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Background Prostate cancer is the most common cancer in men in the developed world, with most deaths caused by advanced and metastatic disease which has no curative options. Here, we identified Mbtps2 alteration to be associated with metastatic disease in an unbiased in vivo screen and demonstrated its regulation of fatty acid and cholesterol metabolism. Methods The Sleeping Beauty transposon system was used to randomly alter gene expression in the PtenNull murine prostate. MBTPS2 was knocked down by siRNA in LNCaP, DU145 and PC3 cell lines, which were then phenotypically investigated. RNA-Seq was performed on LNCaP cells lacking MBTPS2, and pathways validated by qPCR. Cholesterol metabolism was investigated by Filipin III staining. Results Mbtps2 was identified in our transposon-mediated in vivo screen to be associated with metastatic prostate cancer. Silencing of MBTPS2 expression in LNCaP, DU145 and PC3 human prostate cancer cells reduced proliferation and colony forming growth in vitro. Knockdown of MBTPS2 expression in LNCaP cells impaired cholesterol synthesis and uptake along with reduced expression of key regulators of fatty acid synthesis, namely FASN and ACACA. Conclusion MBTPS2 is implicated in progressive prostate cancer and may mechanistically involve its effects on fatty acid and cholesterol metabolism.

To test for evidence of statin-mediated effects in patients with castration-resistant prostate cancer (CRPC) as post-diagnosis use of statins in patients with prostate cancer is associated with favourable survival outcome.The SPECTRE trial was a 6-weeks-long proof-of-concept single-arm Phase II treatment trial, combining atorvastatin and androgen deprivation therapy in patients with CRPC (regardless of metastatic status), designed to test for evidence of statin-mediated effects in patients with CRPC. The primary study endpoint was the proportion of patients achieving a ≥50% drop from baseline in prostate-specific antigen (PSA) levels at any time over the 6-week period of atorvastatin medication (PSA response). Exploratory endpoints include PSA velocity and serum metabolites identified by mass spectrometry .At the scheduled interim analysis, one of 12 patients experienced a ≥50% drop in PSA levels (primary endpoint), with ≥2 patients satisfying the primary endpoint required for further recruitment. All 12 patients experienced substantial falls in serum cholesterol levels following statin treatment. While all patients had comparable pre-study PSA velocities, six of 12 patients showed decreased PSA velocities after statin treatment, suggestive of disease stabilization. Unbiased metabolomics analysis on serial weekly blood samples identified tryptophan to be the dominant metabolite associated with patient response to statin.Data from the SPECTRE study provide the first evidence of statin-mediated effects on CRPC and early sign of disease stabilization. Our data also highlight the possibility of altered tryptophan metabolism being associated with tumour response.

Purpose of Review Review work to create and evaluate educational materials that could serve as a primary prevention strategy to help both providers and patients in Panama, Colombia, and the USA reduce disease burden of Toxoplasma infections. Recent Findings Educational programs had not been evaluated for efficacy in Panama, USA, or Colombia. Summary Educational programs for high school students, pregnant women, medical students and professionals, scientists, and lay personnel were created. In most settings, short-term effects were evaluated. In Panama, Colombia, and USA, all materials showed short-term utility in transmitting information to learners. These educational materials can serve as a component of larger public health programs to lower disease burden from congenital toxoplasmosis. Future priorities include conducting robust longitudinal studies of whether education correlates with reduced adverse disease outcomes, modifying educational materials as new information regarding region-specific risk factors is discovered, and ensuring materials are widely accessible.

Supplementary Data from Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with β-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFκBp65 and β-catenin. Overall, WNT/β-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes.Significance:Targeting of both AR and WNT/β-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression.Significance:Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

The supplementary file contains details of the materials and methods used in the manuscript. File also contains supplementary figures S1 to S3 describing in details the murine model used to study role of activated ß-catenin in prostate cancer progression. Supplementary figure S4 describes the murine orthograft model generated to study mechanistic details. Supplementary figure S5 describes the metabolic profile of primary murine prostate cancer cells with Pten loss and ß-catenin activation and figures S6 and S7 show the effects of combining ADT with inhibition of WNT signalling.

Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression. Significance: Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

Peroxisome Proliferator-Activated Receptor Gamma (PPARG) is one of the three members of the PPAR family of transcription factors. Besides its roles in adipocyte differentiation and lipid metabolism, we recently demonstrated an association between PPARG and metastasis in prostate cancer. In this study a functional effect of PPARG on AKT serine/threonine kinase 3 (AKT3), which ultimately results in a more aggressive disease phenotype was identified. AKT3 has previously been shown to regulate PPARG co-activator 1 alpha (PGC1α) localisation and function through its action on chromosome maintenance region 1 (CRM1). AKT3 promotes PGC1α localisation to the nucleus through its inhibitory effects on CRM1, a known nuclear export protein. Collectively our results demonstrate how PPARG over-expression drives an increase in AKT3 levels, which in turn has the downstream effect of increasing PGC1α localisation within the nucleus, driving mitochondrial biogenesis. Furthermore, this increase in mitochondrial mass provides higher energetic output in the form of elevated ATP levels which may fuel the progression of the tumour cell through epithelial to mesenchymal transition (EMT) and ultimately metastasis.

Purpose of Review Review international efforts to build a global public health initiative focused on toxoplasmosis with spillover benefits to save lives, sight, cognition and motor function benefiting maternal and child health. Recent Findings Multiple countries’ efforts to eliminate toxoplasmosis demonstrate progress and context for this review and new work. Summary Problems with potential solutions proposed include accessibility of accurate, inexpensive diagnostic testing, pre-natal screening and facilitating tools, missed and delayed neonatal diagnosis, restricted access, high costs, delays in obtaining medicines emergently, delayed insurance pre-approvals and high medicare copays taking considerable physician time and effort, harmful shortcuts being taken in methods to prepare medicines in settings where access is restricted, reluctance to perform ventriculoperitoneal shunts promptly when needed without recognition of potential benefit, access to resources for care, especially for marginalized populations, and limited use of recent advances in management of neurologic and retinal disease which can lead to good outcomes.

Purpose of ReviewReview building of programs to eliminateToxoplasmainfections.Recent FindingsMorbidity and mortality from toxoplasmosis led to programs in USA, Panama, and Colombia to facilitate understanding, treatment, prevention, and regional resources, incorporating student work.SummaryStudies foundational for building recent, regional approaches/programs are reviewed. Introduction provides an overview/review of programs in Panamá, the United States, and other countries. High prevalence/risk of exposure led to laws mandating testing in gestation, reporting, and development of broad-based teaching materials aboutToxoplasma.These were tested for efficacy as learning tools for high-school students, pregnant women, medical students, physicians, scientists, public health officials and general public. Digitized, free, smart phone application effectively taught pregnant women about toxoplasmosis prevention. Perinatal infection care programs, identifying true regional risk factors, and point-of-care gestational screening facilitate prevention and care. When implemented fully across all demographics, such programs present opportunities to save lives, sight, and cognition with considerable spillover benefits for individuals and societies.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. In patients, THEM6 expression correlates with progressive disease and is associated with poor survival. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, THEM6 is located at the endoplasmic reticulum (ER) membrane and controls lipid homeostasis by regulating intracellular levels of ether lipids. Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated induction of ATF4. Finally, we show that THEM6 is required for the establishment of the MYC-induced stress response. Thus, similar to PCa, THEM6 loss significantly impairs tumorigenesis in the MYC-dependent subtype of triple negative breast cancer. Altogether, our results highlight THEM6 as a novel component of the treatment-induced stress response and a promising target for the treatment of CRPC and MYC-driven cancer.

Prostate cancer is highly prevalent, being the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of PTEN and SPRY2 (Sprouty 2) tumour suppressors, we identified enhanced creatine metabolism within the phosphagen system in progressive disease. Altered creatine metabolism was validated in in vitro and in vivo prostate cancer models and in clinical cases. Upregulated creatine levels were due to increased uptake through the SLC6A8 creatine transporter and de novo synthesis, resulting in enhanced cellular basal respiration. Treatment with cyclocreatine (a creatine analogue that potently and specifically blocks the phosphagen system) dramatically reduces creatine and phosphocreatine levels. Blockade of creatine biosynthesis by cyclocreatine leads to cellular accumulation of S-adenosyl methionine (SAM), an intermediary of creatine biosynthesis, and suppresses prostate cancer growth in vitro. Furthermore, cyclocreatine treatment impairs cancer progression in our GEMM and in a xenograft liver metastasis model. Hence, by targeting the phosphagen system, cyclocreatine results in anti-tumourigenic effects from both SAM accumulation and suppressed phosphagen system.

