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Title: Structure-based development of Plasmodium hypoxanthine-guanine phosphoribosyltransferase inhibitors: a proof of concept study
Authors: Laporte, Manon
Naesens, Lieve
Hocková, Dana
Keough, Dianne
Guddat, Luke
Issue Date: 3-Nov-2014
Conference: Belgian Society for Microbiology and the National Committee for Microbiology location:Academy Palace, Brussels date:18-11-2014
Abstract: Structure-based development of Plasmodium hypoxanthine-guanine phosphoribosyltransferase inhibitors: a proof of concept study

Manon Laportea, Dana Hockováb, Dianne T. Keoughc, Luke W. Guddatc, Lieve Naesensa
aRega Institute, KU Leuven, Belgium; bInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, cSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia

The malarial parasites Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are purine auxotrophs that rely on the salvage of host purines for their survival and growth. A critical enzyme in this salvage pathway, hypoxanthine guanine phosphoribosyltransferase (HGPRT), is considered a prime target for antimalarial therapy. Our collaboration between the University of Queensland, the Institute of Organic Chemistry and Biochemistry (Prague) and the Rega institute (KU Leuven), is focussed on rational development of novel acyclic nucleoside phosphonates (ANPs) that are structural analogues of the HGPRT nucleotide reaction products IMP and GMP. Based on available HGPRT crystal structures, ANP derivatives with a second phosphonate group attached (bisANPs) were designed. These compounds proved to be particularly strong HGPRT inhibitors with Ki values as low as 30 nM, and displayed antimalarial activity in Pf-infected erythrocytes with IC50 values as low as 3.8 µM (Keough et al. 2013). However, since the Plasmodium parasite possesses several enzymes that could possibly serve as the target for inhibition by the (bis)ANPs, either directly or after metabolic conversion, it remained to be established whether the observed antiparasitic effect indeed results from HGPRT inhibition. We here present a target validation assay to assess, in a cellular environment, the inhibitory effect of (bis)ANPs towards HGPRT. This method complements the enzymatic assays (in which purified HGPRT enzymes are studied in a cell-free environment), and the Plasmodium cell culture assay (that involves replication of the whole Pf parasite). First, we created adenoviral (Ad) vectors containing the cDNA sequences encoding human, Pf or PvHGPRT. Transduction of these Ad vectors into HGPRT-deficient human 1306 cells generated high HGPRT expression levels, as estimated by a tritium release assay with [2,8-3H]hypoxantine (Balzarini and De Clercq, 1992). Several (bis)ANPs were shown to inhibit the HGPRT reaction by the human or Pv enzyme. Our novel assay allows to validate Plasmodium HGPRT inhibitors in cell culture and will be instrumental to guide further development of this new class of antimalarial drugs.
Publication status: submitted
KU Leuven publication type: IMa
Appears in Collections:Laboratory of Virology and Chemotherapy (Rega Institute)

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