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 ...
Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 M, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.
Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 "short" (Tb10.161.3140) than for T. brucei GSK-3 "long" (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei. Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found.
BackgroundThe genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.Methodology/Principal FindingsWe experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.Conclusions/SignificanceThis collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.
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