Falciparum Calcium-dependent Protein Kinase Research Articles

7H-Pyrrolo[2,3-d]pyrimidine-4-amines as Potential Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinases.

A series of pyrrolo[2,3-d]pyrimidines were designed in silico as potential bumped kinase inhibitors targeting P. falciparum calcium dependent protein kinase 4 (PfCDPK4), with the potential to inhibit PfCDPK1 based on earlier studies of the two kinases. A small series of these compounds were prepared and assessed for inhibitory activity against PfCDPK4 and PfCDPK1 in vitro. Four of the compounds displayed promising inhibitory activity against either PfCDPK4 (IC50 =0.210-0.530 μM), or PfCDPK1 (IC50 =0.589 μM). These data will enable optimisation of the molecular model to better predict inhibitory activity against PfCDPK4.

Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome.

Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics.IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.

Targeted Inhibition of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 with a Constrained J Domain-Derived Disruptor Peptide.

To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.

Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum

ABSTRACTThe human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like P. falciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the P. falciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress.

PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection

Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

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

ABSTRACTWe 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 (PfCDPK1). 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.

5-Aminopyrazole-4-carboxamide analogues are selective inhibitors of Plasmodium falciparum microgametocyte exflagellation and potential malaria transmission blocking agents

Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) is essential for the exflagellation of male gametocytes. Inhibition of PfCDPK4 is an effective way of blocking the transmission of malaria by mosquitoes. A series of 5-aminopyrazole-4-carboxamide analogues are demonstrated to be potent inhibitors of PfCDPK4. The compounds are also able to block exflagellation of Plasmodium falciparum male gametocytes without observable toxicity to mammalian cells.

Computational strategies to explore antimalarial thiazine alkaloid lead compounds based on an Australian marine sponge Plakortis Lita

In this work, an attempt was made to propose new leads based on the natural scaffold Thiaplakortone-A active against malaria. The 2D QSAR studies suggested that three descriptors correlate with the anti-malarial activity with an R2 value of 0.814. Robustness, reliability, and predictive power of the model were tested by internal validation, external validation, Y-scrambling, and applicability domain analysis. HQSAR studies were carried out as an additional tool to find the sub-structural fingerprints. The CoMFA and CoMSIA models gave Q2 values of 0.813 and 0.647, and values of 0.994 and 0.984, respectively. Using the 2D-QSAR equation, the activity values of the seven modified compounds were calculated and it was found that three molecules showed good anti-malarial activity. Molecular docking of the 42 Thiaplakortone-A derivatives with Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was carried out to find out protein-ligand interactions. Data mining of the bioassay data-set AID: 504850 using the classifier based on Random Forest of Weka suggested that all of the eight molecules selected and three out of the seven virtual molecules were anti-malarial active. Both the virtual molecules and drug molecules were docked with CYP3A4, indicating that the virtual molecules could metabolize easily. Toxicity studies using Osiris shows that three molecules showed no toxic characters.

Biochemical and antiparasitic properties of inhibitors of the Plasmodium falciparum calcium-dependent protein kinase PfCDPK1.

PfCDPK1 is a Plasmodium falciparum calcium-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-throughput in vitro biochemical 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 growth in vitro. Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition of Plasmodium vivax CDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition of Plasmodium berghei CDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds to in vivo evaluation in the P. berghei rodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

Optimization of an Imidazopyridazine Series of Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 (PfCDPK1)

A structure-guided design approach using a homology model of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was used to improve the potency of a series of imidazopyridazine inhibitors as potential antimalarial agents. This resulted in high affinity compounds with PfCDPK1 enzyme IC50 values less than 10 nM and in vitroP. falciparum antiparasite EC50 values down to 12 nM, although these compounds did not have suitable ADME properties to show in vivo efficacy in a mouse model. Structural modifications designed to address the ADME issues, in particular permeability, were initially accompanied by losses in antiparasite potency, but further optimization allowed a good balance in the compound profile to be achieved. Upon testing in vivo in a murine model of efficacy against malaria, high levels of compound exposure relative to their in vitro activities were achieved, and the modest efficacy that resulted raises questions about the level of effect that is achievable through the targeting of PfCDPK1.

