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.

Imaging of conditional gene silencing in vivo using a bioluminescence-based method with thermo-inducible microRNAs.

RNA interference (RNAi)-based gene therapy has great potential in cancer and infectious disease treatment to correct abnormal up-regulation of gene expression. We show a new original method uses synthetic microRNAs combined with a thermo-inducible promoter to reduce specific gene expression. The targeted gene is the luciferase firefly reporter gene overexpressed in a subcutaneous tumor which allows the RNAi monitoring by bioluminescence imaging (BLI). The inducible inhibition was first demonstrated in vitro using genetically modified cells lines and then in vivo using the corresponding xenograft model in mice. Achieving spatio-temporal control, we demonstrate the feasibility to induce, in vivo, a specific gene inhibition on demand. Future applications of this RNAi-based gene therapy, which can be restricted to pathological tissue, would offer wide-ranging potential for disease treatment.

Inhibiting Firefly Bioluminescence by Chalcones.

Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibition by HTS compounds resulting in the occurrence of "false positives." In the current research, we discovered a series of chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demonstrated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound 3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We believed that our research would contribute to improving scientists' awareness of the Fluc inhibitors, pay attention to the bias results, and even expand the utilization of bioluminescence in life science research.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.(1-3) Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.(4,5) In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

Juan César García: social medicine as project and endeavor / Juan César García: a medicina social como projeto e realização

This paper analyses some aspects of the trajectory of the Argentinian physician and sociologist Juan César García (1932-1984) in the field of Latin American Social Medicine. Three dimensions constituting his basic orientations are highlighted: the elaboration of systematic and reflective social thought; a critical attitude in questioning teaching and professional practices; a commitment to the institutionalization and dissemination of health knowledge.

O artigo analisa aspectos da trajetória de Juan César García (1932-1984), médico e sociólogo argentino, no campo da medicina social latino-americana. Destaca três dimensões que constituem as suas orientações básicas no campo da saúde: a elaboração de um pensamento sobre o social, sistemático e reflexivo; uma atitude crítica na problematização do ensino e das práticas profissionais; um compromisso com a institucionalização e divulgação do saber sanitário.

Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 µM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

Interaction of firefly luciferase and silver nanoparticles and its impact on enzyme activity.

We report on the dose-dependent inhibition of firefly luciferase activity induced by exposure of the enzyme to 20 nm citrate-coated silver nanoparticles (AgNPs). The inhibition mechanism was examined by characterizing the physicochemical properties and biophysical interactions of the enzyme and the AgNPs. Consistently, binding of the enzyme induced an increase in zeta potential from -22 to 6 mV for the AgNPs, triggered a red-shift of 44 nm in the absorbance peak of the AgNPs, and rendered a 'protein corona' of 20 nm in thickness on the nanoparticle surfaces. However, the secondary structures of the enzyme were only marginally affected upon formation of the protein corona, as verified by circular dichroism spectroscopy measurement and multiscale discrete molecular dynamics simulations. Rather, inductively coupled plasma mass spectrometry measurement revealed a significant ion release from the AgNPs. The released silver ions could readily react with the cysteine residues and N-groups of the enzyme to alter the physicochemical environment of their neighboring catalytic site and subsequently impair the enzymatic activity.

Comparison of compound administration methods in biochemical assays: effects on apparent compound potency using either assay-ready compound plates or pin tool-delivered compounds.

Compound sample preparation and delivery are the most critical steps in high-throughput screening (HTS) campaigns. Historically, several methods of compound delivery to assays have been used for HTS, including intermediate plates with prediluted compounds, assay-ready plates (ARPs) using either preplated dried compound films or nanoliter DMSO spots of compounds, as well as pin tool-delivered compounds. We and others have observed differences in apparent compound potency depending on the compound delivery method. To quantitatively measure compound potency differences due to the chosen delivery methods, we conducted a controlled study using a validated biochemical luciferase assay and compared potencies when compounds were delivered in either ARPs (using acoustic dispensed nanoliter spots) or by pin tool. Here we compare hit rates, confirmation rates, false-positive rates, and false-negative rates between the two delivery methods using the luciferase assay. We compared polystyrene (PS) and cyclic olefin copolymer (COC) plates using both delivery methods and examined whether ARPs stored at 4 °C were superior to those stored frozen at -20 °C. The data show that the choice of compound delivery method to the assay has an effect on the apparent IC(50)'s and that pin tool delivery results in more confirmed hits than preplated compounds, resulting in a lower false-negative rate. However, this effect is minimized through the use of COC plates and by obtaining plates in a "just-in-time" mode. Overall, this report provides guidance on using assay-ready compound plates and has affected the way HTS campaigns are using acoustically dispensed plates in our department.

