Ullsten S et al. (2022), Identification of New Purpuroine Analogues from...

ECB-ART-51097

Int J Mol Sci 2022 Dec 13;2324:. doi: 10.3390/ijms232415852.

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Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Ullsten S , Petit GA , Isaksson J , Hansen IKØ , Schneider YK , Jenssen M , Li C , Hansen KØ .

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Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

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	Figure 1. Structures of purpuroine K–M (compound 1–3) isolated as part of this work.
	Figure 2. UHPLC-HR-MS chromatograms of the cytotoxic flash fraction five, as well as the non-cytotoxic fractions four and six of the organic extract of Pteraster militaris. Three halogenated secondary metabolites were exclusively present in flash fraction five, which suggests their involvement in the activity seen towards the human melanoma cell line A2058.
	Figure 3. Key COSY (bold) and HMBC (blue arrows) correlations of purpuroine K (1).
	Figure 4. Cell cycle analysis. (a) MV-4-11 cells are significantly arrested in G0/G1 phase after treatment with 1 at 52 and 104 µM (2 × IC50 and 4 × IC50, respectively) with a corresponding decrease in cells in S and G2/M phase when compared to control (n = 3). (b–e) representative images of flow cytometry data. * denotes p ≤ 0.05.
	Figure 5. Apoptosis assay. Compound 1 (PurK) increased apoptosis in MV-4-11 cells after 24 h (a) and 48 h (b) at 26 µM, 52 µM and 104 µM compound concentration (1 × IC50, 2 × IC50 and 4 × IC50, respectively) when compared to control (n = 3). At 104 µM of compound 1, the percentages of cells in late apoptotic/necrotic phase rather than apoptotic cells increased compared to the lower concentration of 52 µM, indicating induced toxicity or that multiple targets are being affected at this concentration. Representative flow cytometry images (c). * denotes p ≤ 0.05.
	Figure 6. Measurement of binding between 1 and FLT3 variants. Compound 1 (squares) does not bind to either FLT3 WT or FLT-ITD mutant active site at the concentrations tested (up to 10 μM). The emission ratio (normalized signal) remains constant despite increasing compound concentration. As a comparison, quizartinib (solid circles) behave as expected, with IC50 of 8.2 nM against FLT3 WT and 0.7 nM against FLT3-ITD mutant, calculated from fitting the normalized signal with an inhibitory dose response curve (see methods). Error bars represent the standard deviation (n = 3). The X-axis is the Log10 of the compound concentrations in nanomolar (nM), and the Y-axis shows the normalized emission ratio signal.
	Figure 7. Docking of 1 in the active site of FLT3 WT model PDB ID: 6JQR [35]. Compound 1 is placed in the active site pocket (shown as a grey surface) in two different manners: pose 1 (light green carbon atoms) and pose 2 (dark green carbon atoms), with similar docking scores (both around −5.9, suggesting weak binding). Pose 2 is roughly a 180-degree rotation of pose 1 along the long axis of the molecule. However, none of the poses suggest any specific interaction with FLT3 beyond having dimensions that roughly match its active site cavity. FLT3 residues that are in proximity of 1 are displayed as grey sticks and labelled. Color code for non-carbon atoms: hydrogen atoms are shown in white, nitrogen atoms in blue, oxygen atoms in red, sulfur atoms in yellow and bromine atoms in maroon. The orange dashed lines represent too-close contacts.

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Bailon-Moscoso, Natural Compounds as Modulators of Cell Cycle Arrest: Application for Anticancer Chemotherapies. 2017, Pubmed