Development of potent and selective Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) inhibitors that block the transmission of malaria to mosquitoes

Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.

Imidazopyridazines as potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1): Preparation and evaluation of pyrazole linked analogues

The structural diversity and SAR in a series of imidazopyridazine inhibitors of Plasmodium falciparum calcium dependent protein kinase 1 (PfCDPK1) has been explored and extended. The opportunity to further improve key ADME parameters by means of lowering logD was identified, and this was achieved by replacement of a six-membered (hetero)aromatic linker with a pyrazole. A short SAR study has delivered key examples with useful in vitro activity and ADME profiles, good selectivity against a human kinase panel and improved levels of lipophilic ligand efficiency. These new analogues thus provide a credible additional route to further development of the series.

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu(356) to Thr(370)) of the JD in the presence of calcium (Ca(2+)). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.

Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4; PF07_0072)

Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4; PF07_0072)

Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3

Ca2+ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca2+ stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [γ-32P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone IIAS as a representative substrate in a Ca2+-dependent manner at a high Ca2+ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.

Characterization of Plasmodium falciparum calcium-dependent protein kinase 4

In Plasmodium berghei, the orthologous gene of P. falciparum calcium-dependent protein kinase 4 (PfCDPK4) was reported to be essential for the exflagellation of male gametocytes. To elucidate the role of PfCDPK4 in P. falciparum gametogenesis, we characterized the biological function of PfCDPK4 in vitro. PfCDPK4 was purified as a fusion protein that was labeled with [γ-32P]ATP; this labeling was then eliminated by phosphatase. Phosphorylation activity of PfCDPK4 was eliminated when its putative catalytic lysine residue was replaced with alanine. In biochemical analyses, PfCDPK4 was found to have characteristics that were similar to those of homologous proteins from plants. PfCDPK4 phosphorylation was activated when experimental conditions were changed from those characteristic of human blood (37 °C, pH 7.4) to those of the mosquito bloodmeal (at least 5 °C below 37 °C, pH 7.6, with xanthurenic acid (XA)). PfCDPK4 was overexpressed in day 15 gametocytes exposed to XA or human serum. Thus, PfCDPK4 phosphorylation is activated by an increase in Ca2+ concentration or pH and by a decrease in temperature, and is associated with the Ca2+ signals that facilitate P. falciparum gametogenesis.

Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography–tandem mass spectrometry

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.

Dissection of Mechanisms Involved in the Regulation of Plasmodium falciparum Calcium-dependent Protein Kinase 4

Recent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite ( Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117, 503-514 ). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level. We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4. PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family.

Raf kinase inhibitor protein affects activity of Plasmodium falciparum calcium-dependent protein kinase 1

Proteins, such as the raf kinase inhibitory protein (RKIP), serve as modulators of signalling pathways by either promoting or inhibiting the formation of productive signalling complexes through protein–protein interactions. In the present study, the plasmodial RKIP ortholog, PfPE-PB1, was cloned, recombinantly expressed and purified to homogeneity. The purified protein was used to investigate the effect of plasmodial RKIP on the autophosphorylation and substrate phosphorylation activity of Plasmodium falciparum calcium-dependent protein kinase 1, PfCDPK1. Phosphorylation of RKIP by PfCDPK1 in in vitro kinase assays suggests that RKIP may be an in vivo substrate of this kinase, although the specific activity of PfCDPK1 is approximately seven-fold lower when RKIP, instead of casein, an exogenous substrate of this enzyme, is used as a substrate. In addition to the observed phosphorylation of RKIP itself, its presence in the assays greatly enhanced the autophosphorylation capacity of PfCDPK1 by approximately 5.5-fold. This substantial increase in autophosphorylation activity was associated with a diminished substrate phosphorylation activity of PfCDPK1 when casein was used. At the same time, RKIP phosphorylation slightly increased when casein was included into the assays. Thus, RKIP is recognized as a substrate under in vitro conditions and appears to act as a regulator of PfCDPK1 kinase activity, which possibly is one of its actual functions in the parasite.