Neocarzilin A induces apoptosis and mitochondrial disturbance by targeting reticulon 4-mediated endoplasmic reticulum stress

Natural compounds are a valuable source of highly active biomolecules for the discovery of innovative drug targets as well as drug leads. The natural compound neocarzilin A (NCA) exhibits pronounced antiproliferative and antimigratory activity, which we previously ascribed to the target proteins vesicle amine transporter protein 1 (VAT-1) and bone marrow stromal antigen 2 (BST-2). We here additionally demonstrate the perturbation of mitochondrial functions (fragmentation of mitochondrial networks, ultrastructural changes, increased Opa1 splicing, loss of mitochondrial membrane potential, and excessive ROS generation) upon treatment with NCA. We observe impairment of the electron transfer chain and diminished ATP synthesis. Furthermore, NCA triggers apoptosis via activation of caspase-8, enhanced Bid processing, and cytochrome c release from mitochondria into the cytosol, leading to the activation of caspase-3 and -9 and, finally, PARP cleavage and DNA fragmentation. Endoplasmic reticulum (ER) stress is induced by treatment with NCA, and subsequently, the unfolded protein response (UPR) via the protein kinase r-like ER kinase (PERK) branch is prompted. Proteomic ABPP data indicate reticulon 4 (Rtn4, Nogo), an ER-located protein mainly involved in shaping ER tubules and maintaining proper ER function, as a promising hit to explain those effects. This novel molecular target was verified by co-staining of the target probe NC-4 and Rtn4, as well as RNA interference experiments, which resulted in reduced responsiveness of HeLa cells to NCA treatment. We propose NCA as a powerful tool to study the biology of Rtn4, and to develop more specific modulators of reticulons in the future. Furthermore, we introduce-to our knowledge-the first small molecular modulator of reticulon proteins.