[Show abstract][Hide abstract] ABSTRACT: Background The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed
[Show abstract][Hide abstract] ABSTRACT: Controlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers.
All volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed.
All six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9-14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2-69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2-4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited.
The CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model.
PLoS ONE 11/2014; 9(11):e109654. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium parvum is a zoonotic agent that infects humans and animals occasionally causing severe, watery diarrhoea. In immunocompetent hosts, cryptosporidiosis is self-limiting but can have a fatal outcome in immunocompromised individuals. Cryptosporidium is one of the most common causes of waterborne diseases (recreational water and drinking water) in humans, a leading cause of moderate to severe childhood diarrhoea, and a major agent of diarrhoea in calves leading to high economic losses and up to 10 % lethality. So far, available treatment options are insufficient for both veterinary and human clinical disease cases. Here, we report for the first time that the novel bumped kinase inhibitor (BKI) 1294 targeting the calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium is able to reduce the oocyst shedding of C. parvum by calves-its natural host-without obvious side effects.
Parasitology Research 11/2014; · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.
[Show abstract][Hide abstract] ABSTRACT: Toxoplasma gondii is a unicellular parasite that causes severe brain and eye disease. Current drugs for T. gondii are limited by toxicity. Bumped kinase inhibitors (BKI) selectively inhibit calcium-dependent protein kinases of the apicomplexan pathogens T. gondii, Cryptosporidia and Plasmodia. A lead anti-Toxoplasma BKI, 1294, has been developed to be metabolically stable and orally bioavailable. Herein, we demonstrate the oral efficacy of 1294 against toxoplasmosis in vivo.
Antimicrobial Agents and Chemotherapy 03/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.
PLoS ONE 03/2014; 9(3):e92929. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malaria remains a major health concern for a large percentage of the world’s population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitos. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block P. falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.
European Journal of Medicinal Chemistry 03/2014; · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.
Molecular and Biochemical Parasitology 02/2014; 193(1):33-44. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.
[Show abstract][Hide abstract] ABSTRACT: 5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: The 1H, 13C, and 15N chemicals shifts have been extensively assigned for Gl-FKBP, a 109-residue, FKBP-type, peptidyl-proline cis-trans isomerase from Giardia lamblia, an enteric protozoan parasite responsible for giardiasis. These chemical shift assignments were deposited into the BioMagResBank database under the accession number BMRB-17818 and used to determine the solution structure for Gl-FKBP (PDB-ID 2LGO). The core of the Gl-FKBP structure consists of an -helix (I59 – M69) nestled against the face of a six-strand, antiparallel -sheet. The FKBP family of proteins is a potential target class for novel antimicrobials. The chemical shift assignments for Gl-FKBP, in combination with its solution structure (2LGO), will enable backbone dynamics, chemical shift perturbation, and ligand screening studies that will assist employing FKBP-type proteins for antimicrobial drug discovery.
Journal of Biomolecular NMR 11/2013; · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that co-formulation of this transmission-blocking agent with asexual stage anti-malarias such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.
The Journal of Infectious Diseases 10/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fragment based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission blocking agents.
ACS Chemical Biology 09/2013; · 5.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30 000-40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T. brucei GSK3. From this we identified a series of several highly ligand-efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low-nanomolar inhibitors of TbGSK3 that are potent in vitro inhibitors of T. brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from Giardia lamblia (Gl-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of Gl-MIF is compared to the MIF structures from humans (Hs-MIF) and three Plasmodium species (falciparum, berghei, and yoelii). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to Hs-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of Gl-MIF and the Plasmodium-MIFs due primarily to two "gate-keeper" residues in the parasitic MIFs. For the Plasmodium MIFs the gate-keeper residues are at positions 44 (Y⇒R) and 100 (V⇒D) and for Gl-MIF it is at position 100 (V⇒R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins.
Journal of Structural and Functional Genomics 05/2013;
[Show abstract][Hide abstract] ABSTRACT: Malaria, most commonly caused by the parasite Plasmodium falciparum, is a devastating disease that remains a large global health burden. Lack of vaccines and drug resistance necessitate the continual development of new drugs and exploration of new drug targets. Due to their essential role in protein synthesis, aminoacyl-tRNA synthetases are potential anti-malaria drug targets. Here we report the crystal structures of P. falciparum cytosolic tryptophanyl-tRNA synthetase (Pf-cTrpRS) in its ligand-free state and tryptophanyl-adenylate (WAMP)-bound state at 2.34Å and 2.40Å resolutions, respectively. Large conformational changes are observed when the ligand-free protein is bound to WAMP. Multiple residues, completely surrounding the active site pocket, collapsed onto WAMP. Comparison of the structures to those of human cytosolic TrpRS (Hs-cTrpRS) provides information about the possibility of targeting Pf-cTrpRS for inhibitor development. There is a high degree of similarity between Pf-cTrpRS and Hs-cTrpRS within the active site. However, the large motion that Pf-cTrpRS undergoes during transitions between different functional states avails an opportunity to arrive at compounds which selectively perturb the motion, and may provide a starting point for the development of new anti-malaria therapeutics.
Molecular and Biochemical Parasitology 05/2013; · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 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.
We 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.
This 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.
PLoS ONE 01/2013; 8(1):e53851. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2), proline, xylitol, NDSB 201, CTAB and K(2)PO(4)) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.
PLoS ONE 12/2012; 7(12):e52482. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Today, due to great technological advancements, it is possible to study everything at the same time. This ability has given birth to “totality” studies in the fields of genomics, transcriptomics, proteomics, and metabolomics. In turn, the combined study of all these global analyses gave birth to the field of systems biology. Another “totality” field brought to life with new emerging technologies is structural genomics, an effort to determine the three-dimensional structure of every protein encoded in a genome. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a specialized structural genomics effort composed of academic (University of Washington), government (Pacific Northwest National Laboratory), not-for-profit (Seattle BioMed), and commercial (Emerald BioStructures) institutions that is funded by the National Institute of Allergy and Infectious Diseases (Federal Contract: HHSN272200700057C and HHSN27220120025C) to apply genome-scale approaches in solving protein structures from biodefense organisms, as well as those causing emerging and re-emerging disease. In five years over 540 structures have been deposited into the Protein Data Bank (PDB) by SSGICD. About one third of all SSGCID structures contain bound ligands, many of which are metabolites or metabolite analogues present in the cell. These proteins structures are the blueprints for the structure-based design of the next generation of drugs against bacterial pathogens and other infectious diseases. Many of the selected SSGCID targets are annotated enzymes from known metabolomic pathways essential to cellular vitality since selectively “knocking-out” one of the enzymes in an important pathway with a drug may be fatal to the organism. One reason metabolomic pathways are important is because of the small molecules, or metabolites, produced at various steps in these pathways and identified by metabolomic studies. Unlike genomics, transcriptomics, and proteomics that may be influenced by epigenetic, post-transcriptional, and post-translational modifications, respectively, the metabolites present in the cell at any one time represent downstream biochemical endproducts, and therefore, metabolite profiles may be most closely associated with a phenotype and provide valuable information for infectious disease research. Metabolomic data would be even more useful if it could be linked to the vast amount of structural genomics data. Towards this goal SSGCID has created an automated website (http://apps.sbri.org/SSGCIDTargetStatus/Pathway) that assigns selected SSGCID target proteins to MetaCyc pathways (http://metacyc.org/). Details of this website will be provided here. The SSGCID-Pathway website represents a first big step towards linking metabolites and metabolic pathways to structural genomic data with the goal of accelerating the discovery of new agents to battle infectious diseases.