2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Comprehensive assessment of NR ligand polypharmacology by a multiplex reporter NR assay.

Nuclear receptors (NR) are ligand-modulated transcription factors that regulate multiple cell functions and thus represent excellent drug targets. However, due to a considerable NR structural homology, NR ligands often interact with multiple receptors. Here, we describe a multiplex reporter assay (the FACTORIAL NR) that enables parallel assessment of NR ligand activity across all 48 human NRs. The assay comprises one-hybrid GAL4-NR reporter modules transiently transfected into test cells. To evaluate the reporter activity, we assessed their RNA transcripts. We used a homogeneous RNA detection approach that afforded equal detection efficacy and permitted the multiplex detection in a single-well format. For validation, we examined a panel of selective NR ligands and polypharmacological agonists and antagonists of the progestin, estrogen, PPAR, ERR, and ROR receptors. The assay produced highly reproducible NR activity profiles (r > 0.96) permitting quantitative assessment of individual NR responses. The inferred EC50 values agreed with the published data. The assay showed excellent quality ( = 0.73) and low variability ( = 7.2%). Furthermore, the assay permitted distinguishing direct and non-direct NR responses to ligands. Therefore, the FACTORIAL NR enables comprehensive evaluation of NR ligand polypharmacology.

Anti-HIV Compounds from the Deep-Sea-Derived Fungus Chaetomium globosum.

Chemical investigation on the deep-sea-derived fungus Chaetomium globosum led to the isolation of nine compounds. By extensive analyses of the 1D and 2D NMR as well as HR-ESI-MS spectra, their structures were elucidated as xylariol A (1), 1,3-dihydro-4,5,6-trihydroxy-7-methylisobenzofuran (2), epicoccone B (3), epicoccolide B (4), chaetoglobosin G (5), chaetoglobosin Fex (6), cochliodone A (7), cochliodone B (8), and chaetoviridin A (9), assorting as four phenolics (1-4), two cytochalosans (5-6), and three azaplilones (7-9). Compounds 1-3 were firstly reported from C. globosum. Under the concentrations of 20 µg/mL, 1, 2, and 3 exhibited potent in vitro anti-HIV activity with the inhibition rates of 70 %, 75 %, and 88 %, respectively.

Monitoring arsenic using genetically encoded biosensors in vitro: The role of evolved regulatory genes.

Toxic pollutant (TP) detection in situ using analytical instruments or whole-cell biosensors is inconvenient. Designing and developing genetically coded biosensors in vitro for real-world TP detection is a promising alternative. However, because the bioactivity and stability of some key biomolecules are weakened in vitro, the response and regulation of reporter protein become difficult. Here, we established a genetically encoded biosensor in vitro with an arsenical resistance operon repressor (ArsR) and GFP reporter gene. Given that the wildtype ArsR did not respond to arsenic and activate GFP expression in vitro, we found, after screening, an evolved ArsR mutant ep3 could respond to arsenic and exhibited an approximately 3.4-fold fluorescence increase. Arsenic induced expression of both wildtype ArsR and ep3 mutant in vitro, however, only ep3 mutant regulated the expression of reporter gene. Furthermore, the effects of cell extracts, temperature, pH, incubation, and equilibrium time were investigated, and the equilibration of reaction mixtures for 30 min at 37 °C was found to be essential for in vitro arsenic detection prior to treatment with arsenic. Based on our data, we established a standard procedure for arsenic detection in vitro. Our results will facilitate the practical application of genetically encoded biosensors in TP monitoring.

Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain.

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays.

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.

Programmable CRISPR-Cas transcriptional activation in bacteria.

Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by more than 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Quantitative Evaluation of the Transcriptional Activity of Steroid Hormone Receptor Mutants and Variants Using a Single Vector With Two Reporters and a Receptor Expression Cassette.

Although the rapid development of high-throughput sequencing has led to the identification of a large number of truncated or mutated steroid hormone receptor (SHR) variants, their clinical relevance remains to be defined. A platform for functional analysis of these SHR variants in cells would be instrumental for better assessing their impact on normal physiology and SHR-associated diseases. Here we have developed a new reporter system that allows rapid and accurate assessment of the transcriptional activity of SHR variants in cells. The reporter is a single construct containing a firefly luciferase reporter gene, whose expression is under the control of a promoter with multiple steroid hormone responsive elements, and a Renilla luciferase reporter gene, that is constitutively expressed under the control of an internal ribosome entry site (IRES) and is not regulated by steroid hormones. The corresponding SHR (wildtype or mutant/variant) is also expressed from the same construct. Using this improved reporter system, we revealed a large spectrum of transactivation activities within a set of previously identified mutations and variations of the androgen receptor (AR), the estrogen receptor α (ERα) and the glucocorticoid receptor (GR). This novel reporter system enables functional analysis of SHR mutants and variants in physiological and pathological settings, offering valuable preclinical, or diagnostic information for the understanding and treatment of associated diseases.

