Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.Chemistry and Chemical Biolog

Screening cascade for DOS screen (A) and potency comparison of hits from the DOS library between the novel axenic and intramacrophage assay (B).

<p>A: Screening cascade. Numbers of compounds that passed and failed the hit selection criteria are shown respectively in green and red. Numbers marked with an asterisk indicate that the selectivity window is unknown and may be ≥ 10 (no effect seen in HepG2 assay, activity in the cidal axenic assay pEC50<5.3). The cascade starts with screening 9,907 compounds in the novel axenic assay at a single concentration (nov. axenic SP). Identified hits are processed in potency format in both the novel axenic assay (axenic pot., for activity confirmation) and HepG2 assay (HepG2 pot., toxicity information against the human cell line HepG2). Active compounds that provide a ≥ 10-fold toxicity window are processed in the intramacrophage potency assay (intramac. pot.) for hit confirmation, resulting in 24 hits. B: Comparison of novel axenic and intramacrophage mean pEC₅₀ values for 141 compounds. The size of the markers is inversely related to the toxicity against the THP-1 cells (i.e. high toxicity–small symbol). Colour is by series (green: Ortho Azetidine Nitrile, blue: Azetidine Nitrile, orange: Povarov, red: SnAr 8-ortho, yellow: other). Data set represents three biological replicates for the novel axenic assay (with the exception of one compound with two replicates only) and at least two replicates for the intramacrophage assay.</p

Screening cascade.

<p>Novel axenic assay is primary, single concentration (SP) entry to the cascade, followed by confirmation, assessment of potency and selectivity using the novel axenic assay and a mammalian counterscreen assay (HepG2) in potency mode (pot.). Potent and selective hits are then profiled in the intramacrophage assay.</p

Library screen with novel axenic assay, comparison with intracellular results.

<p>15,667 compounds were screened in the novel axenic assay at 15μM. Comparison with the intracellular assay shows that 67 of the hits are also active in the intracellular assay whereas 71 of the hits are not. 280 novel axenic assay inactive compounds were hits in the intracellular assay, 170 of these compounds showed toxicity in the intracellular assay (in single point mode, at 50μM, or less than 3-fold selectivity window in potency assay) and are therefore considered false positives in the intracellular assay. Potencies were determined for 110 non-toxic compounds in the novel axenic and intracellular assays. 13 compounds were inactive in the intracellular assay and are added to the false positive count (13 + 170 = 183). 97 compounds had confirmed activity in the intracellular assay; 42 of these compounds showed activity in the novel axenic assay, while 54 did not. (INMAC = intracellular assay). Hit criteria as described in Table A in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004094#pntd.0004094.s001" target="_blank">S1 Text</a>.</p

Suitability of the <i>Leishmania donovani</i> assays for diversity screening.

<p>Suitability of the <i>Leishmania donovani</i> assays for diversity screening.</p

Limit-of-detection of historic and novel axenic assay and assay models.

<p>A: Detection limit of the historic axenic assay. Black line shows the linear regression (R² = 0.983, p < 0.0001). Dashed line shows average value of blanks. Blue line shows detection limit (3x standard deviation above the blanks value) for this assay. Vertical blue arrow indicates the number of cells at the limit of detection in this single experiment. Data is from a representative experiment of 4 with a minimum of 24 technical replicates. B: Detection limit of the novel axenic assay. Black line shows the linear regression (R² = 0.998, p < 0.0001). Dashed line shows average value of blanks. Red line shows detection limit for this assay (3x standard deviation above the blanks value). Red vertical arrow indicates the number of cells at the limit of detection in this single experiment. Data is from a representative experiment of 4 with a minimum of 24 technical replicates. Inset shows data from a similar range of cell densities as used in 1A. C and D: The coloured areas represent cell densities that can be detected with the respective assay formats (C: historic axenic assay, D: novel axenic assay). Red lines represent different cell-growth inhibition scenarios during the course of this assay: compounds that do not inhibit cell growth (marked “no Inhibition”), compounds that arrest cell growth, without killing cells (marked “static Inhibition”), and compounds that kill cells (marked with “cidal Inhibition”). The black double arrows represent the analysis window. Blue (historic assay) and red (novel assay) arrows show the fold difference between the starting density and detection limit.</p

Potency comparison of a series of structurally distinct compounds between the novel axenic and intramacrophage assay.

<p>In this scatterplot the mean pEC₅₀ values for control compounds representing a range of pharmacophores are compared between the novel and intramacrophage assay. Data set represents three or more replicates and the standard deviation is shown with error bars for each assay. Black line shows equipotency between both assays, blue line shows linear regression for compounds that are active in both assay (with outliers miltefosine and VL-2098 excluded, R² = 0.81).</p

Development and validation of a novel <i>Leishmania donovani</i> screening cascade for high-throughput screening using a novel axenic assay with high predictivity of Leishmanicidal intracellular activity

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery