RESUMO
OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.
Assuntos
Luciferases de Vaga-Lume/antagonistas & inibidores , Cloreto de Sódio/farmacologia , Animais , Escherichia coli , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Luciferases de Vaga-Lume/genética , Medições Luminescentes , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidoresRESUMO
The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1-3 enzymes confer oxygen sensitivity to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of the PHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic inflammation. Several pharmacologic PHD inhibitors (PHIs) are available, but our understanding of their selectivity and its chemical basis is limited. We here report that the PHI JNJ-42041935 (JNJ-1935) is structurally similar to the firefly luciferase substrate D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592 (roxadustat) and FG-2216 (ICA, BIQ, IOX3, YM 311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36⯵M. D-luciferin did not inhibit the PHDs, despite its structural similarity to JNJ-1935. This study provides insights into a previously unknown JNJ-1935 off-target effect as well as into the chemical requirements for firefly luciferase and PHD inhibitors and may inform the development of novel compounds targeting these enzymes.
Assuntos
Luciferases de Vaga-Lume/metabolismo , Inibidores de Prolil-Hidrolase/química , Animais , Benzotiazóis/metabolismo , Ligação Competitiva , Vaga-Lumes/enzimologia , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Isoquinolinas/química , Isoquinolinas/metabolismo , Cinética , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Inibidores de Prolil-Hidrolase/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Renilla/enzimologiaRESUMO
Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40⯱â¯0.15⯵M). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.
Assuntos
Benzoxazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Vaga-Lumes/enzimologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Animais , Benzoxazóis/síntese química , Benzoxazóis/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Luciferases de Vaga-Lume/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Genes Reporter/efeitos dos fármacos , Luciferases de Vaga-Lume/antagonistas & inibidores , MicroRNAs/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Febre , Inativação Gênica , Glioblastoma/patologia , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , MicroRNAs/genética , Imagem Óptica/métodos , Animais , Feminino , Glioblastoma/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Chalconas/farmacologia , Inibidores Enzimáticos/farmacologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Luminescência , Animais , Linhagem Celular Tumoral , Chalconas/química , Inibidores Enzimáticos/química , Feminino , Vaga-Lumes , Luciferases de Vaga-Lume/isolamento & purificação , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Estrutura MolecularRESUMO
The weakness of firefly luciferase is its rapid inactivation. Many studies have been done to develop thermostable luciferases. One of these modifications was LRR mutant in which the Leu300 was substituted with Arg in the E(354)RR(356)Lampyris turkestanicus luciferase as template. LRR was more thermostable than the wild type but with only 0.02% activity. In this study, site-directed mutagenesis was used to change the proposed ionic bond between the Arg and two neighboring residues (Glu270 and Glu271), to understand if the induced interactions were responsible for inactivation in LRR. Our results showed that substitution of Glu270 and 271 with Ala removed the interactions but the activity of enzyme did not return. The E270A mutant was more active than LRR but the E271A and E270A/E271A mutants were inactive. Fluorescence and CD measurements showed that these mutations were accompanied by conformational changes. Extrinsic fluorescence measurement and obtained quenching data by KI and acrylamide also confirmed that the mutants were less compact than the LRR enzyme. In conclusion, in LRR, the interactions between Arg300 and Glu270 and Glu271 were not responsible for the enzyme inactivation and it is proposed that the enzyme inactivation is due to conformational changes of LRR mutant of firefly luciferase.
Assuntos
Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Substituição de Aminoácidos , Animais , Estabilidade Enzimática , Vaga-Lumes/enzimologia , Vaga-Lumes/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/genética , Cinética , Luciferases de Vaga-Lume/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
The luciferase reporter gene assay system is broadly applied in various biomedical aspects, including signaling pathway dissection, transcriptional activity analysis, and genetic toxicity testing. It significantly improves the experimental accuracy and reduces the experimental error by the addition of an internal control. In the current research, we discovered some specific ions that could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase in vitro in the dual-reporter gene assay. We showed that these ionic compounds had a high potential of being utilized as quench-and-activate reagents in the dual-reporter assay. Furthermore, results from kinetic studies on ion-mediated quenching effects indicated that different ions have distinct inhibition modes. Our study is anticipated to guide a more affordable design of quench-and-activate reagents in biomedicine and pharmaceutical analysis.
Assuntos
Vaga-Lumes/enzimologia , Íons/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/metabolismo , Substâncias Luminescentes/metabolismo , Renilla/enzimologia , Animais , Ensaios Enzimáticos , Vaga-Lumes/genética , Genes Reporter , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/antagonistas & inibidores , Luciferases de Renilla/genética , Luminescência , Renilla/genéticaRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Medições Luminescentes/métodos , Mitocôndrias/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Inibidores Enzimáticos/farmacologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Mitocôndrias/fisiologia , Ácido Oxaloacético/farmacologia , Paraquat/farmacologia , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals.
Assuntos
Genes Reporter/efeitos dos fármacos , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Renilla/antagonistas & inibidores , Alcaloides de Pirrolizidina/farmacologia , Animais , Vaga-Lumes , Genes Reporter/genética , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Alcaloides de Pirrolizidina/química , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RenillaRESUMO
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.
