The development of a high-content screening binding assay for the smoothened receptor

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-μM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.     

Structure-function characterization for ethidium photoaffinity labels as mutagens in salmonella

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial findings showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3,8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity.Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.     

Prostate targeting ligands based on N-acetylated alpha-linked acidic dipeptidase

To identify inhibitors of the intrinsic N-acetylated alpha-linked acidic dipeptidase (NAALADase) activity of prostate specific membrane antigen (PSMA) that may be useful for targeting imaging agents or chemotherapeutic drugs to disseminated prostate cancer, analogs of the tetrahedral transition state for hydrolysis of the natural substrate, N-acetylaspartylglutamate (NAAG), were synthesized. These compounds were assayed for their ability to inhibit the membrane-associated enzyme isolated from LNCaP prostate cancer cells. Active inhibitors were further assayed for their cytotoxicity and membrane binding. We have identified nine compounds, including fluorescent and iodine-labeled conjugates, which inhibit NAALADase enzyme activity with IC(50)s at, or below, 120nM. The binding of these compounds to the cell surface of viable LNCaP prostate tumor cells appears to be specific and saturable, and none of the compounds alter the cell cycle kinetics or induce apoptosis in LNCaP cells, suggesting that they are relatively innocuous and are suitable for targeting imaging agents or cytotoxic drugs to disseminated prostate cancer.     

Comparison of ethidium bromide and 4′,6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

Two methods of quantitative, fluorescent detection of DNA bands in agarose gels separated by electrophoresis were evaluated for sensitivity and linearity of response. Comparisons of ethidium bromide staining with a method using 4′,6-diamidino-2-phenylindole (DAPI) developed in this work showed that DAPI is several times more sensitive than ethidium bromide in conditions of comparable background flourescence. Optimum flourescent staining and detection conditions for DNA bands in agarose gels using DAPI are desctribed, and advantages of this method over other fluorescent detection methods are discussed.     

Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.     

Petite induction in Saccharomyces cerevisiae by ethidium analogs. Action on mitochondrial genome

Petite induction of ethidium analogs was examined in both resting and growing yeast cells. All of the analogs used in these experiments were active in dividing cells of Saccharomyces cerevisiae; only the parent ethidium bromide was mutagenic under resting conditions. Incorporation of adenine into mitochondrial DNA appeared to be prevented completely by ethidium and partially inhibited by other analogs. Treatment of growing cells with analogs affected fragmentation of pre-existing DNA as seen by the loss of a mitochondrial antibiotic resistance marker. The rates of elimination of the marker were different; ethidium generated greater loss than the monoamino analogs (3-amino and 8-amino-); and the deaminated analog was least effective. However, in resting yeast the marker was partially eliminated only with treatment of the parent ethidium. The degradation of the mitochondrial DNA by exposure to ethidium compounds was confirmed by agarose gel electrophoresis. Electrophoretic patterns of the mitochondrial DNA treated with each of the analogs under growing conditions and only with ethidium under resting conditions showed degradation of the mitochondrial DNA.     

A fluorescence probe of acetylcholine receptor conformation and local anesthetic binding

The fluorescent dye ethidium bromide binds to the acetylcholine receptor with an apparent K_d of $\text{3 μ}\text{M}$ and a stoichiometry of 1 molecule of ethidium per α-bungarotoxin site. Time dependent fluorescent increases were observed upon addition of carbamylcholine, the amplitude and half-time of which were dependent on the Carb¹ concentration. It appeared that these fluorescence increases resulted from a lowering of the K_d for ethidium as the AcChR-Carb complex underwent an isomerization from low to high affinity form(s) for carb, and more ethidium was bound. Titration with the local anesthetic procaine led to ethidium fluorescence increases at low procaine concentrations, followed by a fluorescence decrease at higher procaine concentrations to that level induced by saturating α-bungarotoxin. Thus it appeared that the ethidium binding site either interacted with or was identical with local anesthetic binding site(s).     

