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
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
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
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
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
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
Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42â¯577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.
RESUMO
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
Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator whose structure is more constrained than the ordinary N-chloroacetyl-α-amino acid initiators. The use of such an initiator has led to a macrocycle library with significantly suppressed population of lariat-shaped species compared with the conventional libraries. We previously used a conventional library and identified a small lariat thioether-macrocycle with a tail peptide with a C-terminal free Cys whose sidechain plays an essential role in potent inhibitory activity against a parasitic model enzyme, phosphoglycerate mutase. On the other hand, the cyclopropane-containing macrocycle library has yielded a larger thioether-macrocycle lacking a free Cys residue, which exhibits potent inhibitory activity to the same enzyme with a different mode of action. This result indicates that such a cyclopropane-containing macrocycle library would allow us to access mechanistically distinct macrocycles.
Assuntos
Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Macrocíclicos/farmacologia , Biblioteca de Peptídeos , Peptídeos/farmacologia , Fosfoglicerato Mutase/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Animais , Caenorhabditis elegans/enzimologia , Ciclopropanos/química , Inibidores Enzimáticos/química , Compostos Macrocíclicos/química , Estrutura Molecular , Peptídeos/química , Fosfoglicerato Mutase/metabolismo , Compostos de Sulfidrila/químicaRESUMO
Immunosuppressants used to treat autoimmunity are often not curative and have many side effects. Our purpose was to identify therapeutics for autoimmunity of the skeletal muscle termed idiopathic inflammatory myopathies (myositis). Recent evidence shows that the pro-inflammatory type I interferons (IFN) and a downstream product major histocompatibility complex (MHC) class I are pathogenic in myositis. We conducted quantitative high-throughput screening on >4500 compounds, including all approved drugs, through a series of cell-based assays to identify those that inhibit the type I IFN-MHC class I pathway in muscle precursor cells (myoblasts). The primary screen utilized CRISPR/Cas9 genome-engineered human myoblasts containing a pro-luminescent reporter HiBit fused to the C-terminus of endogenous MHC class I. Active compounds were counter-screened for cytotoxicity and validated by MHC class I immunofluorescence, Western blot, and RT-qPCR. Actives included Janus kinase inhibitors, with the most potent being ruxolitinib, and epigenetic/transcriptional modulators like histone deacetylase inhibitors and the hypoxia-inducible factor 1 inhibitor echinomycin. Testing in animal models and clinical trials is necessary to translate these therapies to myositis patients. These robust assay technologies can be further utilized to interrogate the basic mechanisms of the type I IFN-MHC class I pathway, identify novel molecular probes, and elucidate possible environmental triggers that may lead to myositis.
Assuntos
Antígenos HLA-B/metabolismo , Fatores Imunológicos/farmacologia , Interferon Tipo I/metabolismo , Mioblastos/efeitos dos fármacos , Linhagem Celular , Antígenos HLA-B/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Interferon Tipo I/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is expressed at high levels in a wide variety of tumor types. This has led to the development of biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more flexible routes of administration. As the first step toward this goal, we report the development of an automated quantitative high-throughput screening (qHTS) assay platform capable of screening large diverse drug-like chemical libraries to discover novel small molecules that inhibit CD47-SIRPα interaction. Using time-resolved Förster resonance energy transfer (TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen), we developed biochemical assays, optimized their performance, and individually tested them in small-molecule library screening. Based on performance and low false positive rate, the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-SIRPα interaction; these compounds will be further characterized and later disclosed. Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other protein-protein interaction modulators.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Imunológicos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antígenos de Diferenciação/química , Biotina/química , Biotina/metabolismo , Antígeno CD47/química , Antígeno CD47/imunologia , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Imunológicos/química , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.
Assuntos
Gânglios Espinais/metabolismo , Prurido/tratamento farmacológico , Receptores do Fator Natriurético Atrial/antagonistas & inibidores , Animais , Comportamento Animal , Sistema Livre de Células , Dermatite de Contato/tratamento farmacológico , Modelos Animais de Doenças , Gânglios Espinais/patologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Prurido/patologia , Receptores do Fator Natriurético Atrial/agonistas , Receptores do Fator Natriurético Atrial/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gαs gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC®1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.
Assuntos
Adenilil Ciclases , Clonagem Molecular/métodos , Schizosaccharomyces/genética , Adenilil Ciclases/biossíntese , Adenilil Ciclases/genética , Animais , Ensaios de Triagem em Larga Escala/métodos , HumanosRESUMO
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3' UTR of a candidate gene, which has been used successfully in our studies of the Pmp22 gene associated with Charcot-Marie-Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Edição de Genes/métodos , Genes Reporter/genética , Ensaios de Triagem em Larga Escala/métodos , Proteínas da Mielina/genética , Bioensaio/instrumentação , Bioensaio/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular , Edição de Genes/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Transfecção/instrumentação , Transfecção/métodosRESUMO
Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.
Assuntos
Bactérias/enzimologia , Inibidores Enzimáticos/farmacologia , Compostos Macrocíclicos/farmacologia , Peptídeos/farmacologia , Fosfoglicerato Mutase/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Biocatálise/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Coenzimas/metabolismo , Cristalografia por Raios X , Cisteína/metabolismo , Compostos Macrocíclicos/química , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Fosfoglicerato Mutase/química , Fosfoglicerato Mutase/metabolismo , Filogenia , Conformação Proteica , Relação Estrutura-Atividade , Compostos de Sulfidrila/metabolismoRESUMO
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
Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
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.