Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis

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.

Wnt-pathway inhibitors with selective activity against triple-negative breast cancer: From thienopyrimidine to quinazoline inhibitors

“…Compound dilutions were prepared by serial dilution in DMSO and diluted with the amount of medium necessary to obtain their final concentrations indicated on the figures and tables and maintain concentration of DMSO of 0.5% in all assay points. After overnight incubation, the supernatant in each well was removed, and the luciferase activity was measured as described ( Dyer et al, 2000 ; Huber et al, 2021 ; Pellissier et al, 2021 ). The culture medium was completely removed from all wells of the plate.…”

Wnt-Independent SARS-CoV-2 Infection in Pulmonary Epithelial Cells

“…Compounds were prepared by serial dilutions in DMSO and further diluted with the medium in amounts needed to obtain their final concentrations as indicated in the text and figures, maintaining the DMSO concentration of 0.5% at all assay points. After overnight incubation, the luciferase activity was measured as follows ( 39 – 41 ): the culture medium was completely removed from all wells of the plate, and the firefly and Renilla luciferase activities were detected sequentially in individual wells of a 384-well plate through injection of the corresponding measurement solutions and immediate reading (400-ms integration time) in an Infinite M Plex multifunctional plate reader equipped with an injection module. The data are presented as a two-point normalized signal with the Wnt3a-induced response set at 100% and the nonstimulated cells representing 0%.…”

Advanced high-resolution chromatographic strategies for efficient isolation of natural products from complex biological matrices: from metabolite profiling to pure chemical entities

“…This matters because large quantities of lipophilic substances, such as lipids and pigments, or highly hydrophilic compounds like saccharides and tannins present in the extract, can significantly affect separation quality and lower the concentration of the target compounds (Ma et al 2018). To remove these unwanted matrix components, methods like vacuum liquid chromatography (VLC) (Houriet et al 2020;Queiroz et al 2014;Saldanha et al 2021), and liquid/liquid partition (LLP) (Alfattani et al 2021(Alfattani et al , 2023Klimenko et al 2021Klimenko et al , 2023Pellissier et al 2021) are typically employed. Applying such a treatment to the sample increases the concentration of the desired compounds in the sample, facilitating their efficient isolation.…”

“…In this approach, the enriched hydroalcoholic fraction of an endophytic fungus Lasiodiplodia venezuelensis was first purified by RP semi-preparative HPLC using a 5 lm high resolution reverse phase stationary phase (Fig. 5b) (Pellissier et al 2021). From this experiment, 15 compounds were successfully purified in a single step, however, the biological activity was concentrated in a fraction still containing a mixture of 3 compounds.…”