TEM analysis of <i>N. caninum</i>-infected HFF cultures treated for 3 days, with 2.5 μM of inhibitor 1294 added at 2 h post-infection.

<p>A and B show micrographs of more or less densely packed parasitophorous vacuoles containing numerous tachyzoites without obvious alterations. C and D show a representative example of a vacuole delineated by a parasitophorous vacuole membrane (pvm) containing parasites displaying a large cytoplasmic mass and aberrant overall morphology. The boxed area in C is enlarged in D, exhibiting the presence of the pvm and rhoptry-like organelles (rho). In many instances, as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092929#pone-0092929-g005" target="_blank">Figure 5E and F</a>, parasitophorous vacuoles contain several parasites exhibiting clear signs of metabolic impairment such as cytoplasmic vacuolization (vac) and electron-dense inclusions (inc). (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092929#pone-0092929-g005" target="_blank">Figure 5E, F</a>). Note that in C–F the matrix has lost its characteristic tubular network structure and is now formed of either granular material or possibly membranous material (C, D), or is even largely missing (E. F). Bars in A = 1 μm; B = 0.9 μm; C = 0.75 μm, D = 0.35 μm; E = 0.3 μm; F = 0.3 μm.</p

TEM analysis of <i>N. caninum-</i>infected HFF cultures in the presence of 2.5 μM of inhibitor 1294 for 5 and 9 days.

<p><b>A–C</b> illustrate two distinct outcomes of treatment with 2.5(PV1 and PV2) in a HFF cell. In <b>B</b>, the higher magnification view of PV1 reveals non-viable remnants of tachyzoite (tach), now largely embedded in a solid matrix (*), and as indicated by arrows, the parasitophorous vacuole membrane has been replaced by a rather amorphous transition zone between PV1 and the host cell cytoplasm. In <b>C</b>, PV2 displays a complex of still viable and presumably proliferating, but non-separating, parasites forming a large multi-nucleated mass, with clearly discernable micronemes (mic), rhoptries (rho), dense granules (dg), and smaller nuclei (nuc). Note the large nuclear mass (NUC) in the center with a large nucleolus marked by the bold arrow. In <b>D</b>, and at a higher magnification in E, a similar multinucleated complex is shown after 9 days of treatment with compound 1294, also displaying a large nuclear mass (NUC), as well as rhoptries, micronemes, dense granules, and an intact parasitophorous vacuole membrane. In <b>F</b>, a non-viable complex with tachyzoite remnants (tach) is shown after 9 days of treatment. Note the difference in electron density of the matrix of PVs containing non-viable parasites (B, F) compared to viable multinucleated complexes (C, D, E). Bars in A = 5 μm; B = 0.8 μm; C = 0.9 μm; D = 2 μm; E = 0.7 μm; F = 0.9 μm.</p

TEM analysis of compound 1294 treated and untreated <i>N. caninum</i>-infected HFF cultures at 3 days post-infection.

<p><b>A</b> shows a HFF cell with inhibitor 1294 (2. 5 μM) added at the time point of infection, <b>B</b> represents a higher magnification view of A. Note the absence of parasites, and the fact that compound 1294 does not induce any obvious ultrastructural alterations within the host cell. <b>C</b> and <b>D</b> show <i>N. caninum</i>-infected HFF cultured in the absence of the inhibitor at 3 days post-invasion. Numerous tachyzoites are located within parasitophorous vacuoles, surrounded by a parasitophorous vacuole membrane (PVM), and embedded in a matrix of a tubular membrane network (PVTN). Apical parts of tachyzoites are characterized by the conoid (con), micronemes (mic), and rhoptries (rho); dg  =  dense granules, nuc  =  tachyzoite nucleus, hcnuc  =  host cell nucleus. The two arrows in C point towards proliferating tachyzoites undergoing endodyogeny. Bars in A = 5 μm; B = 1 μm; C = 0.58 μm; D = 0.28 μm.</p

Compound 1294 treatment reduces cerebral parasite load in <i>N. caninum</i> infected mice.

<p>Balb/c mice (8 animals per experimental group), infected intraperitoneally with 5×10⁶<i>N. caninum</i>-β-gal tachyzoites, were treated with compound 1294 (50 mg/kg in 100 μL honey suspension). Group A (placebo) received honey for 5 days, group B received 1294 treatment starting at 3 days post infection and continuing daily for 5 days; group C received the same as group B but for 10 days. None of the mice exhibited clinical signs at 28 days post-infection, after which the cerebral parasite load was assessed by quantitative real time PCR.</p

a: Binding pose of pyrazolopyrimidine analogues in complexes <i>Nc</i>CDPK1.