Background Prostate cancer is highly prevalent worldwide. Androgen deprivation therapy (ADT) remains the treatment of choice for incurable prostate cancer, but majority of patients develop disease recurrence following ADT. There is therefore an urgent need for early detection of treatment resistance. Methods Isogenic androgen-responsive (CWR22Res) and castration-resistant (22Rv1) human prostate cancer cells were implanted into the anterior lobes of the prostate in CD-1 Nu mice to generate prostate orthografts. Castrated mice bearing CWR22Res and 22Rv1 orthografts mimic clinical prostate cancer following acute and chronic ADT, respectively. 18F-Fluciclovine (1-amino-3-fluorocyclobutane-1-carboxylic acid) with a radiochemical purity of > 99% was produced on a FASTlab synthesiser. Ki67 staining in endpoint orthografts was studied. Western blot, quantitative RT-PCR and next-generation sequencing transcriptomic analyses were performed to assess the expression levels of amino acid transporters (including LAT1 and ASCT2, which have been implicated for Fluciclovine uptake). Longitudinal metabolic imaging with 18F-Fluciclovine-based positron emission tomography (PET) was performed to study tumour response following acute and chronic ADT. Results Both immunohistochemistry analysis of endpoint prostate tumours and longitudinal 18F-Fluciclovine imaging revealed tumour heterogeneity, particularly following ADT, with in vivo 18F-Fluciclovine uptake correlating to viable cancer cells in both androgen-proficient and castrated environment. Highlighting tumour subpopulation following ADT, both SUVpeak and coefficient of variation (CoV) values of 18F-Fluciclovine uptake are consistent with tumour heterogeneity revealed by immunohistochemistry. We studied the expression of amino acid transporters (AATs) for 18F-Fluciclovine, namely LAT1 (SLC7A5 and SLC3A2) and ASCT2 (SLC1A5). SLC7A5 and SLC3A2 were expressed at relatively high levels in 22Rv1 castration-resistant orthografts following chronic ADT (modelling clinical castration-resistant disease), while SLC1A5 was preferentially expression in CWR22Res tumours following acute ADT. Additional AATs such as SLC43A2 (LAT4) were shown to be upregulated following chronic ADT by transcriptomic analysis; their role in Fluciclovine uptake warrants investigation. Conclusion We studied in vivo 18F-Fluciclovine uptake in human prostate cancer orthograft models following acute and chronic ADT. 18F-Fluciclovine uptakes highlight tumour heterogeneity that may explain castration resistance and can be exploited as a clinical biomarker.

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance., Androgen receptor (AR) signalling regulates cellular metabolism in prostate cancer. Here, the authors perform a proteomics and metabolomics characterisation of prostate cancer cells adapted to long-term resistance to AR inhibition and show rewiring of glucose and lipid metabolism, and further identify a signature associated with resistance to AR inhibition.

Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5fl/fl (Erk5fl/fl) mice by ~6–8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with β-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFκBp65 and β-catenin. Overall, WNT/β-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. Significance: Targeting of both AR and WNT/β-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal form of treatment‐resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre‐clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self‐sufficient form of CRPC. Mechanistically, HER2‐IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1‐mediated cholesterol uptake in SPRY2‐deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2‐deficient CRPC is dependent on cholesterol bioavailability and SRB1‐mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2‐deficient CRPC.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: “Orbital-deconvolution” elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. “Cluster-deconvolution” revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, “disease-deconvolution” identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer’s disease, and cancer. This “reconstruction-deconvolution” logic provides templates of progenitor cells’ potentiating effects, and components affecting human brain parasitism and diseases.

Here, we show that spiroindolone, an effective treatment for plasmodia, is also active against Toxoplasma gondii tachyzoites. In vitro , spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC 50 ], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% ( P = 0.002), measured 3 days after the last dose. This inhibition of T. gondii tachyzoites in vitro and in vivo indicates that spiroindolone is a promising lead candidate for further medicine development.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.

We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8+ T cell–eliciting epitopes, a universal CD4+ helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8+ T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8+ T cell–eliciting epitopes in a vaccine that prevents toxoplasmosis.

We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8(+) T cell-eliciting epitopes, a universal CD4(+) helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8(+) T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8(+) T cell-eliciting epitopes in a vaccine that prevents toxoplasmosis.

Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.

