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 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 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 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 Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion

2021 ◽  
Vol 10 ◽  
Author(s):  
Shanshan Liang ◽  
Hui Dong ◽  
Shunhai Zhu ◽  
Qiping Zhao ◽  
Bing Huang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Translation Initiation ◽  
Cell Invasion ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Host Cell Invasion ◽  
Dependent Protein Kinase ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

scholarly journals Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development

2015 ◽  
Vol 60 (3) ◽  
pp. 1464-1475 ◽  
Author(s):  
Judith L. Green ◽  
Robert W. Moon ◽  
David Whalley ◽  
Paul W. Bowyer ◽  
Claire Wallace ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Parasite Growth ◽  
Parasite Line ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Calcium Dependent ◽  
Class 1 ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and ofPlasmodium falciparumparasite growthin vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90.

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).

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