Agrimol B, a natural product from agrimonia pilosa : total synthesis, anticancer activities, and mechanism of action studies

Abstract

With the diversity of compounds and uniqueness of structures, natural products play a crucial role in anticancer drug discovery. Agrimol B (AGB), a trimeric acylphloroglucinol from Agrimonia pilosa, is an important constituent for quality control used in the Chinese Pharmacopoeia. Although AGB was first isolated in 1975, few studies have been performed to evaluate its biological activities. In this project, the total synthesis of AGB was improved and the potential of AGB for anticancer therapy and its underlying mechanisms were investigated for the first time. To overcome the limited availability from low abundance in plants, two improved strategies for the total synthesis of AGB were developed (described in Chapter 2). With the facile generation of the key intermediate 4 (1-(4,6-dihydroxy-2-methoxy-3-methylphenyl) butan-1-one), the total synthesis process was simplified along with significantly increased overall yields. Additionally, five derivatives of AGB were obtained for the structure-activity relationship study. In Chapter 3, the investigation of the anticancer properties of AGB is described. In vitro, the cytotoxicity, colony formation, cell migration, and cancer stem-like renewal ability were examined. It was found that micro-molar levels of AGB selectively inhibited cancer cell proliferation against a panel of cancer cells and inhibited tumorsphere formation in cancer-stem-like cells. In vivo, treatment with 3 mg/kg of AGB elicited marked inhibition of tumor growth in three different mouse xenograft models without observable side effects. Moreover, the maximum tolerable dose of AGB in mice was determined to be 12.8 mg/kg, indicating AGB could be used for anticancer therapy with a safe threshold. Chapter 4 presents the mechanisms of anticancer action of AGB, as determined by quantitative proteomic analysis along with ProTargetMiner analysis. The most specific regulations identified by these approaches were shown as ER stress and mitochondrial dysfunction that induced by AGB. Further studies suggested that AGB treatment induced mitochondrial fission in association with alteration of the mitochondrial dynamics regulatory proteins involving OPA1 (dynamin-like 120 kDa protein) cleavage and DRP1 (dynamin-related protein 1) dephosphorylation and translocation. AGB also acted as a mitochondrial uncoupler, dissipating the proton gradient to depolarize the mitochondrial membrane potential to trigger mitochondria-mediated cell apoptosis. Chapter 5 describes the identification of the molecular targets of AGB by an unbiased thermal proteome profiling method. Heat shock protein 60 (HSP60), citrate synthase (CS), and transaldolase (TADLO1) were found to interact with AGB that was further validated by proteome integral stability alteration assay and cellular thermal shift assays. The direct binding between three identified targets and AGB was measured by protein intrinsic fluorescence quenching experiment and demonstrated in a molecular docking study. Moreover, AGB exhibited significant inhibitory effects on the activity of the mitochondrial chaperone protein, HSP60, resulting in a decrease in protein refolding. Consistent with the targeting of CS and TALDO1, AGB treatment reduced the production of ATP, NADPH, and a variety of biosynthetic precursors that support cancer cell growth.