Leucinostatins fromOphiocordycepsspp. andPurpureocilliumspp. Demonstrate Selective Antiproliferative Effects in Cells Representing the Luminal Androgen Receptor Subtype of Triple Negative Breast Cancer

Abstract

The structures of four leucinostatin analogues (1–4) from Ophiocordyceps spp. and Purpureocillium spp. were determined together with six known leucinostatins [leucinostatins B (5), A (6), B2 (7), A2 (8), F (9), and D (10)]. The structures of the metabolites were established using a combination of analytical methods including HRESIMS and MS/MS experiments, 1D and 2D NMR spectroscopy, chiral HPLC, and advanced Marfey’s analysis of the acid hydrolysate, as well as additional empirical and chemical methods. Compounds 1–10 were evaluated for their biological effects on triple negative breast cancer (TNBC) cells. Leucinostatins 1–10 showed selective cytostatic activities in MDA-MB-453 and SUM185PE cells representing the luminal androgen receptor subtype of TNBC. This selective activity motivated further investigation into the mechanism of action of leucinostatin B (5). The results demonstrate that this peptidic fungal metabolite rapidly inhibits mTORC1 signaling in leucinostatin-sensitive TNBC cell lines, but not in leucinostatin-resistant cells. Leucinostatins have been shown to repress mitochondrial respiration through inhibition of the ATP synthase, and we demonstrated that both the mTORC1 signaling and LAR-selective activities of 5 were recapitulated by oligomycin. Thus, inhibition of the ATP synthase with either leucinostatin B or oligomycin is sufficient to selectively impede mTORC1 signaling and inhibit the growth of LAR-subtype cells.

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents Against Trypanosoma cruzi

“…An iterative bioactivity-guided fraction process led to the identification of an active fraction enriched in putative peptidic natural products. Subjecting this sample to further chromatographic steps led to purification of five natural products, which included three previously reported metabolites (leucinostatins A, B and F) and two leucinostatin analogues (leucinostatins NPDG C and NPDG D) that were recently reported as new natural products 15 (Figure 2). The structures of the metabolites were determined using a combination of spectrometric, spectroscopic, and chemical methods 15 .…”

“…Subjecting this sample to further chromatographic steps led to purification of five natural products, which included three previously reported metabolites (leucinostatins A, B and F) and two leucinostatin analogues (leucinostatins NPDG C and NPDG D) that were recently reported as new natural products 15 (Figure 2). The structures of the metabolites were determined using a combination of spectrometric, spectroscopic, and chemical methods 15 . The leucinostatins and the positive control benznidazole were tested to assess their concentration-dependent antiparasitic and cytotoxic activities (Figures 3 and 4).…”

“…The fungal natural products were obtained via a citizen-science soil-sampling initiative (https://whatsinyourbackyard.org/), as well as a survey of Great Lakes sediments 13,14 . Here, we report leucinostatins A, B and F, as well as novel leucinostatins NPDG C and NPDG D, which we recently characterized 15 , as having potent activity against the replicative, intracellular amastigote form of the parasite and no host cell toxicity up to 1.5 µM using a phenotypic, highcontent imaging assay. We further demonstrated that leucinostatin B prevented parasite growth in an in vivo bioluminescent model of T. cruzi infection.…”

Yuanhuacine Is a Potent and Selective Inhibitor of the Basal-Like 2 Subtype of Triple Negative Breast Cancer with Immunogenic Potential

“…While not anticipated, it is reasonable for PKC activation to be a molecular liability of the BL2 subtype of TNBC, which is characterized in part by a dependency on glycolytic pathways [ 8 ], as some PKC isoforms can directly phosphorylate the insulin receptor substrate 1 (IRS1) leading to starvation of cells with a high glucose dependency [ 39 , 40 ]. The comparative pharmacological approach we used to interrogate the mechanism of action of 1 is similar to the utilization of cancer dependency databases, such as Project Achilles [ 41 ], that can facilitate the identification of the mechanism of action of a compound based on its profile of selectivity among cell lines with distinct genetic liabilities [ 13 ]. These are valuable tools when uncovering the mechanism of action of a particular feature (such as BL2 TNBC subtype selectivity) of natural products like yuanhuacine that have a long and diverse pharmacological history.…”

“…With more refined laser dissection techniques, the subtypes were consolidated into basal-like 1 and 2 (BL1 and BL2), mesenchymal (M) and luminal androgen receptor (LAR) subtypes [ 9 , 10 ]. Our group has previously utilized this molecular subtyping to identify natural products with selective activity against cell lines representing the BL1, M, and LAR subtypes of TNBC to inform on unanticipated druggable targets [ 11 , 12 , 13 , 14 , 15 ].…”