Structure-Based Design of Tetrahydroisoquinoline-7-carboxamides as Selective Discoidin Domain Receptor 1 (DDR1) Inhibitors

The structure-based design of 1, 2, 3, 4-tetrahydroisoquinoline derivatives as selective DDR1 inhibitors is reported. One of the representative compounds, 6j, binds to DDR1 with a Kd value of 4.7 nM and suppresses its kinase activity with an IC50 value of 9.4 nM, but it is significantly less potent for a panel of 400 nonmutated kinases. 6j also demonstrated reasonable pharmacokinetic properties and a promising oral therapeutic effect in a bleomycin-induced mouse pulmonary fibrosis model.


II. In vitro kinase assay
The functional assays of compounds on the kinase activities of Abl were determined using the FRET-based Z′-Lyte assay system according to the manufacturer's instructions (Invitrogen, USA). Tyrosine 2 peptide was used as Abl substrate, and Ser/Thr 6 peptide was used as the substrate for c-Kit. There actions were carried out in 384-well plates in a 10 µL of reaction volume with appropriate amount of kinases in 50 mM HEPES (pH 7.5), 10 mM MgCl 2 , 1 mM EGTA, and 0.01% Brij-35. The reactions were incubated 1h at room temperature in the presence of 2 µM of substrate with 10 µM of ATP (for Abl1 assays) or 300 µM of ATP (kit assay) and in the presence of various concentrations of the compounds. The development reagent was then added for further 2 h room temperature incubation followed by the addition of stop solution. Fluorescence signal ratio of 445 nm (Coumarin)/520 nm (fluorescin) was examined on EnVision Multilabel Reader (Perkin-Elmer, Inc.). The effects of compounds on the kinases DDR1 and DDR2 were assessed by using a LanthaScreen Eu kinase activity assay technology (Invitrogen,USA). Kinase reactionswere performed in an 10 µL volume in low-volume 384-well plates. The kinase reaction buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl 2 , and 1 mM EGTA, and the concentration of Fluorescein-Poly GAT substrate (Invitrogen, USA) in the assay was 100 nM. Kinase reactions were initiated with the addition of 100 nM ATP in the presence of serial dilutions of compounds. The reactions were allowed to proceed for 1 h at room temperature before a 10 µL preparation of EDTA (20 mM) and Eu-labeled antibody (4 nM) in TR-FRET dilution buffer are added. The final concentration of antibody in the assay well was 2 nM, and the final concentration of EDTA was 10 mM. The plate was allowed to incubate at room temperature for one more hour before the TR-FRET emission ratios of 665 nm/340 nm were acquired on a PerkinElmer EnVision multilabel reader (Perkin-Elmer, Inc.). Data analysis and curve fitting were performed using GraphPad Prism4 software.

III. KINOMEscan TM
Kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain.
E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32°C until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 mL. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 µM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
For K d determination, an 11-point 3-fold serial dilution of compound 6j was prepared in 100% DMSO at 100x final test concentration and subsequently diluted to 1x in the assay (final DMSO concentration = 1%). Binding constants (K d values) were calculated with a standard dose-response curve using the Hill equation.
For primary screening, compound 6j was screened at the concentration of 1 µM, and the results are reported as `% Ctrl`.

IV. Protein expression and purification
Recombinant DDR1 protein fused to an N-terminal hexahistidine tag was expressed in Sf9 insect cells and purified as previously described 1 . The tag was removed by overnight TEV cleavage followed by size exclusion chromatography. The purified protein was then passed through a nickel sepharose column to remove the TEV protease. The untagged DDR1 protein was S15 concentrated to 10 mg/mL and kept frozen at -80°C until needed.
Data collection was carried out at the Diamond Light Source I04 and I04-1 beamlines (Harwell, UK). The best dataset was processed with imosflm 2 , scaled up and truncated with Aimless 3 . Initial phases were obtained by molecular replacement using PDB ID: 4CKR as an initial model in Phaser 4 . The DDR1 structure was subject to several cycles of refinement with Refmac 5 coupled with manual building with coot 6 . Ligand restrains were generated with Phenix elbow 7 and final refinement cycles were performed with autoBuster 8 . Geometry of the final model was checked with MOLPROBITY 9 . The excellent electron density maps allowed the modelling of ligands, solvent molecules and most of the polypeptide chain, except for two flexible loops ( Table S5). The figures were generated using PyMol 10 .

