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 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 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 Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1)In Vitroby Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture

2015 ◽  
Vol 60 (1) ◽  
pp. 570-579 ◽  
Author(s):  
Theresa B. Kuhlenschmidt ◽  
Florentine U. Rutaganira ◽  
Shaojun Long ◽  
Keliang Tang ◽  
Kevan M. Shokat ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cryptosporidium Parvum ◽  
Cell Invasion ◽  
Parasite Growth ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

ABSTRACTCryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) ofCryptosporidium parvummight be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibitC. parvumCDPK1 and blockC. parvumgrowth in tissue culturein vitro. Although these compounds potently inhibited kinase activityin vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation inToxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential forC. parvumhost cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets inC. parvumthat are worthy of further exploration.

scholarly journals The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1499-1509 ◽  
Author(s):  
KATELYN R. KEYLOUN ◽  
MOLLY C. REID ◽  
RYAN CHOI ◽  
YIFAN SONG ◽  
ANNA M. W. FOX ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Neospora Caninum ◽  
Babesia Bovis ◽  
Atp Binding ◽  
Calcium Dependent ◽  
Binding Cavity ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases

SUMMARYSpecific 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.

scholarly journals 5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Wenlin Huang ◽  
Ryan Choi ◽  
Matthew A. Hulverson ◽  
Zhongsheng Zhang ◽  
Molly C. McCloskey ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Substantial Reduction ◽  
Parasite Burden ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Calcium Dependent ◽  
Promising Target ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

ABSTRACT Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar CpCDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro. Correlation between anti-CpCDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.

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 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 Biochemical and Antiparasitic Properties of Inhibitors of the Plasmodium falciparum Calcium-Dependent Protein Kinase PfCDPK1

2014 ◽  
Vol 58 (10) ◽  
pp. 6032-6043 ◽  
Author(s):  
Keith H. Ansell ◽  
Hayley M. Jones ◽  
David Whalley ◽  
Alisdair Hearn ◽  
Debra L. Taylor ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Parasite Growth ◽  
Biochemical Assay ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

ABSTRACTPfCDPK1 is aPlasmodium falciparumcalcium-dependent protein kinase, which has been identified as a potential target for novel antimalarial chemotherapeutics. In order to further investigate the role of PfCDPK1, we established a high-throughputin vitrobiochemical assay and used it to screen a library of over 35,000 small molecules. Five chemical series of inhibitors were initially identified from the screen, from which series 1 and 2 were selected for chemical optimization. Indicative of their mechanism of action, enzyme inhibition by these compounds was found to be sensitive to both the ATP concentration and substitution of the amino acid residue present at the “gatekeeper” position at the ATP-binding site of the enzyme. Medicinal chemistry efforts led to a series of PfCDPK1 inhibitors with 50% inhibitory concentrations (IC50s) below 10 nM against PfCDPK1 in a biochemical assay and 50% effective concentrations (EC50s) less than 100 nM for inhibition of parasite growthin vitro. Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition ofPlasmodium vivaxCDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition ofPlasmodium bergheiCDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds toin vivoevaluation in theP. bergheirodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

scholarly journals Use of kinase inhibitors against schistosomes to improve and broaden praziquantel efficacy

Parasitology ◽  
2020 ◽  
Vol 147 (13) ◽  
pp. 1488-1498
Author(s):  
Sujeevi S. K. Nawaratna ◽  
Donald P. McManus ◽  
Robin B. Gasser ◽  
Paul J. Brindley ◽  
Glen M. Boyle ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Lethal Dose ◽  
Alternative Therapies ◽  
Potential Drug ◽  
Dependent Protein Kinase ◽  
Drug Of Choice ◽  
Dependent Protein ◽  
The Impact

AbstractPraziquantel (PZQ) is the drug of choice for schistosomiasis. The potential drug resistance necessitates the search for adjunct or alternative therapies to PZQ. Previous functional genomics has shown that RNAi inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) gene in Schistosoma adult worms significantly improved the effectiveness of PZQ. Here we tested the in vitro efficacy of 15 selective and non-selective CaMK inhibitors against Schistosoma mansoni and showed that PZQ efficacy was improved against refractory juvenile parasites when combined with these CaMK inhibitors. By measuring CaMK activity and the mobility of adult S. mansoni, we identified two non-selective CaMK inhibitors, Staurosporine (STSP) and 1Naphthyl PP1 (1NAPP1), as promising candidates for further study. The impact of STSP and 1NAPP1 was investigated in mice infected with S. mansoni in the presence or absence of a sub-lethal dose of PZQ against 2- and 7-day-old schistosomula and adults. Treatment with STSP/PZQ induced a significant (47–68%) liver egg burden reduction compared with mice treated with PZQ alone. The findings indicate that the combination of STSP and PZQ dosages significantly improved anti-schistosomal activity compared to PZQ alone, demonstrating the potential of selective and non-selective CaMK/kinase inhibitors as a combination therapy with PZQ in treating schistosomiasis.

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