Assessing the effects of adsorptive polymeric resin additions on fungal secondary metabolite chemical diversity

<div><p>Adsorptive polymeric resins have been occasionally described to enhance the production of specific secondary metabolites (SMs) of interest. Methods that induce the expression of new chemical entities in fungal fermentations may lead to the discovery of new bioactive molecules and should be addressed as possible tools for the creation of new microbial chemical libraries for drug lead discovery. Herein, we apply both biological activity and chemical evaluations to assess the use of adsorptive resins as tools for the differential expression of SMs in fungal strain sets. Data automation approaches were applied to ultra high performance liquid chromatography analysis of extracts to evaluate the general influence in generating new chemical entities or in changing the production of specific SMs by fungi grown in the presence of resins and different base media.</p></div

MDN-0066 affects the cell cycle of RCC4-VA/VHL cells.

<p>Cell cycle of RCC4-VA and RCC4-VHL cells after MMDN-0066 treatment. Cell cycle distribution was measured by flow cytometry using a PI Flow Kit and the percent of the cell cycle phase is shown in a table form with the SubGo, G0/G1, S and G2/M phases for the two cell lines in the absence or following 48h incubation with MDN-0066 at 24 uM. Represent results of one of 3 independent experiments.</p

Identification of the Lipodepsipeptide MDN-0066, a Novel Inhibitor of VHL/HIF Pathway Produced by a New <i>Pseudomonas</i> Species

<div><p>Throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. In fact, natural products have largely been –and still are– considered an exceedingly valuable platform for the discovery of new drugs against diverse pathologies. Such value is partly due to their higher complexity and chemical diversity as compared to those of synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau (<i>vhl</i>) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types. The primary cause of morbidity and mortality for these patients arises from progression of Renal Cell Carcinoma (RCC) or end-stage renal disease. Inactivation of the Von Hippel-Lindau (<i>vhl</i>) tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer and is associated with a high degree of vascularization and poor prognosis. Loss of pVHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. In this study, renal carcinoma cell lines with naturally occurring <i>vhl</i> mutations (RCC4 VA) and their genetically matched wild-type <i>vhl</i> (RCC4 VHL) counterparts were seeded onto 96-well plates and treated with a collection of 1,040 organic extracts obtained from 130 bacterial strains belonging to at least 25 genera of the phyla <i>Actinobacteria</i>, <i>Firmicutes</i>, <i>Proteobacteria</i> and <i>Bacteroidetes</i>. This strategy allowed us to identify several extracts obtained from bacterial strain F-278,770^T, the type strain of the recently proposed new species <i>Pseudomonas granadensis</i>, showing biological activities not associated with previously known bioactive metabolites. The fractionation and structural elucidation of one of these extracts led to the discovery of a new lipodepsipeptide (MDN-0066) with specific toxicity in pVHL deficient cells that is not detectable in cells with pVHL expression rescue. This specific toxicity is associated with apoptosis induction in VHL deficient cell line as demonstrated with PARP activation and Annexin V staining. Our study demonstrated the feasibility of selectively targeting the loss of the <i>vhl</i> tumor suppressor gene for potential clinical benefit. Our results may have great impact on the development of new targeted therapies from natural products for the treatment of cancer and other genetic diseases.</p></div

NMR spectra of MDN-0066, DMSO-d₆, 24°C, (500 MHz).

<p>^{a, b, c, d, e} Assignments may be interchangeable.</p><p>NMR spectra of MDN-0066, DMSO-d₆, 24°C, (500 MHz).</p

MDN-0066 cytometry analysis.

<p>Annexin-V staining of RCC4-VA and RCC4-VHL cells cultured after 24 or 48h treatment. Cells were treated with 12.5 or 25 μM MDN-0066. The analysis allows to distinguish in the diagram between living cells (lower left quadrant), early apoptotic cells (lower right quadrant), apoptotic cells (upper right quadrant), and necrotic cells (upper left quadrant). Represent results of one of 3 independent experiments. Table resume all results obtained.</p

MDN-0066 western blot analysis.

<p>Cell lysates were collected from RCC4-VA and RCC4-VHL cells 24h or 48h after MDN-0066 treatment at 25uM for 24hrs or 48hrs. Blots were probed with antibodies targeting either total β-actin, HIF-1α, PKM2, MCT4, C-Myc, Beclin, LC3b, LDHA or cleaved PARP.-, no treatment, <b>+</b> MDN-0066. In Fig 5b we quantified the intensity of the western blot for each bands density using the LI-COR Aerius software.</p