RESUMO
The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.
Assuntos
Aprendizado de Máquina , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Inibidores de beta-Lactamases , beta-Lactamases , beta-Lactamases/metabolismo , beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ensaios de Triagem em Larga EscalaRESUMO
The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.
Assuntos
Proteínas Hedgehog , Humanos , Colesterol/metabolismo , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Ligantes , Proteínas Oncogênicas , EsteróisRESUMO
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure-activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase-phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.
Assuntos
Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fosfoglicerato Mutase/química , Fosfoglicerato Mutase/metabolismo , Animais , Domínio Catalítico , Humanos , Modelos Moleculares , Filogenia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.
Assuntos
Glicoconjugados/farmacologia , Canais de Translocação SEC/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Glicoconjugados/química , Humanos , Estrutura Molecular , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC/metabolismoRESUMO
High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635-637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.
Assuntos
Algoritmos , Rastreamento de Células/métodos , Aprendizado de Máquina , Neuritos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Células Cultivadas , Biologia Computacional/métodos , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Transgênicos , Neuritos/efeitos dos fármacos , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Cultura Primária de Células , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.
Assuntos
MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/fisiologia , Células Ganglionares da Retina/enzimologia , Células Ganglionares da Retina/patologia , Animais , Morte Celular/genética , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Glaucoma/tratamento farmacológico , Glaucoma/etiologia , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Masculino , Camundongos , Doenças do Nervo Óptico/etiologia , Doenças do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/enzimologia , Traumatismos do Nervo Óptico/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Ratos , Ratos Wistar , Células Ganglionares da Retina/efeitos dos fármacos , Transdução de Sinais , Regulação para CimaRESUMO
Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition, and abundance. NPEs can therefore be challenging to evaluate efficiently with high-throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of antimalarial NPEs using a pharmacologically driven, quantitative high-throughput-screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy, and apparent EC50s can be used to rank order NPEs for subsequent organism reculture, extraction, and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species: Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B, belonging to the unusually elaborated nonproteinogenic amino-acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact, 25-epi-actinoramide A, and actinoramides D, E, and F, which are closely related biosynthetic congeners of the previously reported metabolites.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Streptomyces/química , Antimaláricos/química , Produtos Biológicos/química , Costa Rica , Sedimentos Geológicos/química , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Papua Nova Guiné , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Streptomyces/genéticaRESUMO
Transcriptional activation of σ(54)-RNA polymerase holoenzyme (σ(54)-RNAP) in bacteria is dependent on a cis-acting DNA element (bacterial enhancer), which recruits the bacterial enhancer-binding protein to contact the holoenzyme via DNA looping. Using a constructive synthetic biology approach, we recapitulated such process of transcriptional activation by recruitment in a reconstituted cell-free system, assembled entirely from a defined number of purified components. We further engineered the bacterial enhancer-binding protein PspF to create an in vitro two-hybrid system (IVT2H), capable of carrying out gene regulation in response to expressed protein interactions. Compared with genetic systems and other in vitro methods, IVT2H not only allows detection of different types of protein interactions in just a few hours without involving cells but also provides a general correlation of the relative binding strength of the protein interaction with the IVT2H signal. Due to its reconstituted nature, IVT2H provides a biochemical assay platform with a clean and defined background. We demonstrated the proof-of-concept of using IVT2H as an alternative assay for high throughput screening of small-molecule inhibitors of protein-protein interaction.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Sistema Livre de Células , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Genes Reporter/genética , Ensaios de Triagem em Larga Escala , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/metabolismoRESUMO
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
Assuntos
Biopolímeros/metabolismo , Transformação Celular Neoplásica , Ativadores de Enzimas/farmacologia , Piruvato Quinase/metabolismo , Animais , Biopolímeros/química , Western Blotting , Proliferação de Células , Humanos , Camundongos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Piruvato Quinase/químicaRESUMO
PURPOSE: Retinal degenerations are a heterogeneous group of diseases in which there is slow but progressive loss of photoreceptors (PR). There are currently no approved therapies for treating retinal degenerations. In an effort to identify novel small molecules that are (1) neuroprotective and (2) promote PR differentiation, we have developed microscale (1,536 well) cell culture assays using primary retinal neurons. METHODS: Primary murine retinal cells are isolated, seeded, treated with a 1,280 compound chemical library in a 7 point titration and then cultured under conditions developed to assay protection against an introduced stress or enhance PR differentiation. In the protection assays a chemical insult is introduced and viability assessed after 72 h using CellTiterGlo, a single-step chemiluminescent reagent. In the differentiation assay, cells are isolated from the rhodopsin-GFP knock-in mouse and PR differentiation is assessed by fixing cells after 21 days in culture and imaging with the Acumen plate-based laser cytometer (TTP Labtech) to determine number and intensity of GFP-expressing cells. Positive wells are re-imaged at higher resolution with an INCell2000 automated microscope (GE). Concentration-response curves are generated to pharmacologically profile each compound and hits identified by xx. RESULTS: We have developed PR differentiation and neuroprotection assays with a signal to background (S/B) ratios of 11 and 3, and a coefficient of variation (CV) of 20 and 9 %, suitable for chemical screening. Staurosporine has been shown in our differentiation assay to simultaneously increase the number of rhodopsin positive objects while decreasing the mean rhodopsin intensity and punctate rhodopsin fluorescent objects. CONCLUSIONS: Using primary murine retinal cells, we developed high throughput assays to identify small molecules that influence PR development and survival. By screening multiple compound concentrations, dose-response curves can be generated, and the false negative rate minimized. It is hoped that this work will identify both potential preclinical candidates as well as molecular probes that will be useful for analysis of the molecular mechanisms that promote PR differentiation and survival.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Fármacos Neuroprotetores/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Degeneração Retiniana/tratamento farmacológico , Animais , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/citologia , Cultura Primária de Células , Degeneração Retiniana/patologia , Rodopsina/genéticaRESUMO
In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.
