Total Synthesis and Target Identification of the Curcusone Diterpenes

Abstract

The curcusone natural products are complex diterpenes featuring a characteristic [6−7−5] tricyclic carbon skeleton similar to the daphnane and tigliane diterpenes. Among them, curcusones A−D demonstrated potent anticancer activity against a broad spectrum of human cancer cell lines. Prior to this study, no total synthesis of the curcusones was achieved and their anticancer mode of action remained unknown. Herein, we report our synthetic and chemoproteomics studies of the curcusone diterpenes which culminate in the first total synthesis of several curcusone natural products and identification of BRCA1-associated ATM activator 1 (BRAT1) as a cellular target. Our efficient synthesis is highly convergent, builds upon cheap and abundant starting materials, features a thermal [3,3]-sigmatropic rearrangement and a novel FeCl 3 -promoted cascade reaction to rapidly construct the critical cycloheptadienone core of the curcusones, and led us to complete the first total synthesis of curcusones A and B in only 9 steps, C and D in 10 steps, and dimericursone A in 12 steps. The chemical synthesis of dimericursone A from curcusones C and D provided direct evidence to support the proposed Diels−Alder dimerization and cheletropic elimination biosynthetic pathway. Using an alkyne-tagged probe molecule, BRAT1, an important but previously "undruggable" oncoprotein, was identified as a key cellular target via chemoproteomics. We further demonstrate for the first time that BRAT1 can be inhibited by curcusone D, resulting in impaired DNA damage response, reduced cancer cell migration, potentiated activity of the DNA damaging drug etoposide, and other phenotypes similar to BRAT1 knockdown.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals the DNA Repair Protein XRCC5 as a Functional Target of Myrocin G

“…Indeed, probe 7 showed the fastest live cell labeling kinetics among the three tested probes (Figures 3F and S1), with the fluorescent bands appearing as short as one minute after cell treatment. Owing to our interest in pharmacological targeting of biomedically important nuclear proteins, 11,13,54 we also compared the labeling of nuclear cysteines in live HeLa cells. To this end, live HeLa cells were again treated with 100 µM 1, 2 or 7, cellular nuclei were isolated, and the labeling efficiency was analyzed as described above.…”

“…3,[7][8] Functional cysteines tend to display pronounced hyperreactivity and these reactive cysteine hot spots can be precisely mapped in complex proteomes with cysteine-reactive electrophilic probes. These probes are particularly useful for competitive chemoproteomic profiling of cysteines as targets of natural products, [9][10][11][12][13][14] libraries of covalent fragments, [15][16][17] and electrophilic metabolites. 18 Over the past decade, a broad variety of electrophilic probes for cysteine profiling have been reported.…”

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

“…In agreement, DAC treatment triggers the association of BRAT1, one of the most DAC-modified proteins, to ER and nuclear membranes (Figure 3F). Covalent modification of BRAT1 by the natural diterpene curcusone D was recently reported and resulted in BRAT1 degradation and reduced DNA damage response (45). Regarding (S)-DAC, in addition to BRAT1, multiple proteins involved in mechanisms of PQC are also lipoxidized.…”

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals the DNA Repair Protein XRCC5 as a Functional Target of Myrocin G

“…3,[7][8] Functional cysteines tend to display pronounced hyperreactivity and these reactive cysteine hot spots can be precisely mapped in complex proteomes with cysteine-reactive electrophilic probes. These probes are particularly useful for competitive chemoproteomic profiling of cysteines as targets of natural products, [9][10][11][12][13][14] libraries of covalent fragments, [15][16][17] and electrophilic metabolites. 18 Over the past decade, a broad variety of electrophilic probes for cysteine profiling have been reported.…”