Congenital toxoplasmosis is usually diagnosed in the United States when there is fetal loss, prematurity, or severe disease at birth with damage to eyes and brain with lifelong consequences, especially if untreated. This disease also presents as recurrent active retinochoroiditis, loss of cognitive and motor function, and seizures later in life in persons who appeared normal at birth and whose initial disease went unrecognized. Of those who are congenitally infected with Toxoplasma gondii and did not receive treatment, 80%–90% are reported to have eye disease by adolescence [1]. Obstetrical serologic screening is mandated by law in France and Austria, virtually eliminating severe disease in these countries through early treatment of the fetus. In the United States, screening for acute acquired T. gondii infection during gestation is performed only occasionally. The National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS), which has been ongoing since 1981, has allowed careful evaluation of 2 cohorts of persons with congenital toxoplasmosis. There is one cohort of persons, most often diagnosed with substantial disease in the newborn period and treated in the first year of life, and another in which congenital toxoplasmosis was diagnosed after the first year of life. Sera have been collected from all the congenitally infected persons and almost all of their mothers. Genetically disparate parasites behave differently in animal models and tissue culture [2–4]. Type I parasites are more virulent, measured as causing death in mice. Type II parasites are less lethal in mice, produce more cysts in brains of mice, and grow more slowly in tissue culture. Type III parasites are intermediate for these phenotypes. Parasites may also be nonarchetypal, containing mixtures of II or I/III specific alleles, or altogether new alleles. In a small series of persons with substantial ophthalmologic disease, there was an unusual abundance of non-II or atypical parasites, suggesting that disease outcomes in humans infected with different parasite types may differ [3]. Immune responses, including production of interferon γ, dendritic cell responses, and numbers of activated T cells, also differ [3]. Effects of type I, II, and III parasites on transcriptomes of a human neuroepithelial cell line have been shown to differ [4]. In the United States there has been only limited study of distribution of parasite types and diseases they cause, with no analyses of substantial cohorts of congenitally infected persons as described herein. An enzyme-linked immunosorbent assay (ELISA) allows discrimination of infections caused by type II and non-II parasites using a serologic test identifying strain-specific antibodies induced by allelic peptide motifs in dense granule proteins GRA6 and GRA7 [5]. This assay allows us to distinguish strain type (II or not exclusively II [NE-II]) causing congenital toxoplasmosis in our cohorts and to correlate this with demographics of families, manifestations in infants at birth and later in life, and effects of treatment.

(See the Editorial Commentary by Linn, on pages 1090–1.) Background. Congenital toxoplasmosis presents as severe, life-altering disease in North America. If mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. Conversely, if not all risks are identifiable, undetectable risks are suggested. A new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of Toxoplasma gondii infection in 4 North American epidemics and in mothers of children in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS). This novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts. Methods. Acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples. Results. Fifty-nine (78%) of 76 mothers of congenitally infected infants in NCCCTS had primary infection with oocysts. Only 49% of these mothers identified significant risk factors for sporozoite acquisition. Socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors. Conclusions. Undetected contamination of food and water by oocysts frequently causes human infections in North America. Risks are often unrecognized by those infected. Demographic characteristics did not identify oocyst infections. Thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. Only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in North America.

NALP1 is a member of the NOD-like receptor (NLR) family of proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of proinflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines that are important in innate immunity. In this study, susceptibility alleles for human congenital toxoplasmosis were identified in the NALP1 gene. To investigate the role of the NALP1 inflammasome during infection with T. gondii , we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, upregulation of the proinflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-12 upon T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, this is the first report, to our knowledge, on the crucial role of the NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.

The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8(+) T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4(+) epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8(+) T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong proinflammatory responses. Ligation of ATP by purinergic receptor P2X(7), encoded by P2RX7, stimulates proinflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X(7) has a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z-scores +/-2.429; P=0.015) between the derived C(+)G(-) allele (f=0.68; OR=2.06; 95% CI: 1.14-3.75) at single-nucleotide polymorphism (SNP) rs1718119 (1068TC; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical subgroups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0-4.25; 0.004P0.009) when hydrocephalus was removed from the analysis. Association with toxoplasmic retinochoroiditis was replicated (FBAT Z-scores +/-3.089; P=0.002) in a small family-based study (60 families; 68 affected offspring) of acquired infection in Brazil, where the ancestral T(+) allele (f=0.296) at SNP rs1718119 was strongly protective (OR=0.27; 95% CI: 0.09-0.80).

