Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death

This work was supported by Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Ministerio de Economía y Competitividad (CTQ2013-44367-C2-2-P to R.H.-G.) and Diputación General de Aragón (DGA; B89 to R.H.-G.) and the EU Seventh Framework Programme (2007–2013) under BioStruct-X (grant agreement 283570 and BIOSTRUCTX 5186, to R.H.-G.). T.K.S. was supported by the Wellcome Trust grant 093228 and European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED).Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.Publisher PDFPeer reviewe

The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81

Determination of potential scaffolds for human choline kinase α1 by chemical deconvolution studies

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The mechanism of allosteric coupling in choline kinase α1 revealed by the action of a rationally designed inhibitor

5 pags, 4 pags. -- Supporting information for this article (experimental details) is available on the WWW under http://dx.doi.org/10.1002/anie.201209660.Applying a CHOK hold: Combined experimental and computational studies of the binding mode of a rationally designed inhibitor of the dimeric choline kinase α1 (CHOKα1) explain the molecular mechanism of negative cooperativity (see scheme) and how the monomers are connected. The results give insight into how the symmetry of the dimer can be partially conserved despite a lack of conservation in the static crystal structures. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.We thank the Fundación Agencia Aragonesa para la Investigación y el Desarrollo (ARAID, Spain), the Ministerio de Economía y Competitividad (MEC, Spain, grants BFU2010-19504, BFU2010-19451, SAF2009-11955, and BIO2010-20166), the Consejería de Innovación, Ciencia y Empresa of the Junta de Andalucía (grant P07-CTS-03210), the Ministerio de Educación for a FPU fellowship (AP2007-03115) to B.R.-R., and Diputación General de Aragón (Grupo Protein Targets, B89) and the Comunidad Autónoma de Madrid S2010-BMD-2457 (BIPEDD2). This work was granted access to the HPC resources of HECToR, within the Distributed European Computing Initiative (FP7/2007-2013, RI-283493)

A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

69 Pags.- 4 Figs.- Supplementary Information (6 Suppl. Tabls.- 19 Suppl. Figs.). The definitive version is available at: http://www.nature.com/nchembio/index.htmlProtein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.We thank ARAID, MEC (BFU2010-19504, CTQ2013-­‐ 44367-­‐C2-­‐2-­‐P, CTQ2012-36365), NIH (GM061126 and CA123071) and the DGA (B89) for financial support, and BIFI (Memento cluster) for supercomputer support. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement N°283570 and BIOSTRUCTX_5186).Peer reviewe