RESUMEN
While progress has been made in the effort to eradicate malaria, the disease remains a significant threat to global health. Acquired resistance to frontline treatments is emerging in Africa, urging a need for the development of novel antimalarial agents. Repurposing human kinase inhibitors provides a potential expedited route given the availability of a diverse array of kinase-targeting drugs that are approved or in clinical trials. Phenotypic screening of a library of type II human kinase inhibitors identified compound 1 as a lead antimalarial, which was initially developed to target human ephrin type A receptor 2 (EphA2). Here, we report a structure-activity relationship study and lead optimization of compound 1, which led to compound 33, with improved antimalarial activity and selectivity.
Asunto(s)
Antimaláricos , Malaria , Receptor EphA2 , Humanos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Malaria/tratamiento farmacológico , Relación Estructura-Actividad , África , Plasmodium falciparumRESUMEN
Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.
Asunto(s)
Antimaláricos , Humanos , Antimaláricos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa Tipo Polo 1RESUMEN
Cyclic tetrapeptide histone deacetylase inhibitors represent a promising class of antiplasmodial agents that epigenetically disrupt a wide range of cellular processes in Plasmodium falciparum. Unfortunately, certain limitations, including reversible killing effects and host cell toxicity, prevented these inhibitors from further development and clinical use as antimalarials. In this study, we present a series of cyclic tetrapeptide analogues derived primarily from the fungus Wardomyces dimerus that inhibit P. falciparum with low nanomolar potency and high selectivity. This cyclic tetrapeptide scaffold was diversified further via semisynthesis, leading to the identification of several key structural changes that positively impacted the selectivity, potency, and in vitro killing profiles of these compounds. We confirmed their effectiveness as HDAC inhibitors through the inhibition of PfHDAC1 catalytic activity, in silico modeling, and the hyperacetylation of histone H4. Additional analysis revealed the in vitro inhibition of the most active epoxide-containing analogue was plasmodistatic, exhibiting reversible inhibitory effects upon compound withdrawal after 24 or 48 h. In contrast, one of the new diacetyloxy semisynthetic analogues, CTP-NPDG 19, displayed a rapid and irreversible action against the parasite following compound exposure for 24 h.
Asunto(s)
Antimaláricos , Plasmodium falciparum , Antimaláricos/farmacología , Ascomicetos , Inhibidores de Histona Desacetilasas/farmacologíaRESUMEN
BACKGROUND: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. METHODS: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. FINDINGS: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1ß as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1ß-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. INTERPRETATION: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. FUND: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research.
Asunto(s)
Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Sepsis/metabolismo , Sepsis/fisiopatología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Activación Enzimática , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Especies Reactivas de Oxígeno/metabolismo , Sepsis/etiologíaRESUMEN
Double-stranded DNA (dsDNA) constitutes a potent activator of innate immunity, given its ability to bind intracellular pattern recognition receptors during viral infections or sterile tissue damage. While effects of dsDNA in immune cells have been extensively studied, dsDNA signalling and its pathophysiological implications in non-immune cells, such as the vascular endothelium, remain poorly understood. The aim of this study was to characterize prothrombotic effects of dsDNA in vascular endothelial cells. Transfection of cultured human endothelial cells with the synthetic dsDNA poly(dA:dT) induced upregulation of the prothrombotic molecules tissue factor and PAI-1, resulting in accelerated blood clotting in vitro, which was partly dependent on RIG-I signalling. Prothrombotic effects were also observed upon transfection of endothelial cells with hepatitis B virus DNA-containing immunoprecipitates as well human genomic DNA. In addition, dsDNA led to surface expression of von Willebrand factor resulting in increased platelet-endothelium-interactions under flow. Eventually, intrascrotal injection of dsDNA resulted in accelerated thrombus formation upon light/dye-induced endothelial injury in mouse cremaster arterioles and venules in vivo. In conclusion, we show that viral or endogenous dsDNA induces a prothrombotic phenotype in the vascular endothelium. These findings represent a novel link between pathogen- and danger-associated patterns within innate immunity and thrombosis.
Asunto(s)
ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Tromboplastina/biosíntesis , Animales , Coagulación Sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones , Trombosis/inducido químicamente , Trombosis/patología , Factor de von Willebrand/biosíntesisRESUMEN
Intracellular arbuscular mycorrhiza symbiosis between plants and glomeromycotan fungi leads to formation of highly branched fungal arbuscules that release mineral nutrients to the plant host. Their development is regulated in plants by a mechanistically unresolved interplay between symbiosis, nutrient, and hormone (gibberellin) signaling. Using a positional cloning strategy and a retrotransposon insertion line, we identify two novel alleles of Lotus japonicus REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1) encoding a GRAS protein. We confirm that RAM1 is a central regulator of arbuscule development: arbuscule branching is arrested in L. japonicus ram1 mutants, and ectopic expression of RAM1 activates genes critical for arbuscule development in the absence of fungal symbionts. Epistasis analysis places RAM1 downstream of CCaMK, CYCLOPS, and DELLA because ectopic expression of RAM1 restores arbuscule formation in cyclops mutants and in the presence of suppressive gibberellin. The corresponding proteins form a complex that activates RAM1 expression via binding of CYCLOPS to a cis element in the RAM1 promoter. We thus reveal a transcriptional cascade in arbuscule development that employs the promoter of RAM1 as integrator of symbiotic (transmitted via CCaMK and CYCLOPS) and hormonal (gibberellin) signals.