Novel inhibitors of <i>B. malayi</i> iPGAM.

<p>N.D. = Not determined.</p

Dose-response curve of the confirmed hit NCDS-1. NCDS-1 and control inhibitor tannic acid were tested at concentrations from 0.78 µM to 400 µM.

<p>Note that tannic acid may inhibit iPGAM via nonspecific aggregation <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002628#pntd.0002628-Pohjala1" target="_blank">[40]</a>; this would render it unsuitable as a chemical probe, but does not prevent its use as a control inhibitor for comparison of inhibited and uninhibited samples. Nonlinear regression and IC₅₀ value were analyzed with GraphPad Prism software (GraphPad, San Diego, CA, USA). Data are expressed as means ± SEM of at least 3 independent experiments.</p

Robustness of the HTS assays performed at Genzyme (top) and NCDS (bottom).

<p>TOP: scattergram of percent inhibition data from all plates in the Genzyme HTS. Green: 100% inhibition control; red: 50% inhibition control; black: 0% inhibition control; blue: data for test compounds screened at 10 µM. BOTTOM: scattergram of NCDS data representing uninhibited iPGAM (2% DMSO, shown in red) and inhibited iPGAM (200 µM tannic acid, shown in black). 1120 wells for each condition, spread over seventy 384-well plates, are shown. Dashed lines indicate means ±3× standard deviation (SD).</p

Results of NCDS's HTS of 160,000 compounds against the <i>B. malayi</i> iPGAM.

<p>The distribution of compounds is shown as a function of percent inhibition of the enzyme.</p

A summary of the screening workflow at the two HTS sites, along with the number of compounds passing through each step.

<p>See <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002628#s2" target="_blank">Methods</a> for details.</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

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

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

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