The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively., Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD+ and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials.

The triazine WR99210 [4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine] inhibits Toxoplasma gondii in vitro at nanomolar levels ( P < 0.05). The 50% inhibitory concentration (IC 50 ) was approximately 50 nM. It is a potent inhibitor in vitro and is also effective in vivo. Administration of WR99210 parenterally (i.e., intraperitoneally) reduced the mean number of RH strain tachyzoites present in peritoneal fluid substantially 4 days after intraperitoneal infection of mice. There was a mean of approximately 35 million parasites in control mice as contrasted with approximately 2 million parasites in mice treated with 1.25 mg WR99210/kg of body weight in a representative experiment ( P < 0.05). In addition the prodrug PS-15 N′-[3-(2,4, 5-trichlorophenoxy)propyloxy]-N9-(1-methylethyl) imidocarbonimidicdiamide is converted to 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine in vivo when the prodrug is administered orally. PS-15 administered by gavage also reduced intraperitoneal RH strain T. gondii tachyzoite numbers. WR99210 has high efficacy and relatively low toxicity because of its substantial effect on T. gondii dihydrofolate reductase (DHFR) but not the mammalian host DHFR. Amino acid sequences of T. gondii , Plasmodium falciparum , and Homo sapiens DHFRs were compared. It is of interest that of the DHFR amino acids considered to be interacting with WR99210 in P. falciparum within interatomic distances within 3 to 5 Å, four of eight were shared with T. gondii DHFR. H. sapiens also shared four amino acids thought to be interacting with WR99210. Efficacy of intraperitoneal administration of WR99210 and peroral administration of PS-15 demonstrate the potential usefulness of this class of compounds in treatment of toxoplasmosis administered either parenterally or perorally. The recent development program for this class of antimicrobials as antimalarials makes our proof of principle of improved efficacy of triazines (compared with the gold standard treatment, pyrimethamine) against T. gondii especially promising.

The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.

The shikimate pathway is essential for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential conversion of erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then used as a substrate for other pathways that culminate in production of folates, ubiquinone, napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The shikimate pathway is absent from animals and present in the apicomplexan parasites Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway by glyphosate is effective in controlling growth of these parasites. These findings emphasize the potential benefits of developing additional effective inhibitors of the shikimate pathway. Such inhibitors may function as broad-spectrum antimicrobial agents that are effective against bacterial and fungal pathogens and apicomplexan parasites.

Survival of mice during the acute stage of Toxoplasma gondii infection was not influenced by the MHC Class I gene, L(d), but was influenced by the MHC Class II genes, Ia and Ie. As unexplained variability was noted in our initial studies of influence of the L(d) gene on survival, influence of the L(d) gene region on survival in the presence of a number of variables was studied. Although route of administration and dose of parasites, and age and gender of the mice markedly influenced outcome of T. gondii infection, the Class I L(d) gene did not modify survival in any of these circumstances. In separate studies, using mice with a differing genetic background, i.e. H-2(b), C57BL/10 mice, presence of Ia or Ie alone diminished survival even though presence of Ia reduced parasite burden. When neither or both the Ia and Ie genes were present together, survival was greater. In separate analyses of our studies of AxB BxA recombinant inbred mice, similar influences of MHC genes on survival and parasite burden following peroral infection were confirmed. Previously undescribed associations of novel genetic loci and survival and parasite burden also were identified. Genetic loci associated with enhanced survival included D8Mit42, D1Mit3, Iapls1-16, D8Mit14, Hoxb, Mpmv29, Pmv45, and Emv-2; genetic loci associated with reduced parasite burden included H-2, D17Mit62, D17Mit83, D17Mit21, D17Mit34, D17Mit47, D18Mit4, and Gln3-5. These studies demonstrate the importance of MHC region genes (but not L(d)) for survival, and the influence of other novel genes, and endogenous and exogenous variables on survival and parasite burden specified by host genes following T. gondii infection.

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [ P , P , P , P , ALOX12 . In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.

Background:\ud Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2–3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear.\ud \ud Objective:\ud To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC.\ud \ud Design, setting, and participants:\ud RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes.\ud Outcome measurements and statistical analysis:\ud We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model.\ud \ud Results and limitations:\ud We identified ADT-regulated signalling pathways, including the Wnt/β-catenin signalling pathway, and observed overexpression of β-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of 12 (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes β-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches.\ud \ud Conclusions:\ud RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/β-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.

