Treating Cells as Reagents to Design Reproducible Assays.

The reproducibility of high-throughput cell-based assays is dependent on having a consistent source of cells for each experiment. Developing an understanding of the nature of cells growing in vitro and factors that influence their responsiveness to test compounds will contribute to the development of reproducible cell-based assays. Using good cell culture practices and establishing standard operating procedures (SOPs) for handling cultures can eliminate several potential contributors to variability in the responsiveness and performance of cells. The SOPs for handling each cell type must have clear and detailed instructions that can be understood and followed among different laboratories. The SOPs should include documenting the source of cells and authenticating their identity, both of which have become required to achieve peer acceptance of experimental data. Variability caused by biological issues such as phenotypic drift can be reduced by using standardized subculture procedures or using cryopreserved cells to set up experiments. Variability caused by inconsistent dispensing of cells per well and edge effects can be identified by measuring how many cells are present and whether they are alive or dead. Multiplex methods for real-time measurement of viable or dead cell number in each sample can be used for normalizing data and determining if proliferation or cytotoxicity has occurred during the experiment. Following good cell culture practices will go a long way toward executing reproducible cell-based assays. Resources will be included describing good cell culture practices, cell line authentication, and multiplex determination of cell number as an internal control.

Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor.

Monitoring the expression of cell-surface receptors, their interaction with extracellular ligands, and their fate upon ligand binding is important for understanding receptor function and developing new therapies. We describe a cell-based method that utilizes bioluminescent protein complementation technology to interrogate binding of a cellular receptor with its extracellular protein ligand, specifically LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Purified, full-length tagged PCSK9 is added to assay wells containing cells that stably express LDLR with an extracellular complementary tag. When the tagged PCSK9 binds the receptor, a bright luminescence signal is generated. The interaction is detected at the cell membrane with add-and-read simplicity, no wash steps, and flexibility, allowing data to be collected in endpoint format, kinetically, or with bioluminescent imaging. The assay is flexible, is rapid, and reports accurate biology. It is amenable to 96-well and 384-well formats, and the robustness allows for screening of new drug candidates (Z' = 0.83). The assay reports correct potencies for antibody titrations across a 50%-150% potency range and detects potency changes due to heat stress, suggesting that it may be useful during drug development. This assay technology can be broadly applied when studying other receptors with their extracellular ligands, whether protein or small-molecule binding partners.

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors.

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation.

Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

5'-AMP-activated protein kinase (AMPK) supports the growth of aggressive experimental human breast cancer tumors.

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5'-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [(13)C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.

Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

A bioluminescence assay for aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75.

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 novel steroidal inhibitor of estrogen-related receptor alpha (ERR alpha).

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalpha's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

Hsp90 inhibitors block outgrowth of EBV-infected malignant cells in vitro and in vivo through an EBNA1-dependent mechanism.

EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.

Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects the functions of EBNA1. By using electron-transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wild-type EBNA1. This phosphorylation-deficient mutant, however, had a significantly reduced ability to activate transcription and to maintain EBV's plasmids in cells.

Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1)) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

Large-scale Epstein-Barr virus EBNA1 protein purification.

The protein-DNA and protein-protein interactions of Epstein-Barr virus nuclear antigen 1 (EBNA1) are known to play an important role in the many functions of this viral protein. Large quantities of pure EBNA1 protein would be useful in biochemical assays to elucidate such interactions. In particular, the crystal structure of the full-length protein would be important to show possible regions of interaction and/or post-translational modification. Recently, we described a novel approach to overexpress and purify EBNA1 from Escherichia coli; however, it is not ideal for large-scale production of EBNA1. We were able to optimize this protocol by (1) adding a polyethyleneimine precipitation step prior to Ni-NTA chromatography to reduce complexity of the sample and remove nucleic acid, (2) optimizing the Ni-NTA gradient to further separate EBNA1 from impurities, and (3) concluding with a MonoS cation-exchange chromatography step to further purify and concentrate EBNA1. We were able to recover 10-mg quantities of pure EBNA1 protein.

Overproduction in Escherichia coli and purification of Epstein-Barr virus EBNA-1.

Epstein-Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein-Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni-NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).

An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody.

Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.