A reporter cell line for real-time imaging of autophagy and apoptosis.

Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.

Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators.

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides -110 to +30, which is responsible for the constitutive expression of Abcg2. The 5'-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.

Liganded T3 receptor ß2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the ß chain (TSHß). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHß and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRß2, into kidney-derived CV1 cells. We found that liganded TRß2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRß2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LßT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRß2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

Activation of human nuclear receptors by perfluoroalkylated substances (PFAS).

Perfluoralkylated substances (PFAS) such as perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS) are used to produce, e.g., surface coatings with water- and dirt-repellent properties. These substances have been shown to be hepatotoxic in rodents, and the mechanism of action is mostly attributed to the PFAS-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα). In the present study, we investigated by using luciferase-based reporter gene assays whether PFOA, PFOS and six alternative PFAS can activate, in addition to PPARα, eight other human nuclear receptors. All tested PFAS except for perfluorobutanesulfonic acid (PFBS) were able to activate human PPARα. Perfluoro-2-methyl-3-oxahexanoic acid (PMOH) and 3H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP) were weak agonists of human PPARγ. The other human nuclear receptors (PPARδ, CAR, PXR, FXR, LXRα, RXRα and RARα) were not affected by any PFAS tested in this study. Although PMOH was more effective than PFOA in stimulating PPARα in the transactivation assay, it was less effective in stimulating PPARα-dependent target gene expression in human HepG2 hepatocarcinoma cells. Notably, any effect observed in this in vitro study only occurred at concentrations higher than 10 µM of the respective PFAS which is in all cases several magnitudes above the average blood concentration in the Western population. Thus, the results suggest that nuclear receptor activation may only play a minor role in potential PFAS-mediated adverse effects in humans.

Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay.

Interferons (IFNs) are important glycoproteins which can stimulate or inhibit up to three hundred different genes encoding proteins involved in antiviral defense mechanisms, inflammation, adaptive immunity, angiogenesis and among other processes. Nevertheless, different genetic alterations may lead to interferon alpha (IFN-α) overproduction in human autoimmune diseases like systemic lupus erythematosus. As a consequence, IFN-α is a central molecule whose activity must be regulated to block their harmful effect on those disorders where the endogenous cytokine production constitutes the etiology of the illnesses. In this work, we evaluate the biological activity of eighty-eight compounds, from our own chemo-library, to find potential IFN-α inhibitors by using a reporter gene assay (RGA) WISH-Mx2/EGFP. We identified some compounds able to modulate negatively the IFN-α activity. The most active IFN-α inhibitors were further studied achieving promising results. In addition, some combinations of the most active compounds were analyzed accomplishing a stronger effect to decrease the IFN-α activity than each compound alone. Furthermore, the complete inhibition of the cytokine activity was reached with some combinations of compounds.

In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT).

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

Cocaine- and amphetamine-regulated transcript promoter regulated by nicotine in nerve growth factor-treated PC12 cells.

Nicotine and cocaine- and amphetamine-regulated transcripts (CART) have several overlapping functions, such as the regulation of reward, feeding behavior, stress response, and anxiety. Previous studies showed that nicotine regulates CART expression in various brain regions. However, the molecular mechanisms underlying this regulation are not known. This study investigated the regulatory effect of nicotine on promoter activity of the CART gene in PC12 cells, which were differentiated into a neuronal phenotype by nerve growth factor (NGF) treatment. Two vectors containing reporter genes (Gaussia luciferase or mCherry) and the 1,140-bp upstream of the transcriptional start site of the mouse CART gene are used to analyze the CART promoter activity. Transient transfection of PC12 cells with either vector displayed strong promoter activity in both undifferentiated and differentiated PC12 cells. CART promoter activity in the PC12 cell line is increased by forskolin or NGF treatment. In differentiated PC12 cells, exposure to 50 nM nicotine for 6 h increased CART promoter activity. However, treatment with higher nicotine doses for 6 h and treatment with all nicotine doses for 24 h showed no effect. A nicotine concentration of 50 nM is comparable to brain nicotine levels experienced by chronic smokers over long periods of time. Taken together, these data indicate that nicotine may exert some of its actions through the regulation of CART transcription in the brain.

Using the Inducible Caspase-9 Suicide-Safeguard System with iPSC and Bioluminescent Tracking.

