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
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 (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
A comparison of different CDPK inhibitors’ cytotoxicity to human cells.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM is shown for CDPK4 inhibitors in scaffolds D and G (top) and for CDPK1 inhibitors in scaffolds F and H (bottom).</p
Assessment of compound promiscuity with human kinases.
<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
Summary of kinobead competition assays (results reflect two independent experiments).
<p>Summary of kinobead competition assays (results reflect two independent experiments).</p
Human cytotoxicity of inhibitors of 1, 2, or 3 of the <i>P</i>. <i>falciparum</i> kinases studied.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>].</p
Clustering of <i>P</i>. <i>falciparum</i> protein kinase hits into chemical scaffolds.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>]. For some scaffolds, target counts exceed the number of hits because some compounds hit more than one target.</p
Venn diagrams showing overlapping and non-overlapping targets of hit compounds.
<p>225 compounds had IC<sub>50</sub>’s below 1 μM against at least one kinase (left); a subset of 72 compounds had IC<sub>50</sub>’s below 100 nM against at least one kinase (right).</p