VI. Computational Study
All the procedure was performed in Maestro 9.9 (Schrodinger LLC). The crystal structures of DDR1 protein and Abl protein with their corresponding inhibitors were taken from the PDB. The protein was processed using the "Protein Preparation Wizard" workflow in Maestro 9.9 S17 (Schrodinger LLC) to add bond orders and to add hydrogens. All heteroatom (het) residues and crystal water molecules beyond 5 Å from het group were removed. Inhibitors were built in the LigPrep module using the OPLS-2005 force field. Glide module was used as the docking program. The grid-enclosing box was placed on the centroid of the binding ligand in the optimized crystal structure as described above, and a scaling factor of 1.0 was set to van der Waals (VDW) radius of those receptor atoms with partial atomic charges of less than 0.25. Standard precision (SP) approach of Glide was adopted to dock 6a and 6b into DDR1 with the default parameters, and the top-ranking pose was selected for energy minimization using Prime MM-GBSA, under the solvation model of VSGB.
The crystal structures of DDR1 protein with inhibitor 6c were taken from the Protein Data Bank (PDB code: 5FDP). The grid-enclosing box was placed on the centroid of the binding ligand in the optimized crystal structure as described above, and a scaling factor of 1.0 was set to van der Waals (VDW) radius of those receptor atoms with partial atomic charges of less than 0.25. The structures of the designed compounds (6a-6k) were constructed using the LigPrep module using the OPLS-2005 force field. All the solutions of the ligand were aligned with the co-crystal ligand in 5FDP using flexible ligand alignment module. Standard precision (SP) approach of Glide was adopted to dock all the optimized solutions into DDR1 with the default parameters. The docking scores were provided in Table S6, together with the experimentally determined binding affinities for these compounds. The correlation between the DockingScore and log (IC 50 ) is plotted in Figure S2.

Ⅶ Ⅶ Ⅶ Ⅶ. Western blot analysis
Primary human lung fibroblast was cultured in Medium199 (Sigma Aldrich) containing 10% fetal bovine serum (FBS) and maintained at 37 o C in a humidified incubator with 5% CO 2 and 95% air.
Cells were cultured in 100 mm tissue culture dishes in complete media (M199 with 10% FBS) until they reached a high density (~80% confluence). Then cells were cultured in 5 mL of complete media with 4-fold dilution of 6j for 24 hours. DMSO and 1 µM 7rh were added as controls. Cells were lysed and supernatants recovered by centrifugation at 13,000 rpm. Protein concentrations were measured and equal amounts of total protein were separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Bio-Rad; Hercules, CA) followed by blockade for 1 hour in 5% donkey serum in TBS-T. Membranes were incubated overnight at 4 o C with primary antibody phospho-DDR1 (Tyr792, Cell Signaling #11994) and phospho-p38 MAP kinase (Thr180/Tyr182, Cell Signaling #9211). Membranes were incubated with corresponding HRP-conjugated secondary antibody (Pierce Biotechnologies; Rockford, IL) for 1 hour. Specific bands were detected using the enhanced chemiluminescence reagent (ECL, Perkin Elmer Life Sciences; Boston, MA) on antoradiographic film.
The westernblot analysis ( Figure S3) was generally following the procedure provided by Cell signaling Technology Inc. and described before (J. Med. Chem. 2013, 56, 3281-3295). Briefly, after the indicated treatment, cell lysates were collected by using 1X SDS sample buffer. After being sonicated and being boiled, the cell lysate were then loaded to 8-12% SDS-PAGE and separated by electrophoresis. Separated proteins were then electrically transferred to a PVDF film. After being blocked with 1×TBS containing 0.5% Tween-20 and 5% non-fat milk, the film was incubated with corresponding primary antibody and then with HRP-conjugated secondary antibody. And the protein lanes were visualized using ECL Western Blotting Detection Kit (Thermo Scientific, USA).

