scholarly journals Bumped Kinase Inhibitor 1294 Treats Established Toxoplasma gondii Infection

2014 ◽  
Vol 58 (6) ◽  
pp. 3547-3549 ◽  
Author(s):  
J. Stone Doggett ◽  
Kayode K. Ojo ◽  
Erkang Fan ◽  
Dustin J. Maly ◽  
Wesley C. Van Voorhis
Keyword(s):  
Toxoplasma Gondii ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Orally Bioavailable ◽  
Toxoplasma Gondii Infection ◽  
Bumped Kinase Inhibitor

ABSTRACTToxoplasma gondiiis a unicellular parasite that causes severe brain and eye disease. Current drugs forT. gondiiare limited by toxicity. Bumped kinase inhibitors (BKIs) selectively inhibit calcium-dependent protein kinases of the apicomplexan pathogensT. gondii, cryptosporidia, and plasmodia. A lead anti-ToxoplasmaBKI, 1294, has been developed to be metabolically stable and orally bioavailable. Herein, we demonstrate the oral efficacy of 1294 against toxoplasmosisin vivo.

scholarly journals Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

2016 ◽  
Vol 84 (5) ◽  
pp. 1262-1273 ◽  
Author(s):  
Shaojun Long ◽  
Qiuling Wang ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Gene Deletion ◽  
Cyst Formation ◽  
Content Type ◽  
Calcium Dependent ◽  
Targeted Gene Deletion ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.

scholarly journals Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Joachim Müller ◽  
Adriana Aguado-Martínez ◽  
Vreni Balmer ◽  
Dustin J. Maly ◽  
Erkang Fan ◽  
...  
Keyword(s):  
Vertical Transmission ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Neospora Caninum ◽  
Morphological Changes ◽  
Parasite Load ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein

ABSTRACT We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 μM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 μM; however, morphological changes occurred in cultures treated with >5 μM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 μM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 μM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

scholarly journals In VitroandIn VivoEffects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum

2015 ◽  
Vol 59 (10) ◽  
pp. 6361-6374 ◽  
Author(s):  
Pablo Winzer ◽  
Joachim Müller ◽  
Adriana Aguado-Martínez ◽  
Mahbubur Rahman ◽  
Vreni Balmer ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Vertical Transmission ◽  
Kinase Inhibitor ◽  
Neospora Caninum ◽  
Antigen Expression ◽  
Pregnant Mouse ◽  
Content Type ◽  
Elevated Expression ◽  
Bumped Kinase Inhibitor

ABSTRACTWe report on thein vitroeffects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulentNeospora caninumisolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains ofToxoplasma gondii(RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondiiRH) to 360 nM (N. caninumNc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treatedN. caninumbeta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenicT. gondiistrain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. InT. gondiiME49 andN. caninumNc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.

scholarly journals Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

2010 ◽  
Vol 17 (5) ◽  
pp. 602-607 ◽  
Author(s):  
Kayode K Ojo ◽  
Eric T Larson ◽  
Katelyn R Keyloun ◽  
Lisa J Castaneda ◽  
Amy E DeRocher ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Kinase Inhibitors ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

scholarly journals Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

2020 ◽  
Vol 8 (6) ◽  
pp. 801 ◽  
Author(s):  
Pablo Winzer ◽  
Joachim Müller ◽  
Dennis Imhof ◽  
Dominic Ritler ◽  
Anne-Christine Uldry ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Neospora Caninum ◽  
Farm Animals ◽  
Drug Pressure ◽  
Related Sequence ◽  
Drug Candidates ◽  
Calcium Dependent ◽  
Dependent Protein

Background: the apicomplexan parasite Neospora caninum causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of Neospora infections. BKI-1294 exposure of cell cultures infected with N. caninum tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences. Methods: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages. Results: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term “baryzoites” for this stage (from Greek βαρυσ = massive, bulky, heavy, inert).

scholarly journals Evaluation of Immune Responses in Mice after DNA Immunization with Putative Toxoplasma gondii Calcium-Dependent Protein Kinase 5

2014 ◽  
Vol 21 (7) ◽  
pp. 924-929 ◽  
Author(s):  
Nian-Zhang Zhang ◽  
Si-Yang Huang ◽  
Ying Xu ◽  
Jia Chen ◽  
Jin-Lei Wang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Interleukin 2 ◽  
Eukaryotic Expression ◽  
Economic Losses ◽  
Control Groups ◽  
Genotype I ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein

ABSTRACTToxoplasma gondiican cause serious public health problems and economic losses worldwide. Calcium-dependent protein kinases (CDPKs) are key mediators ofT. gondiisignaling pathways and are implicated as important virulence factors. In the present study, we cloned a novelT. gondiiCDPK gene, named TgCDPK5, and constructed the eukaryotic expression vector pVAX-CDPK5. Then, we evaluated the immune protection induced by pVAX-CDPK5 in Kunming mice. After injection of pVAX-CDPK5 intramuscularly, immune responses, determined with lymphoproliferative assays and cytokine and antibody measurements, were monitored, and mouse survival times and brain cyst formation were evaluated following challenges with theT. gondiiRH strain (genotype I) and the PRU strain (genotype II). pVAX-CDPK5 effectively induced immune responses with increased specific antibodies, a predominance of IgG2a production, and a strong lymphocyte proliferative response. The levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), and IL-12(p70) and the percentages of CD3+CD4+and CD3+CD8+cells in mice vaccinated with pVAX-CDPK5 were significantly increased. However, IL-4 and IL-10 were not produced in the vaccinated mice. These results demonstrate that pVAX-CDPK5 can elicit strong humoral and cellular Th1 immune responses. The survival time of immunized mice challenged with theT. gondiiRH strain (8.67 ± 4.34 days) was slightly, but not significantly, longer than that in the control groups within 7 days (P> 0.05). The numbers of brain cysts in the mice in the pVAX-CDPK5 group were reduced by ∼40% compared with those in the control groups (P< 0.05), which provides a foundation for the further development of effective subunit vaccines againstT. gondii.

scholarly journals A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4203
Author(s):  
Héloïse Débare ◽  
Nathalie Moiré ◽  
Firmin Baron ◽  
Louis Lantier ◽  
Bruno Héraut ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Intraperitoneal Administration ◽  
Congenital Toxoplasmosis ◽  
Parasite Burden ◽  
Oral Route ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

scholarly journals Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

scholarly journals Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

2021 ◽  
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Growth Delay ◽  
Hsp90 Inhibitors ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored.  Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

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