RESUMEN
Recent efforts for increasing the success in drug discovery focus on an early, massive, and routine mechanistic and/or kinetic characterization of drug-target engagement as part of a design-make-test-analyze strategy. From an experimental perspective, many mechanistic assays can be translated into a scalable format on automation platforms and thereby enable routine characterization of hundreds or thousands of compounds. However, now the limiting factor to achieve such in-depth characterization at high-throughput becomes the quality-driven data analysis, the sheer scale of which outweighs the time available to the scientific staff of most labs. Therefore, automated analytical workflows are needed to enable such experimental scale-up. We have implemented such a fully automated workflow in Genedata Screener for time-dependent ligand-target binding analysis to characterize non-equilibrium inhibitors. The workflow automates Quality Control (QC) / data modelling and decision-making process in a staged analysis: (1) quality control of raw input data-fluorescence signal-based progress curves - featuring automated rejection of unsuitable measurements; (2) automated model selection - one-step versus two-step binding model - using statistical methods and biological validity rules; (3) result visualization in specific plots and annotated result tables, enabling the scientist to review large result sets efficiently and, at the same time, to rapidly identify and focus on interesting or unusual results; (4) an interactive user interface for immediate adjustment of automated decisions, where necessary. Applying this workflow to first-pass, high-throughput kinetic studies on kinase projects has allowed us to surmount previously rate-limiting manual analysis steps and boost productivity; and is now routinely embedded in a biopharma discovery research process.
Asunto(s)
Análisis de Datos , Descubrimiento de Drogas , Humanos , CinéticaRESUMEN
Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.1.1. Brain tissue was analyzed for tumor volume, invasiveness, hypoxia, vascular density, proliferation, apoptosis, GSCs, and MGCs. Glioma cells were evaluated for CXCR4 expression and polymorphism and POL5551's effects on CXCR4 ligand binding, cell viability, and migration. No CXCR4 mutations were identified. POL5551 inhibited CXCR4 binding to its ligand, stromal cell-derived factor-1α, and reduced hypoxia- and stromal cell-derived factor-1α-mediated migration dose-dependently but minimally affected cell viability. In vivo, B20-4.1.1 increased hypoxic foci and invasiveness, as seen in GBM patients receiving anti-VEGF therapy. Combination of POL5551 and B20-4.1.1 reduced both glioma invasiveness by 16% to 39% and vascular density compared to B20-4.1.1 alone in both glioma models. Reduced populations of GSCs and MGCs were also seen in CT-2A tumors. POL5551 concentrations, evaluated by mass spectrometry, were higher in tumors than in neighboring brain tissues, likely accounting for the results. Inhibition of CXCR4-regulated tumoral, stem cell, and immune mechanisms by adjunctive CXCR4 antagonists may help overcome antiangiogenic therapy resistance, benefiting GBM patients.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos/uso terapéutico , Glioma/tratamiento farmacológico , Receptores CXCR4/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glioma/metabolismo , Glioma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Proteínas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Malaria remains a major killer in many parts of the world. Recently, there has been an increase in the role of public-private partnerships inciting academic and industrial scientists to merge their expertise in drug-target validation and in the early stage of drug discovery to identify potential new medicines. There is a need to identify and characterize new molecules showing high efficacy, low toxicity with low propensity to induce resistance in the parasite. In this context, we have studied the structural requirements of the inhibition of PfCDPK1. This is a calcium-dependent protein kinase expressed in Plasmodium falciparum, which has been genetically confirmed as essential for survival. A primary screening assay has been developed. A total of 54000 compounds were tested, yielding two distinct chemical series of nanomolar small molecule inhibitors. The most potent members of each series were further characterized through enzymatic and biophysical analyses. Dissociation rates of the inhibitor-kinase complexes were shown to be key parameters to differentiate both series. Finally, a homology-based model of the kinase core domain has been built which allows rational design of the next generation of inhibitors.
Asunto(s)
Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Luminiscencia , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Especificidad por SustratoAsunto(s)
Reactivos de Enlaces Cruzados/química , Metilasas de Modificación del ADN/química , Enzimas Reparadoras del ADN/química , Proteínas Recombinantes de Fusión/química , Proteínas Supresoras de Tumor/química , Línea Celular , Metilasas de Modificación del ADN/biosíntesis , Metilasas de Modificación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/efectos de los fármacos , Dimerización , Guanina/análogos & derivados , Guanina/síntesis química , Guanina/química , Guanina/farmacocinética , Humanos , Estructura Molecular , Peso Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/efectos de los fármacos , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/efectos de los fármacosAsunto(s)
Quimera/genética , Ingeniería Genética/métodos , Estreptavidina/química , Animales , Sitios de Unión , Biotina/química , Biotina/metabolismo , Membrana Celular/metabolismo , Humanos , Ligasas/química , Ligasas/metabolismo , Mutación , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estreptavidina/genética , Estreptavidina/metabolismoRESUMEN
We report the functional characterization in Leishmania amazonensis of a soluble pyrophosphatase (LaVSP1) that localizes in acidocalcisomes, a vesicular acidic compartment. LaVSP1 is preferentially expressed in metacyclic forms. Experiments with dominant negative mutants show the requirement of LaVSP1 functional expression for metacyclogenesis and virulence in mice. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in acidocalcisomes, LaVSP1 hydrolyzes either inorganic pyrophosphate (Km = 92 microM, kcat = 125 s(-1)), tripolyphosphate (Km = 1153 microM, kcat = 131 s(-1)), or polyphosphate of 28 residues (Km = 123 microM, kcat = 8 s(-1)). Predicted structural analysis suggests that the structural orientation of the residue Lys78 in LaVSP1 accounts for the observed increase in Km compared with the yeast pyrophosphatase and for the ability of trypanosomatid VSP1 enzymes to hydrolyze polyphosphate. These results make the VSP1 enzyme an attractive drug target against trypanosomatid parasites.
Asunto(s)
Leishmania/enzimología , Polifosfatos/metabolismo , Pirofosfatasas/metabolismo , Animales , Clonación Molecular , Cartilla de ADN , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Leishmania/clasificación , Leishmania/genética , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/metabolismo , Pirofosfatasas/genéticaRESUMEN
Overexpression in Leishmania amazonensis promastigotes of the GTPase-deficient small G protein LdARL-3A-Q70L specifically provokes the loss of the flagella without affecting cell viability and body size. However, motility is lost and, remarkably, cells do not survive in the insect vector Lutzomyia longipalpis gut, leading to interruption of parasite transmission. We report here that overexpression of the same protein in Leishmania major, Leishmania donovani, and Crithidia fasciculata also led to significant alterations of the flagella. Surprisingly, ablation of TbARL-3A expression by RNAi in Trypanosoma brucei brucei also provoked flagella shortening, revealing that overexpression of the GTPase-deficient protein seems functionally equivalent to a drastic reduction in its native counterpart abundance. This renders possible complementary studies of an essential pathway in related organisms. Potential significance for the protein function is discussed as well as future strategies for stopping the transmission of several neglected parasitic diseases.
Asunto(s)
Flagelos/fisiología , Proteínas Protozoarias/fisiología , Trypanosomatina/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , Expresión Génica , Datos de Secuencia Molecular , Movimiento , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia , Trypanosomatina/genéticaRESUMEN
We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg(2+) or Zn(2+), both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PP(i)) (k(cat) = 385 s(-1)) or tripolyP (polyP(3)) and polyphosphate (polyP) of 28 residues (polyP(28)) with k(cat) values of 52 and 3.5 s(-1), respectively. An unusual N-terminal domain of 160 amino acids, containing a putative calcium EF-hand-binding domain, is involved in protein oligomerization. Using double-stranded RNA interference methodology, we produced an inducible bloodstream form (BF) deficient in the TbVSP1 protein (BFiVSP1). The long-chain polyP levels of these mutants were reduced by 60%. Their phenotypes revealed a deficient polyP metabolism, as indicated by their defective response to phosphate starvation and hyposmotic stress. BFiVSP1 did not cause acute virulent infection in mice, demonstrating that TbVSP1 is essential for growth of bloodstream forms in the mammalian host.
Asunto(s)
Endopeptidasas/fisiología , Polifosfatos/metabolismo , Proteínas Protozoarias , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Trypanosoma brucei brucei/patogenicidad , Ácido Anhídrido Hidrolasas/química , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Animales , Western Blotting , Calcio/química , Cromatografía , Cromatografía en Gel , Clonación Molecular , Cósmidos , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Hidrólisis , Immunoblotting , Cinética , Magnesio/farmacología , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Ósmosis , Fenotipo , Fosfatos , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Bicatenario/química , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Factores de Tiempo , Transfección , Virulencia , Zinc/farmacologíaRESUMEN
Vacuolar proton pyrophosphatases (V-H(+)-PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H(+)-PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H(+)-PPase gene by using double-stranded RNA interference methodology to produce inducible V-H(+)-PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H(+)-PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 x 10(5) cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.