Inhibition of uptake of 100 μM [³H] D-glucose (Glc, black bars), 12.5 μM [³H] hypoxanthine (Hyp, stippled bars), 1 μM [³H] uridine (Urd, white bars), and 100 μM [³H] L-proline (Pro, square-hatched bars) by Compounds 1 (A), 7 (B), and 13 (C).

<p>Concentrations of each inhibitor are shown on the x-axis; DMSO represents the control using the DMSO vehicle without any inhibitor. Proline uptake was not measured at 0.1 or 1.0 μM concentrations of inhibitors, hence the cross-hatched bar representing proline uptake does not appear for these concentrations.</p

Analogs of Compound 1 were tested for inhibition of uptake of 100 μM [³H] D-glucose by PfHT and GLUT1, as in Fig 2.

<p>NI indicates no inhibition, and NA indicates does not apply. Other abbreviations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123598#pone.0123598.g002" target="_blank">Fig 2</a>.</p

Dose-response curves for inhibition of glucose uptake by PfHT and GLUT1.

<p>Compounds 1 (A), 7 (B), and 13 (C) were applied over a range from 10^-9–10^-5 M to Δ<i>lmxgt1-3</i> null mutants expressing either PfHT (filled circles) or GLUT1 (open circles), and uptake of 100 μM [³H] D-glucose was measured in a 1 min uptake assay. Results are plotted as the mean and standard deviation (error bars) from 3 replicate uptake determinations. Data were fitted to a sigmoidal inhibition curve.</p

<i>In vitro</i> absorption-distribution-metabolism-excretion (ADME) properties of Compounds 1, 10, 12, 13 and control compounds.

<p>Aqueous solubility, permeation and retention in artificial membrane, metabolic stability against mouse liver microsomes (t_1/2 or half-life), and intrinsic clearance (Clint) were determined as described in Materials and Methods. Values were also determined for albendazole, carbamazepine, ranitidine, and verapamil as control drugs. ND indicates not done.</p><p><i>In vitro</i> absorption-distribution-metabolism-excretion (ADME) properties of Compounds 1, 10, 12, 13 and control compounds.</p

Flow chart for screen of TCAMS library.

<p>The TCAMS library of 13,533 compounds with demonstrated growth inhibitory activity against intraerythrocytic <i>Plasmodium falciparum</i> parasites was screened by sequential criteria. The steps included: 1) proliferation inhibitory screen (>65% inhibition at 3 μM concentration or >20% differential inhibition among the three strains) of PfHT, LmxGT2, and GLUT1 reporter strains to produce 401 primary hits; 2) 96-well plate assays for compounds (20–30 μM) that inhibited uptake of 200 μM [³H] D-glucose by ≥90%; 3) individual uptake assays for compounds (10 μM) that inhibited glucose uptake by ≥50%; 4) individual uptake assays for compounds (10 μM) that inhibited uptake of 100 μM [³H] L-proline by ≤10%; 5) dose-response curves for compounds that selectively inhibited uptake of glucose through PfHT versus GLUT1 (1 compound plus 1 additional hit that emerged from analysis of analogs). Numbers in parentheses represent the number of positive hits obtained after each sequential step.</p

Compounds from the TCAMS (1–6) and Malaria Box (7–9) libraries that were selective inhibitors of glucose, but not proline, transport.

<p>Dose-response curves for inhibition of uptake of 100 μM [³H] D-glucose by each compound were performed for PfHT and GLUT1, and the IC₅₀ values are tabulated. SI_glucose indicates Specificity Index for glucose uptake and is IC₅₀ for GLUT1/IC₅₀ for PfHT. The reported EC₅₀ values (PubChem web site, <a href="http://pubchem.ncbi.nlm.nih.gov/" target="_blank">http://pubchem.ncbi.nlm.nih.gov/</a>) for inhibition of growth of <i>P</i>. <i>falciparum</i> strain 3D7 intraerythrocytic forms by each compound are also tabulated.</p

Inhibition of glucose uptake by PfHT by Compounds 1, 7, and 13.

<p>Substrate saturation curves (A, B, C) were performed for PfHT in the presence of various concentrations of Compounds 1, 7, and 13, respectively. Data represent the mean and standard deviation of 3 replicate uptake assays. Data were fitted to the Michaelis-Menten equation by non-linear regression.</p

Top hits for inhibition of growth of <i>L</i>. <i>mexicana</i> amastigotes.

<p>EC₅₀ values represent the mean ± standard deviation for n = 2 and were calculated from dose-response curves against intracellular amastigotes of <i>L</i>. <i>mexicana</i> and <i>L</i>. <i>donovani</i>, the bloodstream form of <i>T</i>. <i>brucei</i>, and the host macrophage J774A.1. TI was calculated as EC₅₀ J774A.1/EC₅₀ <i>L</i>. <i>mexicana</i> amastigotes. None of the nine compounds inhibited proliferation of normal fibroblasts (BJ cells) at 20 μM. *Exact compound has been previously reported as exhibiting antileishmanial activity. For J774.A5 macrophages, compounds were tested up to 10 μM concentration, and those that showed no inhibition of growth were reported to have an EC₅₀ value of >10μM.</p

Plasma protein binding for compounds 4, 5, and the control drug propranolol.

<p>Plasma protein binding for compounds 4, 5, and the control drug propranolol.</p

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening

<div><p>Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for <i>in vitro</i> absorption, distribution, metabolism, excretion, and <i>in vivo</i> pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC₅₀) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to <i>Leishmania</i> species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant <i>in vivo</i> efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.</p></div