatic representation linking the water-ion coupling ratio to the mode of ion permeation and suggested that the coupling ratio of one may represent the least hydrated ion flux in the single-file pore.     

Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.     

Development and validation of a cell-based high-throughput screening assay for TRPM2 channel modulators

TRPM2 is a member of the transient receptor potential melastatin (TRPM)-related ion channel family. The activation of TRPM2 induced by oxidative/nitrosative stress leads to an increase in intracellular free Ca(2+). Although further progress in understanding TRPM2's role in cell and organism physiology would be facilitated by isolation of compounds able to specifically modulate its function in primary cells or animal models, no cell-based assays for TRPM2 function well suited for high-throughput screening have yet been described. Here, a novel suspension B lymphocyte cell line stably expressing TRPM2 was used to develop a cell-based assay. The assay uses the Ca(2+)-sensitive fluorescence dye, Fluo-4 NW (no wash), to measure TRPM2-dependent Ca(2+) transients induced by H(2)O(2) and N-methyl-N'-nitrosoguanidine in a 96-well plate format. Assay performance was evaluated by statistical analysis of the Z' factor value and was consistently greater than 0.5 under optimal conditions, suggesting that the assay is very robust. For assay validation, the effects of known inhibitors of TRPM2 and TRPM2 gating secondary messenger production were determined. Overall, the authors have developed a cell-based assay that may be used to identify TRPM2 ion channel modulators from large compound libraries.     

Development of a high-throughput purification method and a continuous assay system for chlorophyllase

In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity.     

The regulation of the cardiac potassium channel (HERG) by caveolin-1

Protein-protein interaction plays a key role in the regulation of biological processes. The human potassium (HERG) channel is encoded by the ether-à-go-go-related gene (herg), and its activity may be regulated by association with other cellular proteins. To identify cellular proteins that might play a role in the regulation of the HERG channel, we screened a human heart cDNA library with the N terminus of HERG using a yeast 2-hybrid system, and identified caveolin-1 as a potential HERG partner. The interaction between these 2 proteins was confirmed by coimmunoprecipitation assay, and their overlapping subcellular localization was demonstrated by fluorescence immunocytochemistry. The physiologic implication of the protein-protein interaction was studied in whole-cell patch-clamp electrophysiology experiments. A significant increase in HERG current amplitude and a faster deactivation of tail current were observed in HEK293/HERG cells in a membrane lipid rafts disruption model and caveolin-1 knocked down cells by RNA interference. Alternatively, when caveolin-1 was overexpressed, the HERG current amplitude was significantly reduced and the tail current was deactivated more slowly. Taken together, these data indicate that HERG channels interact with caveolin-1 and are negatively regulated by this interaction. The finding from this study clearly demonstrates the regulatory role of caveolin-1 on HERG channels, and may help to understand biochemical events leading to arrhythmogenesis in the long QT syndrome in cardiac patients.     

Reliable and high-throughput mutation screening for beta-thalassemia by a single-base extension/fluorescence polarization assay

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.