Parasite Enzyme Research Articles

Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds

We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness.

Review of Kudoa-induced myoliquefaction of marine fish species in South Africa and other countries

Myoliquefaction of fish musculature results in customer quality complaints and in huge economic losses, especially with regard to Pacific hake (Merluccius productus), farm-reared Atlantic salmon (Salmo salar), South African pilchards (Sardinops ocellatus) and Cape snoek (Thyrsites atun). Myoliquefaction, or ‘jelly flesh’, is caused by proteolytic enzymes released by the marine myxosporean parasite, Kudoa thyrsites, after the death of the fish. Currently there are no fast methods of detection for this microscopic parasite, and because myoliquefaction is evident only after 38–56 h post-mortem, infected fish inevitably reach the processor and/or consumer. Several methods of detection have been investigated, but most of these methods are time-consuming and/or result in destruction of the fish, and are thus impractical for fishing vessels and fish processors. Limited research is available on possible means of destroying or inhibiting the post-mortem activity of the parasitic proteolytic enzyme. Means such as manipulating post-mortem pH and temperature control have been suggested; leaving opportunities for research into food technology applications such as cold-chain management and ionising radiation.

The mutual influence of proteins from Varroa destructor extracts and from honeybee haemolymph on their proteolytic activity — in vitro study

The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.

Homology Modeling and characterization of Phosphoenolpyruvate Carboxykinase (PEPCK) from Schistosoma japonicum

Phosphoenolpyruvate carboxykinase (PEPCK), a carboxylase enzyme is present in all living organisms. It catalyzes metal-nucleotide coupled reversible decarboxylation and phosphorylation between phosphoenolpyruvate (PEP) and oxaloacetate (OAA) depending on the system and the availability of the intermediates. In fungi, plants and in most bacteria, production of PEP from OAA by PEPCK is the key step during gluconeogenesis. In healthy human cytosolic PEPCK enzyme is present only during glucose starvation; cytosolic PEPCK rapidly disappears on the replenishment of glucose due to hormonal control of the transcription of the cytosolic PEPCK- gene. In some parasitic helminthes like Ascaris suum, nematodes such as Haemonchus contortus, PEPCK carry out the reverse reaction to produce OAA from PEP. In Trypanosoma cruzi and all species of the genus Leishmania, this enzyme is very active even in the presence of high level of carbohydrate. There is a significant functional difference between parasitic PEPCK and mammalian host PEPCK. These differences between the mammalian host and the parasite enzyme strongly support the belief that PEPCK should be further investigated as a possible target for selective chemotherapeutic agents. The experimental 3D structure of PEPCK of Schistosoma japonicum is not available in protein data bank. Therefore, based on the knowledge of the best template (3DT7), model of 3D structure of Schistosoma japonicum PEPCK was prepared using Modeller v9.10 software and processed in to energy minimization, Ramachandran plot analysis, quality assessment and characterization.

Expression profiles of peroxiredoxins in liver stage of the rodent malaria parasite Plasmodium berghei

mRNA and protein expression profiles for three peroxiredoxins (TPx-1, TPx-2 and 1-Cys Prx) of liver stage Plasmodium berghei were examined through quantitative reverse transcription-PCR (RT-PCR) and indirect immunofluorescence microscopy assay (IFA). RT-PCR experiments revealed that mRNA expression for the TPx-1 was detected shortly after the sporozoite infection and kept expressed until the schizont stage. In contrast, the mRNA expression for 1-Cys Prx had begun increasing when the parasite developed into the schizont stage. Using the IFA, TPx-1 and 1-Cys Prx were detected in the cytosol. This finding suggested the developmental stage-specific expression of the cytosolic enzymes in the liver stage parasite. On the other hand, the mRNA expression for TPx-2 had begun increasing at the trophozoite stage and peaked at the schizont stage. In the IFA, TPx-2 was found localized in the mitochondria. The increase of TPx-2 might be explained by the exponential development of the parasite during the schizont stage requiring ATP production which may induce reactive oxygen species (ROS) in the mitochondria.

Homology modeling and docking studies of phosphoenolpyruvate carboxykinase in Schistosoma mansoni

Phosphoenolpyruvate carboxykinase (PEPCK) is an important catalytic enzyme in helminth parasites that catalyses a reverse reaction forming oxaloacetate from phosphoenolpyruvate. Because of its functional significance, present work was undertaken to construct and validate the 3D structure of PEPCK in Schistosoma mansoni. The 3D model protein was generated based on the known crystal structure of rat cytosolic PEPCK as template using Modeller 9v10 software. The resulting models were assessed by Procheck, Anolea, ProSA-web, and Errat. We have also identified all the pockets and voids on the best model protein to provide a detailed delineation of all atoms participating in their formation using CASTp server and to dock with best fitted ligand (guanosine diphosphate, GDP) using AutoDock 4.2. The model protein showed reliable structure with Ramachandran plot: 90.2 % core region, 9.1 % allow, 0.8 % generously allowed and 0.0 % disallowed region amino acid distributions. GDP was found to be docked in its active site, which formed a single H-bond with ASN 524 residue of model protein. Characterisation of PEPCK model protein has also been done to know its various biochemical natures. Therefore, the model structure could prove useful in further functional characterization and may be used to target and design drugs against S. mansoni.

Isolation, cloning and sequence analysis of lactate dehydrogenase gene from Theileria annulata may lead to design of new antitheilerial drugs

Theileriosis is a serious animal disease that is transmitted by ticks. Theileria species are tick-borne obligate intracellular protozoan parasites that cause severe and mild infections in their hosts. Two of them, Theileria annulata and Theileria parva, cause lymphoproliferative disease with high mortality and morbidity in cattle commonly known as tropical theileriosis and East Coast fever, respectively. Currently available antiparasitic drugs are effective in animals but animals may remain carriers and treatment is most effective in the early stages of the disease. The isolation, cloning and analysis of lactate dehydrogenase from T. annulata was the goal of the present study with the ultimate aim of designing new antiparasitic drugs that will hopefully have a wider mode of action in animals. Blood samples were taken from a four year-old Brown Swiss cow showing signs of acute tropical theileriosis and genomic DNA was extracted following the confirmation of the clinical diagnosis. For the first time, in this study, the lactate dehydrogenase sequence was isolated from from a Theileria species. Following extraction from genomic DNA by PCR the sequence was cloned into the vector pGEM-T easy. Sequencing of the whole gene from both directions indicated that the open reading frame was interrupted by two introns. Several single nucleotide exchanges, deletions and insertions were also observed in the T. annulata lactate dehydrogenase sequence compared to the host. The most remarkable difference between the parasite and host enzyme is a five residue insertion in the active site loop region that might be an attractive target for inhibitors of the enzyme. This study opens a new route to further kinetic and structural studies towards the development of novel inhibitors of T. annulata lactate dehydrogenase.  

Phthalazine Derivatives Containing Imidazole Rings Behave as Fe-SOD Inhibitors and Show Remarkable Anti-T. cruzi Activity in Immunodeficient-Mouse Mode of Infection

A series of new phthalazine derivatives 1-4 containing imidazole rings were prepared. The monoalkylamino substituted derivatives 2 and 4 were more active in vitro against T. cruzi and less toxic against Vero cells than both their disubstituted analogues and the reference drug benznidazole. Compounds 2 and 4 highly inhibited the antioxidant parasite enzyme Fe-SOD, and molecular modeling suggested that they interact with the H-bonding system of the iron atom moiety. In vivo tests on the acute phase of Chagas disease gave parasitemia inhibition values twice those of benznidazole, and a remarkable decrease in the reactivation of parasitemia was found in the chronic phase for immunodeficient mice. Glucose metabolism studies showed that compounds 1-4 did not affect the succinate pathway but originated important changes in the excretion of pyruvate metabolites. The morphological alterations found in epimastigotes treated with 1-4 confirmed extensive cytoplasm damage and a high mortality rate of parasites.

