Discovery of Novel, Orally Bioavailable, Antileishmanial Compounds Using Phenotypic Screening

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 in vitro absorption, distribution, metabolism, excretion, and in vivo 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 Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo 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

Unexpected Biotransformation of the HDAC Inhibitor Vorinostat Yields Aniline-Containing Fungal Metabolites

The diversity of genetically encoded small molecules produced by filamentous fungi remains largely unexplored, which makes these fungi an attractive source for the discovery of new compounds. However, accessing their full chemical repertoire under common laboratory culture conditions is a challenge. Epigenetic manipulation of gene expression has become a well-established tool for overcoming this obstacle. Here, we report that perturbation of the endophytic ascomycete <i>Chalara</i> sp. 6661, producer of the isofusidienol class of antibiotics, with the HDAC inhibitor vorinostat resulted in the production of four new modified xanthones. The structures of chalanilines A (<b>1</b>) and B (<b>2</b>) and adenosine-coupled xanthones A (<b>3</b>) and B (<b>4</b>) were determined by extensive NMR spectroscopic analyses, and the bioactivities of <b>1</b>–<b>4</b> were tested in antibiotic and cytotoxicity assays. Incorporation studies with deuterium-labeled vorinostat indicate that the aniline moiety in chalalanine A is derived from vorinostat itself. Our study shows that <i>Chalara</i> sp. is able to metabolize the HDAC inhibitor vorinostat to release aniline. This is a rare report of fungal biotransformation of the popular epigenetic modifier vorinostat into aniline-containing polyketides

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

<i>In vivo</i> pharmacokinetic profiling of compounds 4 and 5.

<p>Murine pharmacokinetic studies for compound <b>4</b> and <b>5</b> delivered <i>per os</i> (25 mg/kg and 50 mg/kg respectively).</p

<i>In vivo</i> efficacy for controlling cutaneous lesion progression in the mouse.

<p>Mice (5 per cohort) were infected with <i>L</i>. <i>mexicana</i> promastigotes on day 0; by week 4 after infection, cutaneous lesions had grown to ~0.5 mm width. Compound <b>4</b> (triangles), compound <b>5</b> (squares), or miltefosine (diamonds) were delivered daily by oral gavage for 10 sequential days. One cohort of mice (circles) received vehicle alone. Measurements are plotted as the mean ± standard deviation.</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

Pharmacokinetic parameters for compound 4 and 5 based on oral administration in mice.

<p>Pharmacokinetic parameters for compound 4 and 5 based on oral administration in mice.</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

Screening flow chart and high throughput primary screen of promastigotes.

<p><b>a.</b> Schematic of the high-throughput screening workflow. SP refers to single point, or single concentration, and DR represents dose-response. The cutoff value of 65% inhibition in the first step was chosen based in ROC analysis, the cutoff of 2 μM and TI > 5 in step 2 was somewhat arbitrary but produced a reasonable number of hits for subsequent analysis, and the cutoff of 1 μM and TI > 10 for the final step is consistent with recommendations for lead identification for leishmaniasis [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006157#pntd.0006157.ref012" target="_blank">12</a>]. <b>b.</b> Scatter plot of primary screen data shown as normalized percent growth inhibition. Each dot represents the activity of one compound. Negative controls (DMSO treated) are in red, positive controls (pentamidine treated) are in green, test compounds are in blue (hits) or black (non-hits). The orange and purple horizontal lines indicate the 95% and 99% quantiles of activity respectively. <b>c.</b> Primary screen quality control: Z-prime value per assay plate screened, lower outlier bound (in purple), yellow lines separate screen runs. <b>d.</b> Receiver operating characteristic (ROC) curve (red); the combined ROC set AUC is 0.893. The blue line represents an AUC of 0.5 that is indicative of an assay with random results. TP is true positive, FP is false positive, FN is false negative, and TN is true negative.</p