High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3' untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.     

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to ‘dial out’ the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.     

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.     

A simple reporter assay for screening claudin-4 modulators

Claudin-4, a member of a tetra-transmembrane protein family that comprises 27 members, is a key functional and structural component of the tight junction-seal in mucosal epithelium. Modulation of the claudin-4-barrier for drug absorption is now of research interest. Disruption of the claudin-4-seal occurs during inflammation. Therefore, claudin-4 modulators (repressors and inducers) are promising candidates for drug development. However, claudin-4 modulators have never been fully developed. Here, we attempted to design a screening system for claudin-4 modulators by using a reporter assay. We prepared a plasmid vector coding a claudin-4 promoter-driven luciferase gene and established stable reporter gene-expressing cells. We identified thiabendazole, carotene and curcumin as claudin-4 inducers, and potassium carbonate as a claudin-4 repressor by using the reporter cells. They also increased or decreased, respectively, the integrity of the tight junction-seal in Caco-2 cells. This simple reporter system will be a powerful tool for the development of claudin-4 modulators.     

L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.     

Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10² parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite’s α-tubulin promoter. Renilla luciferase activity was detected with at least 10² parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.     

Plasmodium falciparum: development of a transgenic line for screening antimalarials using firefly luciferase as the reporter

High-throughput screening (HTS) of small-molecule libraries against pharmacological targets is a key strategy of contemporary drug discovery. This study reports a simple, robust, and cell-based luminescent method for assaying antimalarial drugs. Using transfection technology, we generated a stable Plasmodium falciparum line with high levels of firefly luciferase expression. A luciferase assay based on this parasite line was optimized in a 96-well plate format and used to compare with the standard [³H] hypoxanthine radioisotope method. The 50% inhibitory concentrations (IC₅₀s) of chloroquine, artesunate, artemether, dihydroartemisinin and curcumin obtained by these two methods were not significantly different (P > 0.05, ANOVA). In addition, this assay could be performed conveniently with a luminescence plate reader using unsynchronized stages within as early as 12 h. Furthermore, the luciferase assay is robust with a Z′ score of 0.77-0.92, which suggests the feasibility for further miniaturization and automation.     

Development of a high-throughput cell-based reporter assay for screening of JAK3 inhibitors

JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rβ/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.     

A luciferase reporter assay to investigate the differential selenium-dependent stability of selenoprotein mRNAs

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.     

The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene ( luc ) as a combined marker/reporter gene. Protocorm-likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S-luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low-light video microscopy in combination with a real-time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.     

Functional characterization of human neuropeptide Y receptor subtype five specific antagonists using a luciferase reporter gene assay

Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with alphaMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G(s). In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the alphaMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a 'physiological' level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10(-9) M) that form a new class of active NPY5 receptor antagonists.     

A fluorescence-based rapid screening assay for cytotoxic compounds

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death.     

A reporter gene assay for screening of PDE4 subtype selective inhibitors

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.     

Differential assay for high-throughput screening of antibacterial compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.     

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas

The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5′ untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera. Received: 12 April 1999 / Accepted: 24 June 1999