Assuntos
Antimaláricos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/crescimento & desenvolvimento , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/farmacologia , Humanos , Relação Estrutura-AtividadeRESUMO
Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.
Assuntos
Antineoplásicos , Antagonistas do Ácido Fólico , Neoplasias , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbono , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Metotrexato/farmacologia , Metotrexato/metabolismo , Metotrexato/uso terapêutico , Neoplasias/tratamento farmacológico , Quimera de Direcionamento de Proteólise , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
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.
Assuntos
Luciferases de Vaga-Lume/metabolismo , Modelos Moleculares , Oxidiazóis/metabolismo , Monofosfato de Adenosina/metabolismo , Linhagem Celular , Coenzima A/metabolismo , Cristalografia por Raios X , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Isomerismo , Luciferases de Vaga-Lume/antagonistas & inibidores , Luciferases de Vaga-Lume/química , Oxidiazóis/química , Oxidiazóis/farmacologia , Soluções , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
High throughput screening (HTS) is widely used in drug discovery and chemical biology to identify and characterize agents having pharmacologic properties often by evaluation of large chemical libraries. Standard HTS data can be simply plotted as an x-y graph usually represented as % activity of a compound tested at a single concentration vs compound ID, whereas quantitative HTS (qHTS) data incorporates a third axis represented by concentration. By virtue of the additional data points arising from the compound titration and the incorporation of logistic fit parameters that define the concentration-response curve, such as EC50 and Hill slope, qHTS data has been challenging to display on a single graph. Here we provide a flexible solution to the rapid plotting of complete qHTS data sets to produce a 3-axis plot we call qHTS Waterfall Plots. The software described here can be generally applied to any 3-axis dataset and is available as both an R package and an R shiny application.
RESUMO
Quantitative high-throughput screening (qHTS) pharmacologically evaluates chemical libraries for therapeutic uses, toxicological risk and, increasingly, for academic probe discovery. Phenotypic high-throughput screening assays interrogate molecular pathways, often relying on cell culture systems, historically less focused on multicellular organisms. Caenorhabditis elegans has served as a eukaryotic model organism for human biology by virtue of genetic conservation and experimental tractability. Here, a paradigm enabling C. elegans qHTS using 384-well microtiter plate laser-scanning cytometry is described, in which GFP-expressing organisms revealing phenotype-modifying structure-activity relationships guide subsequent life-stage and proteomic analyses, and Escherichia coli bacterial ghosts, a non-replicating nutrient source, allow compound exposures over two life cycles, mitigating bacterial overgrowth complications. We demonstrate the method with libraries of anti-infective agents, or substances of toxicological concern. Each was tested in seven-point titration to assess the feasibility of nematode-based in vivo qHTS, and examples of follow-up strategies were provided to study organism-based chemotype selectivity and subsequent network perturbations with a physiological impact. We anticipate that this qHTS approach will enable analysis of C. elegans orthologous phenotypes of human pathologies to facilitate drug library profiling for a range of therapeutic indications.
Assuntos
Caenorhabditis elegans , Ensaios de Triagem em Larga Escala , Animais , Humanos , Ensaios de Triagem em Larga Escala/métodos , Caenorhabditis elegans/genética , Proteômica , Descoberta de Drogas/métodos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.
Assuntos
Aurora Quinase A , Ensaios de Triagem em Larga Escala , Humanos , Células HeLa , Ciclo Celular , Glucose/metabolismoRESUMO
Paracatalytic inducers are antagonists that shift the specificity of biological catalysts, resulting in non-native transformations. In this Chapter we describe methods to discover paracatalytic inducers of Hedgehog (Hh) protein autoprocessing. Native autoprocessing uses cholesterol as a substrate nucleophile to assist in cleaving an internal peptide bond within a precursor form of Hh. This unusual reaction is brought about by HhC, an enzymatic domain that resides within the C-terminal region of Hh precursor proteins. Recently, we reported paracatalytic inducers as a novel class of Hh autoprocessing antagonists. These small molecules bind HhC and tilt the substrate specificity away from cholesterol in favor of solvent water. The resulting cholesterol-independent autoproteolysis of the Hh precursor generates a non-native Hh side product with substantially reduced biological signaling activity. Protocols are provided for in vitro FRET-based and in-cell bioluminescence assays to discover and characterize paracatalytic inducers of Drosophila and human hedgehog protein autoprocessing, respectively.
Assuntos
Proteínas de Drosophila , Proteínas Hedgehog , Animais , Humanos , Proteínas Hedgehog/genética , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Proteínas de Drosophila/química , Drosophila/metabolismo , Colesterol/metabolismo , CatáliseRESUMO
Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five- to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.
RESUMO
Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRß with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRß antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRß to disrupt SRC2 association.
Assuntos
Nitrobenzoatos/farmacologia , Coativador 2 de Receptor Nuclear/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Compostos de Anilina/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Metilaminas/farmacologia , Nitrobenzoatos/química , Piperidinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-AtividadeRESUMO
High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87-91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC(50) = 7 +/- 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results.