Histochemical applications of two phenanthridinium compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10(-5) M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10(-5) M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

Histochemical Applications of Two Phenanthridinium Compounds

The fluorescent compounds ethidium monoazide and ethidium bromide were found to react intensely with nucleic acids of fixed, paraffin embedded tissues of rat and mouse. For routine staining, 10^-5 M solutions of ethidium bromide and its monoazide analogue were virtually identical in their reactions. Fresh frozen sections of the tissues reacted in the same manner as fixed, paraffin embedded samples. Fluorescence of DNA and RNA in rat pancreas could be selectively abolished by taking advantage of the greater sensitivity of RNA to acid hydrolysis. Hydrolysis in aqueous solutions (1 N HCl at 55-60 C) abolished RNA fluorescence in 5 min, whereas 20 min or longer were required to destroy DNA fluorescence. DNA fluorescence was selectively abolished by 3 hr in 0.1 N HCl in anhydrous methanol while the RNA remained unaffected. Rat pancreas stained with the 10^-5 M ethidium compounds below pH 5.0 showed reduced RNA fluorescence, but the DNA continued to fluoresce brightly at pH 0.6. Reducing the pH of the staining solution to pH 1.0, therefore, was an additional method of selectively abolishing RNA fluorescence. Ethidium solutions in 5.0 M NaCl at pH 5.0 had little effect on DNA or RNA fluorescence. This new method of examining nucleic acids in fixed tissue samples opens new approaches to the histochemistry of these substances. The method also offers new possibilities for the study of mutagenic drug-DNA interactions.     

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.     

Ligand polarizability contributes to tau fibril binding affinity

Whole brain imaging of tau-bearing neurofibrillary lesions has the potential to improve the premortem diagnosis and staging of Alzheimer's disease. Diverse compounds with high affinity for tau aggregates have been reported from high-throughput screens, but the affinity driving features common among them have not been determined. To identify these features, analogs of compounds discovered by high-throughput screening, including phenothiazine, triarylmethine, benzothiazole, and oxindole derivatives, were tested for their ability to displace fluorescent thioflavin dyes from filaments made from recombinant tau protein or authentic paired helical filaments purified from Alzheimer's disease tissue. When representative members of all scaffolds were assayed, the rank order of binding affinity determined for synthetic and authentic filaments correlated strongly, indicating that synthetic filaments have predictive utility for ligand development. Within individual scaffold families, binding affinity was found to correlate with compound polarizability, consistent with a role for dispersion forces in mediating ligand binding. Overall, the data indicate that polarizability is an important commonality among structurally diverse tau binding ligands, and that affinity for tau aggregates can be maximized by integrating formal assessment of this parameter into ligand discovery efforts.     

Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence

The fluorogenic properties of quercetin and similar flavonoids common in plants were exploited to analyse their interaction with target proteins. Quercetin produced a strong fluorescent signal upon binding to bovine serum albumin (BSA) and insulin. The fluorescent signal showed saturation kinetics with increasing flavonoid concentrations indicating the presence of defined peptide binding motifs. Other tested proteins showed no fluorescence with the flavonoids. In a comparative study including 22 flavonoids the compounds with fluorogenic properties were identified using our model proteins BSA and insulin and the structural requirements for the fluorogenic property were defined. Only flavones with a high degree of hydroxylation were able to elicit fluorescence. The emitted fluorescence was strongly enhanced at alkaline pH. Finally, an attempt was made to identify intracellular target molecules in live cells. Drosophila follicles showed a distinct staining pattern thus giving evidence that high concentrations of quercetin binding proteins are present in the nuclei and are associated with the ring canals. The presented biochemical and cytological data show that the interaction of the studied flavonoids with target proteins is specific and this finding opens up new experimental possibilities to systematically identify the cellular proteins with specific binding motifs for quercetin or other fluorogenic compounds of medical interest.     

Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.     

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.     

