Plasmodium Falciparum Calcium-dependent Protein Kinase Research Articles

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.

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.

Exploring the structural requirements in multiple chemical scaffolds for the selective inhibition of Plasmodium falciparum calcium-dependent protein kinase-1 (PfCDPK-1) by 3D-pharmacophore modelling, and docking studies

Current research on antimalarial protein kinases has provided an opportunity to design kinase-based antimalarial drugs. We have developed a common feature-based pharmacophore model from a set of multiple chemical scaffolds including derivatives of 3,6-imidazopyridazines, pyrazolo[2,3-d]pyrimidines and imidazo[1,5-a]pyrazines, in order to incorporate the maximum structural diversity information in the model for the Plasmodium falciparum calcium-dependent protein kinase-1 (PfCDPK-1) target. The best pharmacophore model (Hypo-1) with the essential features of two hydrogen bond donors (HBD), one hydrophobic aromatic (HYAr) and one ring aromatic (RA) showed the classification accuracies of 86.27%, 78.43% and 100.00% in labelling the training and test set (test set-1 and test set-2) compounds into more active and less active classes. In order to identify the crucial interaction between multiple scaffold ligands and the target protein, we first developed the homology model using a template structure of P. bergheii (PbCDPK1; PDB ID: 3Q5I), and thereafter performed the docking studies. The residues such as Lys85, Phe147, Tyr148, Leu198, Val211, and Asp212 were found to be the most important interacting residues for possessing PfCDPK-1 inhibitory activity.

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.

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.

Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under invitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

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.

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.

Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.

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.

Export of Plasmodium falciparum calcium‐dependent protein kinase 1 to the parasitophorous vacuole is dependent on three N‐terminal membrane anchor motifs

Calcium-dependent protein kinases play a pivotal role in calcium signalling in plants and some protozoa, including the malaria parasites. They are found in various subcellular locations, suggesting an involvement in multiple signal transduction pathways. Recently, Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) has been found in the membrane and organelle fraction of the parasite. The kinase contains three motifs for membrane binding at its N-terminus, a consensus sequence for myristoylation, a putative palmitoylation site and a basic motif. Endogenous PfCDPK1 and the in vitro translated kinase were both shown to be myristoylated. The supposed membrane attachment function of the basic cluster was experimentally verified and shown to participate together with N-myristoylation in membrane anchoring of the kinase. Using immunogold electron microscopy, the protein was detected in the parasitophorous vacuole and the tubovesicular system of the parasite. Mutagenesis of the predicted acylated residues and the basic motif confirmed that dual acylation and the basic cluster are required for correct targeting of Aequorea victoria green fluorescent protein to the parasitophorous vacuole, suggesting that PfCDPK1 as the leishmanial hydrophilic acylated surface protein B is a representative of a novel class of proteins whose export is dependent on a 'non-classical' pathway involving N-myristoylation/palmitoylation.

Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane

The unusual Ca 2+-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62000, is activated by Ca 2+ ( K 0.5 = 15 μM) in the presence of Mg 2+ or Mn 2+, and can associate with 45Ca 2+. The activation by Ca 2+ could be partially replaced by Mn 2+, but not by Zn 2+ or Mg 2+. PfCPK preferentially phosphorylated casein and histone H1. The K m and V max for Mg 2+ ATP were 26 μM and 70 nmol min −1 mg −1, respectively, with casein as substrate; and 34 μM and 143 nmol min −1 mg −1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.