Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives

Abstract

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Figure 1

Compound structures a) chemical structure of cordyheptapeptide A (1). b) Effect of 1 on cell proliferation in HCT 116 cells after 72 h. Error bars are one SD. N =3

Figure 2

Molecular dynamics results comparing cordyheptapeptide A (1) and 11 a – b) Principal component (PC) analysis of free energy landscapes in chloroform (CHCl₃) and water (H₂O) for a) 1 and b) 11. The triangle in cyan labeled with “A” denotes the mirror-imaged X-ray structure (CCDC 287376). Green numbers within each plot indicate representative structures in each solvent. c) Major MD simulated conformer of 1 in chloroform. d) Overlay of the major conformer in water for 1 (in green) and 11 (in purple). e) Major MD simulated conformer of 11 in chloroform.

Figure 3

COMPARE results and cytological profiling dendrogram fingerprint a) Top ten compounds with known MOA^{, -} from COMPARE analysis ordered by COMPARE correlation to 1 b) Cytological profiling results of 1 compared to DMSO controls, normalized with features represented on a scale from −1 (blue, below DMSO control) to +1 (yellow, above DMSO control) with black indicating no difference between compound and DMSO for that feature.

Figure 4

Effects of cordyheptapeptide A on protein synthesis and DNA synthesis a – b) Representative images of HeLa cells stained for the following features: nuclei (Hoechst, blue), protein synthesis (BONCAT, green) after a 24-h incubation with a) DMSO orb) 100 nM 1. c) Effect of 1 on normalized cell average BONCAT intensity in HeLa cells. d – e) Representative images of HeLa cells stained for the following features: nuclei (Hoechst, blue), and S-phase cells (EdU, yellow) d) DMSO or e) 0.6 μM 1. f) Effect of 1 on normalized EdU intensity in HeLa cells. Scale bars: 211 μm. Error bars are one SD.

Figure 5. Target identification

eEF1A WT HCT 116 cells and eEF1A A399V HCT 116 cells were incubated with 1 for 72 hours. Cell proliferation was measured with and Alamar blue assay. Error bars are one SD.