• Resúmenes - - - - - - - - - - - - - - - - - Instituto de Parasitología y Biomedicina "López-Neyra" CSIC - - - - - - - - - - - - - - - - - - - -
    • Fotos Jornadas 2012
    • Jose M Rodríguez-Vargas - LAB105
    • Cristina Romero López - LAB112
    • Maria Morell - LAB202
    • Jenny Campos-Salinas - LAB213
    • Paula M. Sánchez Carrasco - 103/104
    • Francisco Macías Huete - LAB212
    • Michael Caraballo - LAB210
    • Beatriz del Blanco - LAB110
    • Juan Pablo Muñoz-Cobo Belart - LAB109
    • Diana Lopez-Farfan - LAB102
    • Perandrés López, Rubén - LAB209
    • Samuel Prieto Vega - LAB111
    • Agustina Arias - LAB211

Paula M. Sánchez Carrasco. LAB104

Validation and exploitation of nucleotide metabolism enzymes as therapeutic targets in Plasmodium

Malaria, with approximately 210 million cases per year and more than 500.000 attributed deaths reported globally in 2010, remains a devastating global health problem. The disease in humans is caused by the infection of 5 different Plasmodium species, amongst which Plasmodium falciparum causes most mortality, mainly in children below the age of 5. It is transmitted via a mosquito vector. Due to the appearance of resistance to the current antimalarial drugs, there is a need for new drugs to treat the disease.
During its 48 h intraerythrocytic life cycle, P. falciparum replicates its 23 Mb genomic DNA 16–32 times. Given the large requirement for nucleotides for DNA synthesis, considerable interest has focused on the purine salvage pathway and de novo pyrimidine biosynthesis as a possible drug target. In this work, we describe our efforts to (i) validate thymidylate kinase (PfTMPK), which catalyzes the phosphorylation ofdeoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), as a drug target and (ii) the screening performed against purine nucleoside phosphorylase (PfPNP), which catalyzes the phosphorolysis of inosine to hypoxanthine, to discover new inhibitors with potential antiplasmodial activity. Both enzymes were selected because of their unique kinetic features that make them differ from their human homologues.
For the validation of PfTMPK as a drug target, deletion of the gene has been attempted in P. falciparum using different strategies that include the single cross‐over recombination of the endogenous gene sequence. Different deleted versions of the gene were cloned into an appropriate vector, leading to the disruption or regeneration of the coding sequence upon single cross‐over recombination of the plasmid in the gene locus. Transfected parasite lines were taken through several drug selection cycles in order to select for parasites where the Pftmpk locus had been disrupted by the transfection plasmid. Genomic DNA were isolated and analysed by diagnostic Southern blotting in order to establish integration events into the parasite genome. After 3 drug cycles, integration can be detected in the case of those plasmids that give rise to a functional copy but not for the ones that render non functional truncated versions. Gene disruption is also underway in parasites overexpressing an heterologous copy of the tmpk gene (human tmpk).
A part of the MEDINA’s natural product collection was employed to carry out a screening against PfPNP. The importance of this collection lies in the great chemical diversity of natural products which has not been yet explored in the search for new antimalarials. For the screening against PfPNP, a previously described PNP activity assay, the measure of hypoxanthine production via xanthine oxidase, was adapted to a 384‐plate format, a more suitable format for high throughput screening. After assaying more than 18240 extracts, 2 new families of PfPNP inhibitors with in vitro antimalarial properties have been discovered.
In conclusion, the results indicate that (i) TMPK is an essential enzyme in the intraerythrocytic form of the Plasmodium parasite and (ii) the new families of PfPNP inhibitors discovered can be useful as a starting point to design specific inhibitors against this enzyme with antimalarial activity.