For scientists working within the field of induced pluripotent stem cells (iPSCs), this protocol will provide a thorough walk-through on how to conduct in vitro and in vivo experiments that validate the function of a particular safeguard system technology. In short, we provide instructions on how to generate inducible Caspase-9 (iC9) safeguard system with human iPSCs that act as normal or abnormal models of the cells for therapeutics to be tried after differentiation. These iC9-iPSCs should be modified prior to beginning this protocol by constitutively expressing luciferase, an enzyme capable of generating bioluminescent signals through the oxidation of the substrate luciferin. Monitoring the bioluminescent signal over time provides the information on whether a safeguard system is working or not.

Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening.

Most studies using high-throughput in vitro cell-based bioassays tested chemicals up to a certain fixed concentration. It would be more appropriate to test up to concentrations predicted to elicit baseline toxicity because this is the minimal toxicity of every chemical. Baseline toxicity is also called narcosis and refers to nonspecific intercalation of chemicals in biological membranes, leading to loss of membrane structure and impaired functioning of membrane-related processes such as mitochondrial respiration. In cells, baseline toxicity manifests as cytotoxicity, which was quantified by a robust live-cell imaging method. Inhibitory concentrations for baseline toxicity varied by orders of magnitude between chemicals and were described by a simple quantitative structure activity relationship (QSAR) with the liposome-water partition constant as a sole descriptor. The QSAR equations were remarkably similar for eight reporter gene cell lines of different cellular origin, six of which were used in Tox21. Mass-balance models indicated constant critical membrane concentrations for all cells and all chemicals with a mean of 69 mmol·kglip-1(95% CI: 49-89), which is in the same range as for bacteria and aquatic organisms and consistent with the theory of critical membrane burden of narcosis. The challenge of developing baseline QSARs for cell lines is that many confirmed baseline toxicants are rather volatile. We deduced from cytotoxicity experiments with semi-volatile chemicals that only chemicals with medium-air partition constants >10,000 L/L can be tested in standard robotic setups without appreciable loss of effect. Chemicals just below that cutoff showed crossover effects in neighboring wells, whereas the effects of chemicals with lower medium-air partition constants were plainly lost. Applying the "volatility cut-off" to >8000 chemicals tested in Tox21 indicated that approximately 20% of Tox21 chemicals could have partially been lost during the experiments. We recommend applying the baseline QSARs together with volatility cut-offs for experimental planning of reporter gene assays, that is, to dose only chemicals with medium-air partition constants >10,000 at concentrations up to the baseline toxicity level.

Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono-methylchrysenes.

Polycyclic aromatic hydrocarbons (PAHs) comprise a large family of toxic compounds that come from natural and anthropogenic sources. Chrysene is a PAH with multiple effects, but the toxic potentials of mono-methylchrysenes are less characterized. A comparison of chrysene and six mono-methylchrysenes was performed using assays for cytotoxicity, human aryl hydrocarbon receptor (AhR) reporter gene signaling, and AhR-regulated target gene and protein expression. Sulforhodamine B and trypan blue dye binding assays revealed these chrysenes to be similar in their cytotoxic effects on HepG2 cells. A yeast-based reporter assay detecting human AhR-mediated gene expression identified 4-methylchrysene as being six times more potent and 5-methylchrysene about one-third as potent as chrysene. Other methylchrysenes were more similar to chrysene in the ability to act as AhR ligands. The mono-methylchrysenes all strongly induced CYP1A1 mRNA and protein and moderately induced CYP1B1 expression in HepG2 cells. Levels of CYP1A2 mRNA were induced at higher concentrations of the chrysenes, but protein expression was not significantly altered. The PCR-based gene expression and immunoblotting analyses indicated induced expression differences across the chrysene members were similar to each other. Overall, the effects of methylated chrysenes were comparable to unsubstituted chrysene, suggesting members of this group may be considered approximately equivalent in their effects. © 2019 Wiley Periodicals, Inc.

The dual luciferase reporter system and RT-qPCR strategies for screening of MicroRNA-21 small-molecule inhibitors.

The therapeutic potential of microRNA-21 (miR-21) small-molecule inhibitors has been of particular interest to medicinal chemists. Moreover, the development of more facile screening methods is lacking. In the present study, two potential screening strategies for miR-21 small-molecule inhibitor including the stem-loop reverse transcription-quantitative PCR and dual luciferase reporter assay system were demonstrated and discussed in detail. A pmirGLO-miR21cswt plasmid and its two different mutants were constructed for dual luciferase reporter assay system. In addition, the sensitivity and specificity of these two methods were validated. Our results demonstrated that both strategies are decent choices for the screening of small-molecule inhibitors for miR-21 and possibly other miRNAs. Eventually, we applied our optimized strategy to discover and characterize several promising compounds such as azobenzene derivate A, enoxacin, and norfloxacin for their potential impact on intracellular miR-21 concentration.