Temperate Zone Plant Natural Products-A Novel Resource for Activity against Tropical Parasitic Diseases

Abstract

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite's growth by at least 50% at 1-2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC₅₀ < 1 μM and a selectivity index of >10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

Keywords: African trypanosomiasis; Surra; drug discovery; leishmaniasis; malaria; natural products; neglected tropical diseases; temperate zone.

Figure 1

Heatmap of PhytoQuest Phytopure temperate natural product screen against protozoan parasites. In total, 634 compounds were screened against L. mexicana (L.m) axenic amastigotes, T. brucei (T.b) bloodstream form and P. falciparum (P.f) intraerythrocytic trophozoites at 2 μM and T. evansi (T.e) bloodstream form parasites at 1 μM. Survival (reported as % of DMSO control) of each parasite cell line is represented as a spectrum from black to light grey (see scale to right), with the black cells reporting hits taken forward in this study. Blank cells indicate where no data were collected for that compound.

Figure 2

Structures of compounds which had <1 μM activity against the relevant parasite cell line, with an SI of ≥10.

Figure 3

Comparison of potency and cytotoxicity of hit compounds. Comparison of the EC₅₀ to the selectivity index (SI) of hit compounds in (a) L. mexicana, (b) T. brucei, (c) T. evansi and (d) P. falciparum. The SI reports the CC₅₀ of the compound in HepG2 cell line divided by the EC₅₀ of the reported parasite line. The grey dotted lines indicate the preferred potency and SI (≥10) thresholds, with hits taken forward from the top left quadrant only. The green box highlights the potential lead compounds that fall within these thresholds.

Figure 4

Sterol activity against two Leishmania species and an activated monocyte cell line. (a) Mean EC₅₀ (μM) of the reported sterol compounds against L. donovani and L. mexicana axenic amastigotes. Error bars represent upper limits of the 95% confidence interval. (b) EC₅₀ values (left axis) and selectivity index (SI, right axis) of the same sterols against an activated monocyte cell line (THP-1). Error bars represent upper limits of the 95% confidence interval for the EC₅₀ and the SI represents the selectivity of these compounds against L. mexicana compared to THP-1.

Figure 5

Potency and rate of kill of bisobolane sesquiterpene glycoside hits against Plasmodium falciparum. (a) Comparison of EC₅₀ values of bisobolane sesquiterpene glycoside hits against intraerythrocytic P. falciparum Dd2^Luc and 3D7 cell lines. Time course of rate of kill for (b) control antimalarial compounds atovaquone (ATQ) representing a slow rate of kill and dihydroartemisinin (DHA) representing a fast rate of kill. against (c) the bisobolane sesquiterpene glycoside hits. Time course data (3, 6 and 48 h) show the normalized bioluminescence signal (compared to an untreated control at the same timepoint) following exposure to a fold-EC₅₀ exposure of the indicated compound. Error bars represent upper and lower limits of the StDev (n = 9). See Table S4 for concentrations used.

Figure 6

Comparison between wildtype and 700022-resistant (r22) L. mexicana cell lines. Log concentration versus normalised (compared to untreated control) growth curves for wildtype (WT) and corresponding 700022-selected (r22) L. mexicana (a) promastigotes and (b) axenic amastigotes. Error bars represent upper and lower limits of the StDev (n = 9). (c) Average EC₅₀ of WT and r22 after 8 weeks of 700022 selection pressure against 700022 and structurally related sterol hits against L. mexicana. The resistance index (EC₅₀ in r22/EC₅₀ in WT) are shown for each compound. (d) Scanning electron micrographs of WT and r22 promastigotes illustrating the shortened flagellum in r22 parasites. (e) Analysis of flagellum length in WT and r22 promastigotes (n = 287 of each strain) reported using a box and whisker plot (boxes represents the 25–75th percent distribution with the mean as a horizontal line, whiskers represent the distribution of all values) with a statistically significant difference (Mann–Whitney U test, p-value < 2.2 × 10⁻¹⁶). (f) Mean EC₅₀ of a panel of indicated compounds against WT and r22 after 28 weeks of 700022-selection pressure. Error bars represent upper and lower limits of the StDev (n = 9). Miltefosine, Milt (concentration range used against WT and r22 was 0.16–20 μM and 1.6–50 μM, respectively); Pentamidine, Pent (concentration range used against both WT and r22 was 0.78–50 μM); Amphotericin B, Amp B (concentration range used against both WT and r22 was 0.078–1.3 μM). The resistance index (EC₅₀ in r22/EC₅₀ in WT) are shown for each compound.

Figure 7

Comparison of the PhytoQuest Phytopure library hits across the four parasite species investigated. All compounds shown have an EC₅₀ ≤ 1 μM in the respective species reported here. Compounds reported in black indicates a high selectivity (SI > 20) and/or a low cytotoxicity in HepG2 (CC₅₀ > 20 μM). Compounds in grey indicates a low selectivity (SI ≤ 20) and/or toxicity in HepG2 (CC₅₀ ≤ 20 μM).