Thioredoxin Glutathione Reductase-Dependent Redox Networks in Platyhelminth Parasites

Platyhelminth parasites cause chronic infections that are a major cause of disability, mortality, and economic losses in developing countries. Maintaining redox homeostasis is a major adaptive problem faced by parasites and its disruption can shift the biochemical balance toward the host. Platyhelminth parasites possess a streamlined thiol-based redox system in which a single enzyme, thioredoxin glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase (TR) domains, supplies electrons to oxidized glutathione (GSSG) and thioredoxin (Trx). TGR has been validated as a drug target for schistosomiasis. In addition to glutathione (GSH) and Trx reduction, TGR supports GSH-independent deglutathionylation conferring an additional advantage to the TGR redox array. Biochemical and structural studies have shown that the TR activity does not require the Grx domain, while the glutathione reductase and deglutathionylase activities depend on the Grx domain, which receives electrons from the TR domains. The search for TGR inhibitors has identified promising drug leads, notably oxadiazole N-oxides. A conspicuous feature of platyhelminth TGRs is that their Grx-dependent activities are temporarily inhibited at high GSSG concentrations. The mechanism underlying the phenomenon and its biological relevance are not completely understood. The functional diversity of Trxs and Grxs encoded in platyhelminth genomes remains to be further assessed to thoroughly understand the TGR-dependent redox network. Optimization of TGR inhibitors and identification of compounds targeting other parasite redox enzymes are good options to clinically develop relevant drugs for these neglected, but important diseases.

A Bioinformatics Approach for Homology Modeling Binding Site Identification of Triosephosphate Isomerase from Plasmodium falciparum 3D7

Malaria is a major public health concern, and malarial parasites have developed resistance against the commonly available drugs. So now a days it is a major concern to find out a new target for drug therapy. Plasmodium falciparum 3D7, one of the strains of plasmodium species also lacks in a functional tricarboxylic acid cycle and solely dependent on glycolysis for its energy supply like other plasmodium species. Although enzymes of malarial parasite have been considered as potential antimalarial drug targets, a little is known about their structural biology. The tertiary structure of triose phosphate isomerase of P. falciparum 3D7 was determined by means of homology modeling through multiple alignment followed by intensive optimization and validation. The modeling was done by Swiss-Model Workspace. The obtained model was verified with the structure validation programs such as, PROCHECK, Verify3D, and QMEAN for reliability. The verify3D value of 0.69 indicates that the environment profile of the model is good. A self-optimized prediction method with alignment or SOPMA is employed for calculation of the secondary structural features of triose phosphate isomerase. The secondary structure indicates that the predicted 3D structure of triosephosphate isomerase of P. falciparum 3D7 contains 48.37% α-helix, 29.27% random coil, and 16.67% extended strand. Active site determination through CASTp suggests that this protein can be utilized as a potential drug target. However, these will further be tested by wet lab studies for a targeted vaccine design against P. falciparum 3D7.

Structure- and Ligand-Based Structure-Activity Relationships for a Series of Inhibitors of Aldolase

Aldolase has emerged as a promising molecular target for the treatment of human African trypanosomiasis. Over the last years, due to the increasing number of patients infected with Trypanosoma brucei, there is an urgent need for new drugs to treat this neglected disease. In the present study, two-dimensional fragment-based quantitative-structure activity relationship (QSAR) models were generated for a series of inhibitors of aldolase. Through the application of leave-one-out and leave-many-out cross-validation procedures, significant correlation coefficients were obtained (r²=0.98 and q²=0.77) as an indication of the statistical internal and external consistency of the models. The best model was employed to predict pKi values for a series of test set compounds, and the predicted values were in good agreement with the experimental results, showing the power of the model for untested compounds. Moreover, structure-based molecular modeling studies were performed to investigate the binding mode of the inhibitors in the active site of the parasitic target enzyme. The structural and QSAR results provided useful molecular information for the design of new aldolase inhibitors within this structural class.