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

A lymphocyte cell surface molecule that is antigenically related to actin

When viable murine lymphocytes are incubated sequentially with a saturating amount of affinity-purified, rabbit anti-actin and highly conjugated FITC-goat anti-rabbit Ig, about 52% of mesenteric lymph node (MLN) lymphocytes and 36% of thymocytes exhibit a faint, but sharply punctate surface fluorescence. Cell surface actin (CSA) can be distinguished from staining of cytoplasmic actin in permeable cells, which are identified by their uptake of ethidium bromide. Staining of actin in ethidium bromide-permeable cells is 10-fold more intense than staining of actin on ethidium bromide-impermeable cells and is seen as uniformly fluorescent rings or crescents at the periphery of the cell and as dimmer, diffuse fluorescence centrally. Binding of rabbit anti-actin and goat anti-rabbit Ig to the lymphocyte cell surface is not mediated by Fc receptors; F(ab')2 fragments of these antibodies detect the same number of positive cells as do the intact molecules, and affinity-purified anti-KLH does not bind significantly. The cell surface stain, measured by flow cytometry or fluorescence microscopy, can be absorbed by pretreatment of the anti-actin with immobilized actin but not with IgG-Sepharose. Double-label experiments show that about 70% of the non-B cells and 30% of the MLN B cells bear detectable CSA. Although we have not ascertained the origin of CSA, we find that the number and brightness of cells exhibiting CSA cannot be increased by preincubating the cells with exogenous native skeletal muscle actin or with supernatant from dissociated MLN, indicating that there are no free binding sites for exogenous actin. The findings imply that either there is a developmentally expressed binding site(s) for actin, or that at various stages of development lymphocytes express a protein antigenically related to actin on their surface.     

Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide

Both ethidium bromide and propidium iodide stain growing yeast. As visualized in the fluorescence microscope, ethidium stains the nucleus and cytoplasm in wild type yeast and in those grown in 10% dextrose, with brightly fluorescent cytoplasmic granules being present in both. Under the latter conditions, the mitochondria are repressed but not absent. In rho 0 cells, in which the mitochondrial DNA is absent, ethidium appears to bind to the cell wall or membrane preferentially with no cytoplasmic granules being visible. In all cell types, propidium appears to bind the cell wall or membrane with no cytoplasmic granules being visible in any cell. The staining patterns thus suggest greater differences in the binding of these two types to mitochondrial DNA in situ than is suggested by their in vitro behavior. These differences in binding could explain their different mutagenic capacities..     

The structure-activity relationship of the series of non-peptide small antagonists for p56lck SH2 domain

The antagonists for the SH2 domain are regarded as novel therapeutic candidates for cancer, autoimmune disease, and chronic inflammatory disease. Previously, we identified rosmarinic acid (alpha-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RosA) from Prunella vulgaris as an antagonist for the p56lck SH2 domain by screening natural products. RosA not containing phosphotyrosine surrogate had a considerable inhibitory activity for T-cell antigen receptor (TCR)-induced interleukin (IL)-2 expression, and subsequent T-cell proliferation in vitro cell assay. To investigate the structure-activity relationship of RosA and to identify a novel p56lck SH2 antagonist with more potent in vitro T-cell inhibitory activity, we synthesized several analogs of RosA by using rational design. All synthesized compounds were tested in vitro binding activity for the SH2 domain and in vitro T-cell inhibitory activity. All four hydroxyl groups of RosA were essential for binding with the p56lck SH2 domain and T-cell inhibitory activity. Unexpectedly, conformationally less constrained analogs 4 and 9 showed a more potent binding affinity for the SH2 domain than that of RosA, and chirality of the analog did not play an important role in protein binding. We successfully identified several RosA analogs with a more potent T-cell inhibitory activity than that of RosA. Overall results revealed important structural requirements of the p56lck SH2 antagonists for in vitro T-cell inhibitory activity and in vitro protein binding activity.