Congenital transmission of Toxoplasma gondii from a mother who was apparently immunologically competent and who had toxoplasmic lymphadenitis 2 months before conception is described. Since no T. gondii-specific serological data were available for this mother from the time her lymph node biopsy specimen was obtained, the specimen was studied by polymerase chain reaction (PCR) to determine whether the T. gondii B1 gene was present. The predictive diagnostic value of histologic findings previously considered to be classic signs of T. gondii lymphadenitis also was studied. This was done by correlation of serological tests diagnostic of acute acquired T. gondii infection and presence of characteristic findings in biopsy specimens from persons without known immunocompromise. Both PCR and review of the characteristic features of her lymph node biopsy specimen confirmed the diagnosis of preconceptual infection in the mother. We also discuss two other cases in which apparently immunologically competent mothers with preconceptually acquired infection transmitted this parasite to their fetuses.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a member of the IRAK family and has an important role in inducing the production of inflammatory mediators. This kinase is downstream of MyD88, an adaptor protein essential for Toll-like receptor (TLR) function. We investigated the role of this kinase in IRAK4-deficient mice orally infected with the cystogenic ME49 strain of Toxoplasma gondii . IRAK4 −/− mice displayed higher morbidity, tissue parasitism, and accelerated mortality than the control mice. The lymphoid follicles and germinal centers from infected IRAK4 −/− mice were significantly smaller. We consistently found that IRAK4 −/− mice showed a defect in splenic B cell activation and expansion as well as diminished production of gamma interferon (IFN-γ) by T lymphocytes. The myeloid compartment was also affected. Both the frequency and ability of dendritic cells (DCs) and monocytes/macrophages to produce IL-12 were significantly decreased, and resistance to infection with Toxoplasma was rescued by treating IRAK4 −/− mice with recombinant IL-12 (rIL-12). Additionally, we report the association of IRAK4 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital toxoplasmosis in infected individuals (rs1461567 and rs4251513, P < 0.023 and P < 0.045, respectively). Thus, signaling via IRAK4 is essential for the activation of innate immune cells, development of parasite-specific acquired immunity, and host resistance to infection with T. gondii .

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii ’s fluorescence and luminescence. PPMO directed against T. gondii ’s dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO‘s specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

In our efforts to identify novel chemical scaffolds for the development of new antiprotozoal drugs, a compound library was screened against Toxoplasma gondii tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2-hydroxybenzamide 1a against T. gondii as described elsewhere. Synthesis of a compound set was guided by T. gondii SAR with 1r found to be superior for T. gondii , also active against Thai and Sierra Leone strains of Plasmodium falciparum , and with superior ADMET properties as described elsewhere. Herein, synthesis methods and details of the chemical analysis of the compounds in this series are described. Further, this series of N-benzoyl-2-hydroxybenzamides was repurposed for testing against four other protozoan parasites: Trypanosoma brucei rhodesiense , Trypanosoma cruzi , Leishmania donovani , and P. falciparum (K1 isolate). Structure-activity analyses led to the identification of compounds in this set with excellent antileishmanial activity (compound 1d). Overall, compound 1r was the best and had activity 21-fold superior to that of the standard antimalarial drug chloroquine against the K1 P. falciparum isolate.

Toxoplasma (T.) gondii, the causative agent of toxoplasmosis, is a ubiquitous opportunistic pathogen that infects individuals worldwide, and is a leading cause of severe congenital neurologic and ocular disease in humans. No vaccine to protect humans is available, and hypersensitivity and toxicity limit the use of the few available medicines. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. Using the Hybrid Structure Based (HSB) method, we have previously identified small molecule inhibitors of P. falciparum that seem to target a novel protein-protein interaction between the Myosin tail interacting protein and myosin light chain. This pathway has been hypothesized to be involved in invasion of host erythrocytes by the parasite and is broadly conserved among the apicomplexans. Guided by similar computational drug design approaches, we investigated this series of small molecules as potential inhibitors of T. gondii. Compound C3-21, identified as the most active inhibitor in this series, exhibited an IC(50) value ~500 nM against T. gondii. Among the 16 structural analogs of C3-21 tested thus far, nine additional compounds were identified with IC(50) values

Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)sor = 6 microM without toxicity to host cells, three compounds have IC(90)s45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.