scholarly journals Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hala E. Hussein ◽  
Wendell C. Johnson ◽  
Naomi S. Taus ◽  
Janaina Capelli-Peixoto ◽  
Carlos E. Suarez ◽  
...  
Keyword(s):  
Babesia Bovis ◽  
Xanthurenic Acid ◽  
Bovine Babesiosis ◽  
Dependent Protein Kinase ◽  
Tick Vector ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Sexual Stages ◽  
Calcium Dependent Protein Kinase

Abstract Background Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. Results In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. Conclusions Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis. Graphical abstract

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 Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yi-Ju Lu ◽  
Pai Li ◽  
Masaki Shimono ◽  
Alex Corrion ◽  
Takumi Higaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Biology ◽  
Stomatal Closure ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Cytoskeleton Organization ◽  
Effector Triggered Immunity ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

AbstractPattern-triggered immunity and effector-triggered immunity are two primary forms of innate immunity in land plants. The molecular components and connecting nodes of pattern-triggered immunity and effector-triggered immunity are not fully understood. Here, we report that the Arabidopsis calcium-dependent protein kinase CPK3 is a key regulator of both pattern-triggered immunity and effector-triggered immunity. In vitro and in vivo phosphorylation assays, coupled with genetic and cell biology-based analyses, show that actin-depolymerization factor 4 (ADF4) is a physiological substrate of CPK3, and that phosphorylation of ADF4 by CPK3 governs actin cytoskeletal organization associated with pattern-triggered immunity. CPK3 regulates stomatal closure induced by flg22 and is required for resistance to Pst DC3000. Our data further demonstrates that CPK3 is required for resistance to Pst DC3000 carrying the effector AvrPphB. These results suggest that CPK3 is a missing link between cytoskeleton organization, pattern-triggered immunity and effector-triggered immunity.

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 Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Abhisheka Bansal ◽  
Kayode K. Ojo ◽  
Jianbing Mu ◽  
Dustin J. Maly ◽  
Wesley C. Van Voorhis ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Protein Kinase G ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Reduced Activity ◽  
Calcium Dependent Protein Kinase

ABSTRACT We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 ( Pf CDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes. IMPORTANCE Protein kinases of Plasmodium falciparum are being actively pursued as drug targets to treat malaria. However, compensatory mechanisms may reverse the drug activity against a kinase. In this study, we show that replacement of the wild-type threonine gatekeeper residue with methionine reduces the transphosphorylation activity of CDPK1. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. This study highlights that targeting one enzyme may lead to changes in transcript expression of other kinases that compensate for its function and may select for mutants that are less dependent on the target enzyme activity. Thus, inhibiting two kinases is a better strategy to protect the antimalarial activity of each, similar to artemisinin combination therapy or malarone (atovaquone and proguanil).

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 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.

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