Pharmacokinetics study in ICR mice:
Compound 6j was dissolved in EtOH and a bit of 0.1 mol/L HCl was added to make it a clear S20

Pulmonary fibrosis experiments in mice:
To induce pulmonary damage, 6-to 8-week-old sex-and age-matched wild type or slie mice (at least five animals per group) were intranasally dropped with bleomycin (Nippon Kayaku, Tokyo, Japan) at 5mg/kg BW as described previously 11 . The inhibitors were dissolved in water at a concentration of 5 mg/mL and given to the mice orally by gavage twice a day.
All animal studies were performed according to the protocols and guidelines of the institutional care and use committee.

Determination of hydroxyproline content
Hydroxyproline accounts for 13.4% of the total amino acids of collagen; thus its content can be used to reflect the severity of fibrosis. A commercial hydroxyproline kit from Jiancheng Institute of Biotechnology (Nanjing, China) was used following the provider's instructions. Briefly, fresh lung tissues were weighted and hydrolyzed to release hydroxyproline. After a series of chemical reactions, a pink color solution was formed and then subjected to measurement of absorbance at 560 nm. The hydroxyproline content of each sample was calculated by comparing with the standards. Results were expressed as micrograms of hydroxyproline per gram wet lung weight.

Histological analysis
Mice were anaesthetized, and the right heart received perfusion with calcium and magnesium-free buffered saline containing heparin. The lungs were dissected and then fixed in 10% neutral formalin for 24 h. Paraffin tissue sections (4 µm) were prepared for H&E and Masson's trichrome staining or immunohistochemistry. Masson's trichrome staining was performed according to the manufacturer's instructions (Baso, Zhuhai, China) Ⅸ Ⅸ Ⅸ Ⅸ. Antitumor activity of compound 6j Liquid colony forming assay Panc-1 pancreatic cancer cells were cultured in 6-well tissue culture plates at low density (250 cells per well) in 2 ml of media with 5% FBS with or without 6j for approximately 1.5-2 weeks, or until significant colony formation. 6j was added in 4, 4-fold dilutions, to respective wells, a DMSO control was added to respective wells to demonstrate the vehicle-independent effect. Cells were then fixed with 10% formalin and stained with crystal violet. Images were analyzed with Image J.

Wound healing (scratch) assay
Panc-1 pancreatic cancer cells were cultured in 6-well tissue culture plates at high density (~90% confluence) in 2 ml of media with 5% FBS. Uniform scratches were made down the center of each well with a p20 pipette tip, cells were gently washed with PBS to remove the loose cell debris, and drug (6j) was added in media containing 5% FBS. 6j was added in four, 4-fold dilutions, to respective wells, a DMSO control was added to respective wells to demonstrate the vehicle-independent effect. Cells were plated on respective culture conditions and images from the S21 center of each well were taken at times 0, 10, 20, and 30 hours. The wound width (µm) was measured using NIS Elements AR 2.30 software. The initial wound width was used to verify consistency in scratches. Figure S4. Inhibition of DDR1 reduces colony formation (A) and migration of cancer cells (B). Panc-1 cells were plated at low density in media in the presence or absence of controls or the indicated concentration of 6j. Colony formation was evaluated after 1.5-2 weeks by fixing and staining with crystal violet. The effect of 6j on cell migration was determined through a 'scratch' assay. Panc-1 cells were grown to confluence in a 6 well dish. A scratch was made using a p20 pipette tip and cell migration into the wound was determined at 12, 24, 48, 60, and