Assuntos
Animais , Anestésicos Gerais/farmacologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Anisotropia , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Cristalografia por Raios X , Álcoois Graxos/farmacologia , Halotano/farmacologia , Técnicas In Vitro , Luciferases de Vaga-Lume/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
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.
Assuntos
Proteínas de Transporte/química , Vaga-Lumes/enzimologia , Isoxazóis/química , Isoxazóis/farmacologia , Luminescência , Animais , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/farmacocinética , Cinética , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de Tempo , Distribuição TecidualRESUMO
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.
Assuntos
Luciferases de Vaga-Lume/metabolismo , Nanopartículas Metálicas/química , Prata/metabolismo , Ouro/análise , Íons , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/química , Nanopartículas Metálicas/ultraestrutura , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Prata/análise , Espectrofotometria Ultravioleta , Eletricidade EstáticaRESUMO
Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and ß-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 µM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for ß-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Enzimas/genética , Genes Reporter/efeitos dos fármacos , Medições Luminescentes/métodos , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade Enzimática , Enzimas/metabolismo , Humanos , Luciferases/antagonistas & inibidores , Luciferases/genética , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/antagonistas & inibidores , Luciferases de Renilla/genética , Luminescência , Mutação , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Inibidores de beta-Lactamases , beta-Lactamases/genéticaRESUMO
Small interfering RNAs (siRNAs) can trigger potent gene silencing through the RNA interference (RNAi) pathway. The RNA-induced silencing complex (RISC) is key to this targeted mRNA degradation, and the human Argonaute2 (hAGO2) endonuclease component of RISC is responsible for the actual mRNA cleavage event. During RNAi, hAGO2 becomes loaded with the siRNA guide strand, making several key nucleic acid-enzyme interactions. Chemically modified siRNAs are now widely used in place of natural double-stranded RNAs, and understanding the effects chemical modifications have on guide strand-hAGO2 interactions has become particularly important. Here, interactions between the 5' nucleotide binding domain of hAGO2, MID, and chemically modified nucleotide analogues are investigated. Measured dissociation constants reveal that hAGO2 does not discriminate between nucleotide analogues during binding, regardless of the preferred sugar conformation of the nucleotide analogues. These results correlate well with cell-based gene silencing results employing siRNAs with 5'-modified guide strands. Additionally, chemical modification with 2'-deoxy-2'-fluoroarabino nucleic acid (2'F-ANA) and 2'-deoxy-2'-fluororibonucleic acid (2'F-RNA) at the passenger strand cleavage site of siRNAs has been shown to prevent hAGO2-mediated strand cleavage, an observation that appears to have little impact on overall gene silencing potency.
Assuntos
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , RNA Interferente Pequeno/metabolismo , Sequência de Bases , Sítios de Ligação , Células HeLa , Humanos , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genéticaRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/instrumentação , Trifosfato de Adenosina/química , Benzotiazóis/química , Ensaios Enzimáticos/instrumentação , Hidrólise , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/química , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , SoluçõesRESUMO
2-Substituted pyrrolo[2,3-b]quinoxalines having free NH were prepared directly from 3-alkynyl-2-chloroquinoxalines in a single pot by using readily available and inexpensive methane sulfonamide (or p-toluene sulfonamide) as an ammonia surrogate. The reaction proceeded in the presence of Cu(OAc)(2) affording the desired product in moderate yield. The crystal structure analysis of a representative compound and its supramolecular interactions are presented. Some of the compounds synthesized exhibited inhibitory activities against luciferase that was supported by the predictive binding mode of these compounds with luciferase enzyme through molecular docking studies. The key observations disclosed here can alert users of luciferase reporter gene assays for possible false positive results due to the direct inhibition of luciferase.
Assuntos
Vaga-Lumes/enzimologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Quinoxalinas/química , Quinoxalinas/farmacologia , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Genes Reporter/efeitos dos fármacos , Modelos Moleculares , Pirróis/síntese química , Pirróis/química , Pirróis/farmacologia , Quinoxalinas/síntese químicaRESUMO
Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays, owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes, including FLuc-catalyzed formation of multisubstrate adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays, compounds acting as FLuc inhibitors yield paradoxical luminescence increases, thus data on compounds acquired from FLuc-dependent assays require careful analysis as described here.
Assuntos
Inibidores Enzimáticos/química , Genes Reporter , Luciferases de Vaga-Lume/antagonistas & inibidores , Ácido Benzoico/química , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Compostos Químicos , Ensaios Enzimáticos , Cinética , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Especificidade por Substrato , Análise Serial de TecidosRESUMO
A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based NF-κB luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus pyralis with an IC(50) value of 0.36 ± 0.05 µM. Kinetic analysis of 3a inhibition showed that it is predominantly competitive with respect to D-luciferin and uncompetitive with respect to ATP. Therefore, several pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relationship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC(50) value of 0.06 ± 0.01 µM. In addition, molecular docking studies suggested that both 3a and 4c could be accommodated in the D-luciferin binding pocket, which is expected for a predominantly competitive inhibitor with respect to D-luciferin.