The nuclear factor κB inhibitor (E)-2-fluoro-4'-methoxystilbene inhibits firefly luciferase.

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4'-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4'-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4'-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4'-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3)H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule) whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

Luciferase inhibition by a novel naphthoquinone.

The novel naphthoquinone 12,13-dihydro-N-methyl-6,11,13-trioxo-5H-benzo[4,5]cyclohepta[1,2-b]naphthalen-5,12-imine (hereafter called TU100) was created as a potential chemotherapeutic agent. Previous work showed it is an irreversible inhibitor of type I and II topoisomerases that alkylates specific enzyme thiols. While analyzing the effect of TU100 on cancer cells, we discovered it is a potent inhibitor of luciferase derived from both Photinus pyralis (fireflies) and Renilla reniformis (sea pansy). Pre-incubation experiments showed that TU100 does not irreversibly inactivate luciferase, indicating its mechanism is different from that observed with topoisomerases. Firefly luciferase generates light using ATP and luciferin as substrates (bioluminescence). An examination of TU100 inhibition at varying substrate concentrations revealed the drug is uncompetitive with respect to ATP and competitive with respect to luciferin. The TU100 binding constant (K(I)) is 2.5±0.7 µM as determined by Dixon plot analysis. These data suggest TU100 specifically binds the luciferase-ATP complex and prevents its interaction with luciferin. Given the novel structure of TU100, unique mechanism of action, and ability to target luciferase from different species, these results identify TU100 as an important new reagent for investigating and regulating bioluminescent enzymes.

Firefly luciferase inhibition.

Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124.

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution.

We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quantitative high-throughput screening (qHTS) was used to determine IC(50)s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC(50) < 10 microM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.

A potentially general method for the in vivo selection of inhibitory peptides targeted at a specific protein using yeast.

Although invaluable for biology and medicine, general methods for the selection of inhibitors directed against any protein activity are still missing. To test whether the fitness-based interferential genetics (FIG) approach performed in yeast might contribute to changing this situation, we used this method for the selection of artificial-gene-encoded peptides targeted at firefly luciferase, a foreign protein which was expressed in yeast. Some of these peptides were shown to inhibit the light-producing activity of luciferase in vitro. These results obtained within a totally artificial setting provide a direct demonstration of FIG selection for antagonistic components. Moreover, they open the way for FIG as a simple and general approach for selecting peptides against any specific protein activity expressed in a cellular environment, thus yielding compounds of potential scientific, medical and therapeutic value. Conditions for the development of such valuable compounds in the future using FIG are discussed.

Resveratrol inhibits firefly luciferase.

The potential therapeutic value of resveratrol in age-related disease settings including cancer, diabetes, and Alzheimer's has emerged from a rapidly growing body of experimental evidence. Protection from oxidative stress appears to be a common feature of resveratrol that may be mediated through SirT1, though more specific molecular mechanisms by which resveratrol mediates its effects remain unclear. This has prompted an upsurge in cell-based mechanistic studies, often incorporating reporter assays for pathway elucidation in response to resveratrol treatment. Here, we report that resveratrol potently inhibits firefly luciferase with a K(i) value of 2microM, and caution that this confounding element may lead to compromised data interpretation.

Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase.

In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5'-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.

Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides.

Firefly luciferase (EC 1.13.12.5) (FL) is the key enzyme in the firefly bioluminescence method (FB), which is widely used to determine the viability of living cells. The FB method can also be applied to monitoring the influence of different pollutants, such as pesticides. Firefly luciferase is a hydrophobic enzyme and its activity depends on the type of solvent, pH and substances present in the reaction mixture. The influence of three aromatic pesticides, including fenoxaprop-p-ethyl (I), diclofop-methyl (II) and metsulfuron methyl (III), on the enzyme activity was indirectly evaluated through the measurement of emitted light in the bioluminescence reaction, expressed in relative luminescence units (RLU). The reaction mixture used in the bioluminescence measurements consisted of: Tris buffer (pH 7.75), adenosine triphosphate (ATP) and ATP monitoring reagent, where FL is present. Ethanol-water solutions of each pesticide were then added at concentrations of 2.4 x 10(-4)-2.4 x 10(-8) mol/L. The FL activity inhibition factors (FL In%) were determined. The FL activity was maximally inhibited in the presence of all pesticides under study at a concentration of 2.4 x 10(-4) mol/L and was lowered by about 15-26% for pesticide I at concentrations of 2.4 x 10(-5)-2.4 x 10(-8) mol/L, whereas pesticides II and III, applied in the same concentration range, showed smaller FL inhibition values (5.3-20%). The pesticide degradation products (obtained after a 1 month period), measured in the same experimental conditions, in most cases exhibited a much less inhibitory effect on the enzyme activity than the corresponding initial pesticide.