Targeting the Substrate Preference of a Type I Nitroreductase To Develop Antitrypanosomal Quinone-Based Prodrugs

Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, having K(cat)/K(m) values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound.

Purification and characterization of phosphoenolpyruvate carboxykinase fromRaillietina echinobothrida, a cestode parasite of the domestic fowl

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is an essential regulatory enzyme of glycolysis in helminths in contrast to its role in gluconeogenesis in their host. Previously we have reported that phytochemicals from Flemingia vestita (Family: Fabaceae), genistein in particular, have vermifugal action and are known to affect carbohydrate metabolism in the cestode, Raillietina echinobothrida. In order to determine the functional differences of PEPCK from the parasite and its avian host (Gallus domesticus), we purified the parasite enzyme apparently to homogeneity, and characterized it. The native PEPCK is a monomer with a subunit molecular weight of 65 kDa. The purified enzyme displayed standard Michaelis-Menten kinetics with Km value of 42·52 μM for its substrate PEP. The Ki for the competitive inhibitors GTP, GMP, ITP and IMP for the carboxylation reaction were determined and discussed. In order to identify putative modulators from plant sources, phytochemicals from F. vestita and Stephania glabra were tested on the purified PEPCK, which resulted in alteration of its activity. From our results, we hypothesize that PEPCK may be a potential target site for anthelmintic action.

The human Aurora kinase inhibitor danusertib is a lead compound for anti-trypanosomal drug discovery via target repurposing

New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet drug discovery efforts are not often focused on this area due to cost. Target repurposing, achieved by the matching of essential parasite enzymes to those human enzymes that have been successfully inhibited by small molecule drugs, provides an attractive means by which new drug optimization programs can be pragmatically initiated. In this report we describe our results in repurposing an established class of human Aurora kinase inhibitors, typified by danusertib (1), which we have observed to be an inhibitor of trypanosomal Aurora kinase 1 (TbAUK1) and effective in parasite killing in vitro. Informed by homology modeling and docking, a series of analogs of 1 were prepared that explored the scope of the chemotype and provided a nearly 25-fold improvement in cellular selectivity for parasite cells over human cells.

Urea-Based Inhibitors of Trypanosoma brucei Methionyl-tRNA Synthetase: Selectivity and in Vivo Characterization

Urea-based methionyl-tRNA synthetase inhibitors were designed, synthesized, and evaluated for their potential toward treating human African trypanosomiasis (HAT). With the aid of a homology model and a structure-activity-relationship approach, low nM inhibitors were discovered that show high selectivity toward the parasite enzyme over the closest human homologue. These compounds inhibit parasite growth with EC(50) values as low as 0.15 μM while having low toxicity to mammalian cells. Two compounds (2 and 26) showed excellent membrane permeation in the MDR1-MDCKII model and encouraging oral pharmacokinetic properties in mice. Compound 2 was confirmed to enter the CNS in mice. Compound 26 had modest suppressive activity against Trpanosoma brucei rhodesiense in the mouse model, suggesting that more potent analogues or compounds with higher exposures need to be developed. The urea-based inhibitors are thus a promising starting point for further optimization toward the discovery of orally available and CNS active drugs to treat HAT.

The Obligate Intracellular Parasite Toxoplasma gondii Secretes a Soluble Phosphatidylserine Decarboxylase

Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 μM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ∼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ∼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives

Chromatin structure plays a vital role in epigenetic regulation of protozoan parasite gene expression. Epigenetic gene regulation impacts upon parasite virulence, differentiation and cell-cycle control. Recent work in many laboratories has elucidated the functions of proteins that regulate parasite gene expression by chemical modification of constituent nucleosomes. A major focus of investigation has been the characterization of post-translational modifications (PTMs) of histones and the identification of the enzymes responsible. Despite conserved features and specificity common to all eukaryotes, parasite enzymes involved in chromatin modification have unique functions that regulate unique aspects of parasite biology.

A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani.

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N €-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ∼42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ∼40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.

Development of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) inhibitors with potent anti-toxoplasma activity.

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated in part by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.