Fig 1.
Limit-of-detection of historic and novel axenic assay and assay models.
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.
Table 1.
Comparison of the novel versus the historic axenic Leishmania donovani assay.
Fig 2.
Library screen with novel axenic assay, comparison with intracellular results.
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 S1 Text.
Fig 3.
Potency comparison of a series of structurally distinct compounds between the novel axenic and intramacrophage assay.
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).
Fig 4.
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.
Table 2.
Suitability of the Leishmania donovani assays for diversity screening.
Fig 5.
Screening cascade for DOS screen (A) and potency comparison of hits from the DOS library between the novel axenic and intramacrophage assay (B).
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.
Fig 6.
Compound series from the diversity-oriented synthesis library show stereochemistry-dependent selectivity as well as traditional SAR.
Compounds from two chemical scaffolds showed reproducible activity in both the novel axenic and intracellular assays. A. Analysis with the Broad's stereochemical structure activity (SSAR) viewer (see Fig E in S1 Text for an example) showed preferential activity with the RSS (C2, C3, C4) stereoisomer of Series 1 compounds. Activity of all available stereoisomers of BRD6650 indicated that the RSS isomer was the most active. B. Analogs of BRD6650 also show SAR around the original hit. Substitution on the phenyl group at the C-3 position of the azetidine ring was tolerated, as was minor variation on the urea functionality (see text for details). C. For compounds from Series 2, preferential activity was observed for the RSS stereoisomer in both thenovel axenic assay screen and the follow-up intracellular assay. D. SAR around both R1 and R2 was also observed for this series; the C-3 phenyl group could be varied with a number of ortho substituents, and variation on the urea was also tolerated.