Publications by authors named "Iván Caballero"

5 Publications

  • Page 1 of 1

Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.

Antimicrob Agents Chemother 2016 10 23;60(10):5949-56. Epub 2016 Sep 23.

Platform Technology and Science, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Spain.

The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.
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http://dx.doi.org/10.1128/AAC.00433-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038259PMC
October 2016

A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

Anal Biochem 2016 08 29;506:13-21. Epub 2016 Apr 29.

Platform Technology and Science Tres Cantos, GlaxoSmithKline, Centro de Investigación Básica, 28760, Tres Cantos, Spain. Electronic address:

Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.
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http://dx.doi.org/10.1016/j.ab.2016.04.013DOI Listing
August 2016

Discovery of Enhancers of the Secretion of Leukemia Inhibitory Factor for the Treatment of Multiple Sclerosis.

J Biomol Screen 2016 Jun 16;21(5):437-45. Epub 2016 Mar 16.

Centro de Investigación Básica, GSK, Tres Cantos, Madrid, Spain.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease that involves activation of T cells, microglia, and astrocytes. There is a clear unmet medical need for MS, as current therapies reduce the relapse rate, but are unable to prevent the neurological deterioration. Leukemia inhibitory factor (LIF) is a proinflammatory cytokine that can also positively modulate the immune response, by inducing the inhibition of myelin-reactive TH17 differentiation, and by promoting oligodendrocyte-mediated myelination. The aim of this project was to find central nervous system (CNS)-permeable and orally available small molecules that upregulate production of endogenous LIF. We describe here the development of a phenotypic assay and screening of 1.7 million compounds to identify LIF enhancers using U87 MG cells. Five chemically tractable series of compounds and a few singletons were selected for further progression. Some of them were also active in a different LIF-expressing cell line and in primary rat astrocytes. Although further studies would be required to deconvolute the targets involved in LIF induction and to confirm activity of hits in more disease-relevant assays, our results have demonstrated the potential of the phenotypic approach to identify specific and chemically tractable small molecules that trigger the production of LIF in relevant cell lines.
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http://dx.doi.org/10.1177/1087057116638821DOI Listing
June 2016

Development of a Fluorescence-based Trypanosoma cruzi CYP51 Inhibition Assay for Effective Compound Triaging in Drug Discovery Programmes for Chagas Disease.

PLoS Negl Trop Dis 2015 Sep 22;9(9):e0004014. Epub 2015 Sep 22.

Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51.
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http://dx.doi.org/10.1371/journal.pntd.0004014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578769PMC
September 2015

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set.

Biochemistry 2012 Jun 1;51(23):4764-71. Epub 2012 Jun 1.

GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, UK.

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.
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http://dx.doi.org/10.1021/bi3005076DOI Listing
June 2012