New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.The food-borne apicomplexan protozoan Toxoplasma gondii is the causative agent of toxoplasmosis and may be the most common infectious eukaryotic parasite of humans, based Correspondence should be addressed to W.C.V.V. (wesley@u.washington.edu) or E.A.M. (merritt@u.washington.edu). Accession codes. Protein Data Bank: Atomic coordinates and structure factors have been deposited with accession numbers 3I79 (apo), 3I7c (NA-PP2 complex) and 3I7b (NM-PP1 complex).Note: Supplementary information is available on the Nature Structural & Molecular Biology website. AUTHOR CONTRIBUTIONSK.K.O., K.R.K., K.K.I. and W.C.V.V. were involved in the biochemical characterization and testing of inhibitors of TgCDPK1; L.J.C., K.K.O., K.R.K., A.J.N., C.L.M.J.V., F.S.B. and W.C.V.V. selected, cloned and purified the recombinant wild-type and mutant TgCDPK1 protein; E.T.L., J.E.K., T.L.A., L.Z., W.G.J.H. and E.A.M. crystallized and solved the structure of TgCDPK1; R.M. and D.J.M. synthesized the inhibitors; A.E.D. and M.P. performed the cellular T. gondii experiments; K.K.O., E.T.L., A.E.D., D.J.M., M.P., E.A.M. and W.C.V.V. wrote the paper; all authors reviewed and edited the paper. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. 10 . These drugs are problematic in that they can cause rash, leucopenia and nephrotoxicity11, and sulfadiazine and pyrimethamine can result in complications during pregnancy. New therapeutics against T. gondii are needed. NIH Public AccessCalcium levels have long been associated with T. gondii's interrelated processes of invasion, gliding motility and secretion 12 . The intracellular Ca 2+ level oscillates during gliding motility and is promptly dampened upon cell invasion, preventing T. gondii from immediately gliding out of cells 13 . Calcium oscillations control many targets in the cell, and the mediation of invasion, micronemal secretion and gliding motility is thought to be largely due to T. gondii calcium-dependent protein kinases (TgCDPKs) 12,14 .Protein ...

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for P. falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5,655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC50 values below 1.25 μM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5′-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.

The single tyrosyl tRNA-synthetase (TyrRS) gene in trypanosomatid genomes codes for a protein that is twice the length of TyrRS from virtually all other organisms. Each half of the double-length TyrRS contains a catalytic domain and an anticodon-binding domain, however the two halves retain only 17% sequence identity to each other. The structural and functional consequences of this duplication and divergence are unclear. TyrRS normally forms a homodimer in which the active site of one monomer pairs with the anticodon-binding domain from the other. However, crystal structures of Leishmania major TyrRS show that instead the two halves of a single molecule form a pseudo-dimer resembling the canonical TyrRS dimer. Curiously, the C-terminal copy of the catalytic domain has lost the catalytically important HIGH and KMSKS motifs characteristic of Class I aminoacyl-tRNA synthetases. Thus the pseudo-dimer contains only one functional active site, contributed by the N-terminal half, and only one functional anticodon recognition site, contributed by the C-terminal half. Despite biochemical evidence for negative cooperativity between the two active sites of the usual TyrRS homodimer, previous structures have captured a crystallographically-imposed symmetric state. As the L. major TyrRS pseudo-dimer is inherently asymmetric, conformational variations observed near the active site may be relevant to understanding how the state of a single active site is communicated across the dimer interface. Furthermore, substantial differences between trypanosomal TyrRS and human homologs are promising for the design of inhibitors that selectively target the parasite enzyme.

In the last decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. Here we present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. We conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal.