9 research outputs found

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

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    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

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    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

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    <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 (IC<sub>50</sub> < 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

    Assessment of compound promiscuity with human kinases.

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    <p>Kinobeads were incubated with K562 cell extract either in the presence of vehicle (DMSO) or TCAMS compound, respectively (20 μM-0.03 μM). Protein kinases captured by the beads (140–150 kinases per experiment) were quantified following tryptic digestion, isobaric peptide tagging, and LC-MS/MS analysis. Kinases were identified as potential targets by virtue of their reduced capture in the presence of excess TCAMS compounds. Apparent dissociation constants (K<sub>d</sub>’s) were calculated from the extent to which capture of each kinase was reduced at each compound concentration. K<sub>d</sub> values from duplicate experiments generally agreed with each other quite well (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.s002" target="_blank">S2 Fig</a>). Colored bands indicate kinase-ligand complexes with apparent pK<sub>d</sub>’s of ≥6, with darker shades denoting higher pK<sub>d</sub>’s. Kinases that did not have an apparent pK<sub>d</sub> of ≥6 for any of the compounds are not represented; only names of every other targeted kinase are shown due to space limitations. These results are summarized numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.t003" target="_blank">Table 3</a>.</p
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