DOI: 10.1002/cbic.201200620 Selective Inhibition of an Apicoplastic Aminoacyl-tRNA Synthetase from Plasmodium falciparum Rob Hoen, [a] Eva Maria Novoa, [b] Alba López, [a] Noelia Camacho, [b] Laia Cubells, [b] Pedro Vieira, [d] Manuel Santos, [d] Patricia Marin-Garcia, [e] Jose Maria Bautista, [e] Alfred CortØs, [b] Lluís Ribas de Pouplana,* [b, c] and Miriam Royo* [a] Introduction Malaria remains one of the most important infectious diseases in the world, causing acute illness in more than 100 million people and leading to approximately 1 million deaths annual- ly. [1] In addition to its human cost, malaria represents a massive economic burden, contributing substantially to poverty in the developing world. Effective antimalarial drugs are available, but their efficacy is compromised by emerging resistance. [2] There is therefore a broad consensus about the need to devel- op new antimalarial drugs. Malaria is caused by Plasmodium, a genus of parasitic protists. At the moment there are over 200 species known of this genus, of which at least 11 can infect humans. Of these, Plasmodium falciparum causes the most severe form of malaria, being responsible for 90 % of the deaths. [1] The P. falciparum genome project revealed many new poten- tial drug targets, [3–9] several of which are enzymes that act in the apicoplast, a relict plastid derived from secondary endo- symbiosis of cyanobacteria, [10] which is essential for the para- site’s survival. [11, 12] Many of its bacterial-like enzymes are sub- stantially different from their mammalian homologues, [13–15] and this makes them excellent drug target candidates. Several antibacterial drugs that are clinically used for the treatment of malaria and toxoplasmosis (e.g., doxycycline, clindamycin, and spiramycin) act on apicoplastic targets. These drugs typically display a “delayed death” phenotype, which is characterized by the inhibition of parasite growth on the second erythrocytic cycle after the drug treatment. [16–20] Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antimicrobial drug targets, [21, 22] and so represent interesting new targets for antimalarial drug discov- ery. [23] They perform a central role in the translation of the ge- netic code by catalyzing the attachment of each amino acid to its cognate transfer RNA (tRNA). Although these enzymes differ widely in size, sequence, and oligomeric state, they all carry out similar two-step reactions. [22] In a first step, the ARS catalyz- es the activation of the amino acid, and in a second step the aminoacyl-adenylate intermediate (AA-AMP) is transferred to the tRNA. 1) AA + ATP !AA-AMP + PP i 2) AA-AMP + tRNA !AA-tRNA + AMP Currently, ARS inhibition is the mechanism of action of one commercially available antibiotic: pseudomonic acid or mupir- ocin (GlaxoSmithKline), a natural product that inhibits bacterial isoleucyl-tRNA synthetases with an 8000-fold selectivity over The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial thera- pies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antibacterial targets and so con- stitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplas- tic ARSs remain unexplored. We have characterized the lysyla- tion system of Plasmodium falciparum, and designed, synthe- sized, and tested a set of inhibitors based on the structure of the natural substrate intermediate : lysyl-adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically in- hibit Plasmodium apicoplastic lysyl-tRNA synthetase and show antimalarial activities in the micromolar range. [a] Dr. R. Hoen, + A. López, Dr. M. Royo Combinatorial Chemistry Unit, Barcelona Science Park University of Barcelona C/Baldiri Reixac 10, 08028 Barcelona, Catalonia (Spain) E-mail : mroyo@pcb.ub.cat [b] Dr. E. M. Novoa, + N. Camacho, Dr. L. Cubells, Prof. A. CortØs, Prof. L. Ribas de Pouplana Institute for Research in Biomedicine (IRB Barcelona) C/Baldiri Reixac 10, 08028 Barcelona, Catalonia (Spain) E-mail : lluis.ribas@irbbarcelona.org [c] Prof. L. Ribas de Pouplana ICREA Passeig Lluís Companys 1, 08010 Barcelona, Catalonia (Spain) [d] P. Vieira, Prof. M. Santos RNA Biology Laboratory, Department of Biology and Centre for Environmental and Marine Studies (CESAM), University of Aveiro 3810-193 Aveiro (Portugal) [e] P. Marin-Garcia, Prof. J. M. Bautista Department of Biochemistry and Molecular Biology Complutense University of Madrid 28040 Madrid (Spain) [ + ] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201200620: includes Figures S1–S6, Table S1, and supplementary methods.  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 499 – 509 499 CHEMBIOCHEM FULL PAPERS