Abstract
The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.
Highlights
The ever-increasing threat of cancer is one of the greatest challenges of modern medicine (Kunnumakkara et al, 2019)
Endophytes are described as a fascinating group of microorganisms that reside in living plant tissues during a considerable part of their life cycle, without causing any symptoms (Petrini et al, 1991; Schulz et al, 2006)
The EtOAc extract of the strain A02 identified as Lasiodiplodia venezuelensis exhibited a clear specific inhibitory activity (Wnt IC50 of 180 ± 56 μg/ml) and low cytotoxicity (Renilla inhibition IC50 > 500 μg/ ml)
Summary
The ever-increasing threat of cancer is one of the greatest challenges of modern medicine (Kunnumakkara et al, 2019). Endophytes are described as a fascinating group of microorganisms that reside in living plant tissues (beneath the epidermal cell layer) during a considerable part of their life cycle, without causing any symptoms (Petrini et al, 1991; Schulz et al, 2006) They occupy millions of unique biological niches, growing in biodiverse environments; almost all plant species studied until now have been found to host at least one to hundreds of endophytes (Arnold et al, 2000). The highly competitive rainforest ecosystem makes the selection pressure intense, probably prompting microbes associated with a tropical host to produce potent secondary metabolites (Rosa et al, 2011) Previous work on this plant model has shown, in another collection of endophytes, that it contained some cytotoxic and antimicrobial strains in which some bioactive compounds have been identified (Barthélemy et al, 2019; Donald et al, 2019; Barthélemy et al, 2020). In the frame of the present study, the anti-Wnt metabolites are characterized, and detailed profiling of this strain is provided to document its metabolite composition
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