The oleocanthal-based homovanillyl sinapate as a novel c-Met inhibitor

The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) signaling axis has gained considerable attention as an attractive molecular target for therapeutic blockade of cancer. Inspired by the chemical structure of S (−)-oleocanthal, a natural secoiridoid from extra-virgin olive oil with documented anticancer activity against c-Met-dependent malignancies, the research presented herein reports on the discovery of the novel olive-derived homovanillyl sinapate (HVS) as a promising c-Met inhibitor. HVS was distinguished for its remarkable potency against wild-type c-Met and its oncogenic variant in cell-free assays and confirmed by in silico docking studies. Furthermore, HVS substantially impaired the c-Met-mediated growth across a broad spectrum of breast cancer cells, while similar treatment doses had no effect on the non-tumorigenic mammary epithelial cell growth. In addition, HVS caused a dose-dependent inhibition of HGF-induced, but not epidermal growth factor (EGF)-induced, cell scattering in addition to HGF-mediated migration, invasion, and 3-dimensional (3D) proliferation of tumor cell spheroids. HVS treatment effects were mediated via inhibition of ligand-mediated c-Met activation and its downstream mitogenic signaling and blocking molecular mediators involved in cellular motility across different cellular contexts. An interesting feature of HVS is its good selectivity for c-Met and Abelson murine leukemia viral oncogene homolog 1 (ABL1) when profiled against a panel of kinases. Docking studies revealed interactions likely to impart high dual affinity for both ABL1 and c-Met kinases. HVS markedly reduced tumor growth, showed excellent pharmacodynamics, and suppressed cell proliferation and microvessel density in an orthotopic model of triple negative breast cancer. Collectively, the present findings suggested that the oleocanthal-based HVS is a promising c-Met inhibitor lead entity with excellent therapeutic potential to control malignancies with aberrant c-Met activity.

HVS was prepared in 60% yield by the reaction of 168 mg of homovanillyl alcohol with 224 mg of sinapic acid. White amorphous powder; 1 H and 13 C NMR, see Table S1 and Figures S1 and S2; HRESIMS m/z 373.1285 [M−H] − (calcd for C 20 H 21 O 7 373.1287). 1 H and 13 C NMR spectra of HVS were recorded in deuterated chloroform (CDCl 3 ), using tetramethylsilane (TMS) as an internal standard, on a JEOL Eclipse-ECS NMR spectrometer operating at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz (Table S1). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublet), and coupling constants. High-resolution ESIMS experiments were conducted using a JEOL JMS-T100 LP AccuTOF LC-Plus, equipped with an ESI source (JEOL Co. Ltd., Tokyo, Japan). ESI-MS detection was set using negative ion mode; needle voltage set at -2,000 V; and the ring lens and orifice 1 and 2 voltages set at -10, -35, and -7 V, respectively. Nitrogen was used as the nebulizing and desolvation gas, and pressure was maintained constant at 0.608 MPa. Desolvation chamber and orifice 1 temperatures were set to 250°C and 120°C, respectively. Results were obtained using Mass Center software, MS-56010MP (JEOL).

Molecular modeling experimental
The in silico experiments were carried out using Schrödinger molecular modeling software package installed on an iMac 27-inch Z0PG workstation with a 3.5 GHz Quad-core Intel Core i7, Turbo Boost up to 3.9 GHz, processor and 16 GB RAM (Apple, Cupertino, CA).

Protein structure preparation
Two X-ray crystal structures of c-Met tyrosine kinase domain; PDB codes: 4XYF [1] and 2RFS [2] of the wild-and mutant-types, respectively, were retrieved from the Protein Data Bank (www.rcsb.org). In order to identify the structural basis which has contributed to differences in affinity among c-Met, ABL1 and IGF1R, the crystal structures of the ABL1 (PDB code: 3OXZ [3]) and IGF1R (PDB code: 1JQH [4]) have been also obtained from the Protein Data Bank. The Protein Preparation Wizard was implemented to prepare the kinase domain of each protein. The protein was reprocessed by assigning bond orders, adding hydrogens, creating disulfide bonds and optimizing H-bonding networks using PROPKA (Jensen Research Group, Denmark). Finally, energy minimization with RMSD value of 0.30°A was applied using an Optimized Potentials for Liquid Simulation (OPLS_2005, Schrödinger) force field.

Ligand structure preparation
The chemical structure of HVS was sketched on the Maestro 9.3 panel interface (Maestro, version 9.3, 2012, Schrödinger). The LigPrep 2.3 module (LigPrep, version 2.3, 2012, Schrödinger) was implemented to generate the 3D structure and to search for different conformers. The OPLS (OPLS_2005, Schrödinger) force field was applied to geometrically optimize the ligand structure and to compute partial atomic charges. Finally, 32 poses per ligand were generated with different steric features for subsequent docking studies.

Molecular docking
The prepared X-ray crystal structures of each protein were used to generate receptor energy grids applying the default value of the protein atomic scale (1.0°A) within the cubic box centered on the co-crystallized ligand of each crystal structure. HVS was then docked using the Glide 5.8 module (Glide, version 5.8, 2012, Schrödinger) in extra-precision (XP) mode. Modeling scores were generated using the Glide-Dock program's empirical scoring functions.