Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.
The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAStransformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic.
An efficient one‐pot method is described for the synthesis of a variety of di‐ and trisubstituted 4‐pyrones based on the gold‐catalyzed cascade reactions between cyclic or acyclic diazodicarbonyl compounds and vinyl ethers. The key strategy used involves the gold‐catalyzed Wolff rearrangement of diazo compounds followed by [4+2] cycloaddition and elimination. This methodology is also applied to the synthesis of tetrasubstituted 4‐pyrones.
This study describes an investigation into secondary metabolites that are produced by a marine red alga, Symphyocladia latiuscula, which was collected from coastal waters off Qingdao, China. A combination of normal, reversed phase, and gel chromatography was used to isolate six citric acid derived natural products, aconitates A–F (1–6), together with two known and ten new polybrominated phenols, symphyocladins C/D (7a/b), and symphyocladins H–Q (8a/b, 9a/b and 10–15), respectively. Structure elucidation was achieved by detailed spectroscopic (including X-ray crystallographic) analysis. We propose a plausible and convergent biosynthetic pathway involving a key quinone methide intermediate, linking aconitates and symphyocladins.
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