Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus

Abstract

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus-a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC₅₀) = 26.6 ± 0.74 µM) and L4 motility (IC₅₀ = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC₅₀ ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

Keywords: Haemonchus contortus; Haliclona sp.; Monanchora unguiculata; alkaloid; anthelmintic; fromiamycalin; halaminol A; marine.

Figure 1

In vitro activity of extracts on the inhibition of xL3 motility and/or L4 development of Haemonchus contortus. Dose-response curves for the extract (Mu-1) from Monanchora unguiculata, and two extracts (Ha-1 and Ha-2) from Haliclona sp. from two geographic locations, for the inhibition of xL3 motility (panel A) and of L4 development (panel B). Representative light microscopy images of xL3s treated with extract Mu-1 showing a ‘circular’ phenotype, and ‘straight’ phenotype of xL3s treated separately with Ha-1 and Ha-2, compared with unaffected larvae in the untreated (U) control, and ‘coiled’ and ‘circular’ xL3s treated with monepantel (MON) and moxidectin (MOX), respectively. White scale bar: 100 µm; 20x magnification (panel C).

Figure 2

The chromatograms and L4 development (%) for C₁₈ semi-preparative fractions of the three active extracts compared with that of control compounds; untreated (U, LB* + 1.25% DMSO) and positive controls MON and MOX. For extract Mu-1, fractions 44–46 elicited the most significant inhibition (panel A), whereas fraction 43 elicited the highest and significant inhibition for extracts Ha-1 and Ha-2 (panels B and C). The five individual chromatograms in panels A–C display the ultraviolet (UV)-absorbance of the eluting fractions at 254 nm (green), 280 nm (red), 320 nm (blue), 350 nm (yellow) and 380 nm (pink). Fraction/s with the highest L4 developmental inhibition are indicated by red rectangles in panels A–C. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 3

Chemical structures of the compounds purified in this study and other related alkaloids. Chemical strutures of fromiamycalin (1) purified from the extract of Monanchora unguiculata and other related pentacyclic guanidine alkaloids, crambescidin 800 (2), celeromycalin (3) as well as the hydroxyspermidine derivative (4) isolated in the original study of fromiamycalin purification [38], and halaminol A (5) purified from the extract of Haliclona sp.

Figure 4

In vitro activity of fromiamycalin (1) on larval stages of Haemonchus contortus. Dose-response curves for fromiamycalin (1) on xL3 motility (panel A), L4 motility (panels B–D) and L4 development (panel E).