RESUMEN
The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.
Asunto(s)
Antimaláricos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Hígado/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/química , Evaluación Preclínica de Medicamentos/métodos , Células Hep G2 , Humanos , Hígado/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/fisiología , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Tiadiazinas/química , Tiadiazinas/farmacologíaRESUMEN
BACKGROUND: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. PRINCIPAL FINDINGS: High-throughput screening of a â¼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. CONCLUSIONS: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.
Asunto(s)
Histonas/metabolismo , Hidroxiquinolinas/farmacología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Biocatálisis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Hidroxiquinolinas/química , Histona Demetilasas con Dominio de Jumonji/genética , Espectrometría de Masas , Metilación/efectos de los fármacos , Estructura MolecularRESUMEN
The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.
Asunto(s)
Artefactos , Inhibidores de Cisteína Proteinasa/análisis , Detergentes/farmacología , Evaluación Preclínica de Medicamentos/métodos , Fluorescencia , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Bacterianas/antagonistas & inhibidores , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas , beta-LactamasasRESUMEN
A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure-activity relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4, and Dyrk1A.