Modified Hederagenin Derivatives Demonstrate Ex Vivo Anthelmintic Activity against Fasciola hepatica

Abstract

Infection with Fasciola hepatica (liver fluke) causes fasciolosis (or fascioliasis) and poses a considerable economic as well as welfare burden to both the agricultural and animal health sectors. Here, we explore the ex vivo anthelmintic potential of synthetic derivatives of hederagenin, isolated in bulk from Hedera helix. Thirty-six compounds were initially screened against F. hepatica newly excysted juveniles (NEJs) of the Italian strain. Eleven of these compounds were active against NEJs and were selected for further study, using adult F. hepatica derived from a local abattoir (provenance unknown). From these eleven compounds, six demonstrated activity and were further assessed against immature liver flukes of the Italian strain. Subsequently, the most active compounds (n = 5) were further evaluated in ex vivo dose response experiments against adult Italian strain liver flukes. Overall, MC042 was identified as the most active molecule and the EC₅₀ obtained from immature and adult liver fluke assays (at 24 h post co-culture) are estimated as 1.07 μM and 13.02 μM, respectively. When compared to the in vitro cytotoxicity of MDBK bovine cell line, MC042 demonstrated the highest anthelmintic selectivity (44.37 for immature and 3.64 for adult flukes). These data indicate that modified hederagenins display properties suitable for further investigations as candidate flukicides.

Keywords: Fasciola hepatica; Hedera helix; anthelmintic drug discovery; saponins and hederagenin.

Figure 1

Hederagenin and its short-hand structure showing the three main positions used for derivatization. The full structure (left) and a simplified template (right) of hederagenin are illustrated, showing the three functional groups targeted for derivatization.

Figure 2

Synthesis of the ureate derivative from protected hederagenin. The acetal protected hederagenin (MC014) was converted into the isocyanate (MC015) using diphenylphosphoryl azide in toluene following a known procedure (Methods S1). Addition of a set of amines then gave the different ureates (MC0XX).

Figure 3

Flowchart describing the screening strategy for H. helix derived modified hederagenins. The initial 36 compounds were passed through a series of assays; the first four assays consisted of co-culturing liver flukes ex vivo with different concentrations of compound (10 µM or 40 µM for single concentration assays; 40 µM, 13.3 µM and 4.4 µM for dose response assays). The laboratory reared F. hepatica Italian strain was originally obtained from Campania, Italy, whereas the F. hepatica wild strain (provenance unknown) was obtained from Randall Parker Foods, Llanidloes (Wales), UK. The final assay was used to assess overt cytotoxicity on the bovine MDBK cell line.

Figure 4

Hederagenin derivatives differentially affect adult F. hepatica (wild strain) viability and motility. Adult F. hepatica flukes (wild strain; n = 4) were co-cultured with TCBZ (40 µM in 0.4% DMSO), the DMSO solvent (0.4%), medium only (RPMI 1640) or the 11 prioritized hederagenin derivatives (40 µM in 0.4% DMSO) for 72 h. Each parasite was carefully observed using bright field microscopy and scored accordingly (from 1 for normal movement to 6 for no movement or death). The individual fluke score is represented by a circle, means of the population are represented in black and error bars are represented by red, and green circles represent population of TCBZ treated parasites only. Statistically significant differences in compound-mediated motility were calculated by multiple comparisons (uncorrected Dunn’s test) to adult flukes co-cultured in 0.4% DMSO. *, ** and *** indicate p < 0.05, p < 0.001 and p < 0.0001, respectively.

Figure 5

Prioritized hederagenin derivatives kill F. hepatica (Italian strain) immature flukes. Immature F. hepatica flukes (n = 4) were co-cultured with TCBZ (40 µM in 0.4% DMSO), medium containing the DMSO solvent (0.4%) or the six prioritized hederagenin derivatives (40 µM in 0.4% DMSO) for 72 h. Each parasite was carefully observed using bright field microscopy and scored accordingly (1 for normal movement, 5 for no movement or death). Each parasite was scored at three time points: 24, 48 and 72 h. Except for MC057, all parasites treated with TCBZ (green circles) and modified saponins were completely immobile by 72 h of co-incubation. Significance was calculated by multiple comparisons (Dunn’s test) with an individual fluke score represented by a circle, means of the population are represented in black and error bars are represented by red. Statistically significant difference in motility, compared to adult flukes co-cultured in 0.4% DMSO, are indicated (***, p < 0.0001).

Figure 6

Structures of the five lead molecules selected for dose response studies. MC014 is the acetonide of hederagenin. The four remaining molecules are ureates derived by addition of particular cyclic secondary amines to isocyanate MC015, as shown in Figure 2.

Figure 7

Dose response titrations of MC014, MC035, MC042, MC055 and MC062 on adult F. hepatica flukes (Italian strain). The heat maps represent means of dose response scores (40 µM, 13.3 µM, 4.4 µM and 0 µM) of adult F. hepatica flukes (Italian strain; n = 3) assayed at: (a) 24 h, (b) 48 h and (c) 72 h. White squares represent the lowest motility score (1) and black squares represent the highest motility scores (6). Squares containing grey scales represent mean of intermediate motility scores.