Target fishing and mechanistic insights of the natural anticancer drug candidate chlorogenic acid

Abstract

Chlorogenic acid (CGA) is a natural product that effectively inhibits tumor growth, demonstrated in many preclinical models, and phase II clinical trials for patients with glioma. However, its direct proteomic targets and anticancer molecular mechanisms remain unknown. Herein, we developed a novel bi-functional photo-affinity probe PAL/CGA and discovered mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) was one of the main target proteins of CGA by using affinity-based protein profiling (AfBPP) chemical proteomic approach. We performed in-depth studies on ACAT1/CGA interactions via multiple assays including SPR, ITC, and cryo-EM. Importantly, we demonstrated that CGA impaired cancer cell proliferation by inhibiting the phosphorylation of tetrameric ACAT1 on Y407 residue through a novel mode of action in vitro and in vivo. Our study highlights the use of AfBPP platforms in uncovering unique druggable modalities accessed by natural products. And identifying the molecular target of CGA sheds light on the future clinical application of CGA for cancer therapy.

Keywords: ACAT1; AfBPP; Chlorogenic acid; Natural product; Probe.

Graphical abstract

Scheme 1

Structures of CGA and PAL/CGA.

Figure 1

Identification of ACAT1 as the natural product CGA binding protein by a photoaffinity purification approach. (a) PAL/CGA suppressed the proliferative ability of melanoma cancer cells; (b) Identification of ACAT1 as the binding protein of PAL/CGA (10 μmol/L) in mitochondria of the human cancer A375 tumors, which was competed off by CGA (50 μmol/L) preincubation. Coomassie blue staining of the PAL/CGA complex. The red arrow represented the position of ACAT1; (c) Validation of ACAT1 pulled down from mitochondria of the human cancer A375 tumors with PAL/CGA by Western blot; (d) Efficacy of CGA treatment in inhibition of purified recombinant ACAT1. IC₅₀ values were determined. The bar graphs show the mean ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 2

Characterization of CGA interactions with ACAT1 in vitro. (a) Characterization of the binding affinity between CGA and ACAT1 by a SPR assay; (b) Binding affinity of CGA to ACAT1 determined by ITC; (c) Thermal shift assay using purified recombinant ACAT1, which were exposed to CGA (1.0 μmol/L) and AH (40 μmol/L); (d) CGA promotes target protein ACAT1 resistant to proteases (DARTS); data are representative of three independent experiments.

Figure 3

Identification of ACAT1 as a CGA-binding protein in live cells. (a, c) Fluorescence images of A375 cells, NCI-H1299 cells and knockdown of endogenous ACAT1 cells incubated with CGA (10 μmol/L) for 2 h; (b, d) Quantification of fluorescence intensities from images above obtained for CGA in the A375 cells, NCI-H1299 cells and knockdown of endogenous ACAT1 cells. The bar graphs show the mean ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 4

Cryo-EM structure of ACAT1 in complex with inhibitor CGA. (a) Negative stain micrograph of ACAT1/CGA complex; (b) FSC curve of the map of ACAT1/CGA complex; (c) Local resolution of the map of ACAT1/CGA complex; (d) The superposition of cryo-EM structure of the ACAT1/CGA complex; (e) The crystallographic structure of the ACAT1/CGA complex; (f) Schematic drawing of the interactions between CGA and ACAT1.

Figure 5

CGA is a no-covalent ACAT1 Inhibitor, disrupting active ACAT1 function by eliminating Y407 phosphorylation without hindering tetramer reformation. (a, b) Western blot assay of phospho-ACAT1(Y407) and ACAT1 expression in the A375 cells and NCI-H1299 cells; (c, d) Western blot assay of tetramer ACAT1/Phospho-ACAT1(Y407) proteins in the A375 cells treated with or without CGA by native page.

Figure 6

CGA significantly reduces Y407 phosphorylation, inhibiting cancer cell proliferation and tumor growth. (a–c) Effects of CGA administered via intraperitoneal injection in A375 xenograft nude mice on body weight, tumor growth rate and mass; (d) Western blot assay of phospho-ACAT1(Y407) expression in the A375 tumor; (e) Phospho-ACAT1(Y407) immunohistochemical staining of A375 tumor. Scale bar, 50 μmol/L; (f) Quantification of intensities from phospho-ACAT1(Y407) immunohistochemical staining, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. Data present as mean ± SD.