Structure-activity exploration of a small-molecule allosteric inhibitor of T790M/L858R double mutant EGFR

Abstract EGFR is a protein kinase whose aberrant activity is frequently involved in the development of non-small lung cancer (NSCLC) drug resistant forms. The allosteric inhibition of this enzyme is currently one among the most attractive approaches to design and develop anticancer drugs. In a previous study, we reported the identification of a hit compound acting as type III allosteric inhibitor of the L858R/T790M double mutant EGFR. Herein, we report the design, synthesis and in vitro testing of a series of analogues of the previously identified hit with the aim of exploring the structure-activity relationships (SAR) around this scaffold. The performed analyses allowed us to identify two compounds 15 and 18 showing improved inhibition of double mutant EGFR with respect to the original hit, as well as interesting antiproliferative activity against H1975 NSCLC cancer cells expressing double mutant EGFR. The newly discovered compounds represent promising starting points for further hit-to-lead optimisation.


Chemical synthesis
All available chemicals and solvents were purchased from commercial sources and used without any further purification. Thin layer chromatography (TLC) was performed using 0.25 mm silica gel precoated plates Si 60-F254 (Merck, Darmstadt, Germany) visualized by UV-254 light and CAM staining. Purification by flash column chromatography (FCC) was conducted by using silica gel Si 60, 230-400 mesh, 0.040-0.063 mm (Merck). 1

Synthesis
General procedure for the one-pot ACF reaction A mixture of aldehyde 2a-i (1.0 mmol) and aniline 1a-c (0.95 mmol) in dry toluene (4.0 mL) was stirred at 80 °C for 4 h. Then, the reaction was cooled to 25 °C and indole 3a-e (0.95 mmol) was added. The resulting solution was stirred at 25 °C for 24 hours, diluted with ethyl acetate (10 mL), washed with saturated NaHCO3 solution (3×10 mL), and brine (10 mL), dried over NA2SO4, and filtered. After evaporation of the solvent under vacuum (RV), the crude was purified following Procedure a or b.
Procedure a -The residue was purified by FCC to afford compounds 4-14.
Procedure b -Acetate deprotection procedure: The residue was dissolved in methanol (5.0 mL).
Then, NaOH (27 mg, 1.5 mmol) and water (2.0 mL) were added, and the mixture was stirred at 25 °C for 6 hours. After evaporation of the solvent under vacuum (RV), diethyl ether was added.
The solid formed was filtered, washed whit diethyl ether and purified by FCC affording compounds 15-20.

S4
Starting material, product, yield, chromatographic eluent, physical and analytical data of compounds 4-20 are as follows.
The IC50 value is defined as the concentration of compound that inhibits 50% enzyme activity.
Compound 4 and EAI045 were used as reference compounds in all experiments.
For cytotoxicity assays, compounds were added to cells in 96-well plates for 72 hours. Cell viability was measured by MTS assay using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay System (Promega, Madison, WI, USA) according to instructions. Inhibition of EGFR was assessed by Western blotting after incubating the cells with the compounds for 8 hours. Cells were then harvested, lysed and equal total protein lysates were loaded on SDS-PAGE gels and probed with phospho-EGFR (Tyr1068), total EGFR and actin specific antibodies (Cell Signaling).

Molecular docking analyses
All molecules were drawn by using Maestro of the Schrödinger 2021-1 suite 1 . Then, the LigPrep tool was used to generate all possible protomers and tautomers potentially present in the pH range of 6-8, and to obtain minimized structures suitable for molecular docking.

S11
The PDB structure of mutant EGFR complexed with EAI001 (PDB ID: 5D41) 2 was downloaded from the Protein Data Bank website. The chain B of the downloaded PDB complex was deleted, as it has not been co-crystallized with EAI001 allosteric ligand. 2 Chain A was processed by using the Protein Preparation Wizard toolkit 3 to fix atom types and bond connectivity issues, to rebuild missing residue side chains and to add hydrogen atoms. Finally, a restrained minimization was performed on the structure according to the OPLS4 force field, to relieve steric clashes, until a final root means square deviation (RMSD) of 0.3 Å, with respect to the input protein coordinates.
Afterwards, all water molecules were removed from the minimized complex, except one (residue number 1285) which was previously investigated for its importance in the binding mode of EAI001 and EAI045 4 .
A receptor grid was then built on the prepared structure by centering the enclosing box (default dimensions of 10 Å × 10 Å × 10 Å) at the centroid of the workspace ligand. Molecular docking calculations were performed by using the Extra Precision (XP) mode of the Glide software (Schrödinger 2021-1) 5 . The protocol reported in our previously published work was applied by using the same settings, with enhancement of the conformational space of ligand sampling by a factor of 4, and 50 poses per ligand submitted to post-docking minimization 6 . All other parameters were set to their default values.
To account for binding site flexibility, additional docking experiments were also performed with the Induced Fit Docking (IFD) protocol (Schrödinger Suite 2021-1) 7 . IFD calculations were conducted with the standard settings. All poses were visually inspected and manually clustered according to the obtained binding modes. S12 Figure S1: Dose-response curves of compounds 15 (panel A) and 18 (panel B) against H1975 (Lung adenocarcinoma, double mutant EGFR), H3122 (Lung adenocarcinoma, wild type EGFR) and HL60 (Acute myeloid leukemia, negative EGFR) cell lines. S13 Table S1: In vitro inhibitory activity of the most relevant compounds obtained through the EGFR(T790M/L858R) Kinase Assay Kit. Compounds 4 and EAI045 were used as references.