<p>The crystallographically observed binding poses of inhibitors RM-1-132 (15n) and 1294 (15o). The bulky naphthalene substituent is accommodated by the presence of a small (glycine) gatekeeper residue. The piperidine substituents extend into the ribose binding pocket of the kinase active site. The pose and interactions of the PP scaffold with the protein backbone are highly consistent for members of this series of BKI compounds ²⁴. b: Conformational states of <i>Nc</i>CDPK1 in solution using SAXS. The SAXS profiles measured for the low calcium (green) and high calcium (black) states of <i>NcCDPK1</i> in solution. The inset shows the corresponding low resolution 3D models generated from the respective profiles, superimposed onto crystal structures of the low calcium state of <i>NcCDPK1</i> (this work; PDB 4m97) and a high calcium state of <i>TgCDPK1</i> (PDB 4hx4). The crystallographic and SAXS models agree better for the low calcium state (left) than for the high calcium state (right). The model for the high calcium state derived from the observed SAXS profile (gray solid) is more extended, i.e. has a larger radius of gyration, than the crystal structure.</p

<i>In vitro</i> activity of BKIs against <i>Nc</i>CDPK1 activity and <i>N</i>. <i>caninum</i> proliferation.

<p>⁺IC₅₀ represents the concentration that inhibits protein kinase activity by 50%. *EC₅₀ represents the concentration that inhibits cell growth by 50%.</p

Compound 1294 interferes in host cell invasion.

<p>(A) Measurement of β-galactosidase activities in HFF during the invasion process. HFF monolayers were infected by incubating them with 2×10⁴<i>N. caninum (b-Gal)</i> tachyzoites for 2 hours. Prior to this 2 hour invasion phase (timepoint 0′), tachyzoites were suspended either in DMSO (DMSO 0′), in 5 μM compound 1266 (1266 0′) or in 5 μM 1294 (1294 0′). In additional assays, compound 1294 was added at 60 min after initiation of invasion (1294 60′), 90 min (1294 90′) or 115 min (1294 115′) prior to removal of the drug by washing. Cultures were then incubated for an additional 60 min before β-galactosidase activity was measured. There was a significant decrease in β-galactosidase activity (t-test; p<0.001) when compound 1294 was added at the beginning of infection (1294 0′), but not when the compound was added at later timepoints, and no changes occurred when 1294 was added after the infection phase (1294 60′ post-infection. (B–D) Scanning electron micrographs of HFF infected with <i>N. caninum</i> for 3 days. Cultures were maintained in the absence of 1294 (B), in the presence of 2.5 μM 1294 added 2 h post-invasion (C), and in the presence of 2.5 μM 1294 added already at the time point of invasion (D). Cultures were processed for SEM analysis after 3 days. Note the presence of parasitophorous vacuoles in B and C (arrows), and respective absence in D.</p

Development of <i>Toxoplasma gondii</i> Calcium-Dependent Protein Kinase 1 (<i>Tg</i>CDPK1) Inhibitors with Potent Anti-<i>Toxoplasma</i> Activity

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite <i>Toxoplasma gondii</i>. Proliferation of <i>T. gondii</i> 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 <i>Tg</i>CDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in <i>Tg</i>CDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent <i>Tg</i>CDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics

Multiple Determinants for Selective Inhibition of Apicomplexan Calcium-Dependent Protein Kinase CDPK1

Diseases caused by the apicomplexan protozoans Toxoplasma gondii and Cryptosporidium parvum are a major health concern. The life cycle of these parasites is regulated by a family of calcium-dependent protein kinases (CDPKs) that have no direct homologues in the human host. Fortuitously, CDPK1 from both parasites contains a rare glycine gatekeeper residue adjacent to the ATP-binding pocket. This has allowed creation of a series of C3-substituted pyrazolopyrimidine compounds that are potent inhibitors selective for CDPK1 over a panel of human kinases. Here we demonstrate that selectivity is further enhanced by modification of the scaffold at the C1 position. The explanation for this unexpected result is provided by crystal structures of the inhibitors bound to CDPK1 and the human kinase c-SRC. Furthermore, the insight gained from these studies was applied to transform an alternative ATP-competitive scaffold lacking potency and selectivity for CDPK1 into a low nanomolar inhibitor of this enzyme with no activity against SRC

Biochemical Screening of Five Protein Kinases from <i>Plasmodium falciparum</i> against 14,000 Cell-Active Compounds

<div><p>In 2010 the identities of thousands of anti-<i>Plasmodium</i> compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of <i>Plasmodium falciparum</i> calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple <i>Plasmodium</i> kinase targets without harming human cells is challenging but feasible.</p></div