Identification of the novel antitumor targets of Lagunamide A and Neocarzilin A

Hu, Yudong (2023): Identification of the novel antitumor targets of Lagunamide A and Neocarzilin A. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Natural products are a rich source of bioactive compounds and have traditionally contributed to the discovery and exploration of prospective drug development targets. Novel targets, particularly for cancer, are desperately needed to study cancer biology and develop precision therapeutics. Inspired by the promising anticancer activities of the marine cyanobacterial product Lagunamide A (Lag A) and the streptomyces carzinostaticusin product Neocarzilin A (NCA), we performed in depth studies on their modes of action. For Lagunamide A, a biologically active natural compound, the cellular target is yet unidentified. With an affinity-based approach (ABPP), we failed to identify a target protein. In contrast, by using thermal proteome profiling, we found three proteins EYA3, SMC1A and NDC80 as putative targets. For validation of the proposed targets, genetic depletion of EYA3 showed only limited impact on antagonizing the anti-proliferative and pro-apoptotic effect of Lag A. However, EYA3 turned out to be important for DNA damage repair since incubation with Lag A sensitized cancer cells to doxorubicin treatment. Thus, this work not only shows that Lag A might serve as a chemosensitizing agent in the tumor context, but also pinpoints that depending on the target proteins, different target identification techniques are required. Besides the chemoresistance machinery, cell proliferation is another hotspot in the field of cancer research. Our previous study showed that the natural compound NCA exhibited potent anti-migratory and anti-proliferative effects on cancer cells. We have previously identified VAT-1 as a putative anti-migratory target of NCA. However, the target and mechanism for the anti-proliferative effect of NCA were unknown to date. Affinity-based protein profiling (ABPP) based on an NCA-derived probe, and subsequent mass spectrometry were employed to identify additional potential target proteins of NCA. For validation of the proposed targets, we used genetic knockdown (with siRNA) or knockout (with CRISPR/Cas9) and over-expression approaches. By applying ABPP, Bone Marrow Stromal Antigen 2 (BST-2) was identified as it was highly enriched by binding to an NCA-derived probe. Binding of BST-2 to the probe was outcompeted by NCA itself, proposing BST-2 as a target of NCA. Silencing or knockout of BST-2 decreased the anti-proliferative action of NCA against HeLa cells, while overexpression or reconstitution of BST-2 increased sensitivity of cells towards NCA, thereby supporting the hypothesis of BST-2 as an anti-proliferative target of NCA. In-depth mechanistic studies revealed that NCA promoted BST-2 degradation via the lysosomal pathway and influenced the EGFR signaling pathway via disturbing lipid rafts, which in turn regulated cell proliferation. In conclusion, we identify BST-2 as an innovative target of NCA, and NCA as an interesting lead structure for inhibiting cancer cell proliferation.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Lagunamide A, EYA3, Neocarzilin A, BST-2, Target
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. Juli 2023
• 1. Bericht­erstatter:in: Vollmar, Angelika