JIAN GAO

Method(s)

Molecular docking and dynamic simulation:
The molecular docking was performed using Ligand Docking implanted in Maestro 11.9. Parameters were maintained at the default configuration. The docked structure of the compounds complexed within the active pocket of 4GV1 was used as the initial structures for MD calculations using Amber. ff99SB and gaff2 force fields were applied to the complex and the resulting system was subjected to a double-fold minimization (5000 cycles of steepest descent minimization and 5000 cycles of conjugate gradient minimization) protocol for three times, with cartesian restraints imposed on heavy atoms, Cα atoms of the complex and cartesian restraints released respectively. The system was heated from 0 K to 300 K under cartesian restraints over a period of 100 ps and subsequently equilibrated for 100 ps with cartesian restraints and 100 ps without with cartesian restraints. Starting from the last frame of the equilibration, a production simulation was performed for 10 ns using the Berendsen barostat and Langevin thermostat under a constant temperature of 300 K and pressure of 1 atm. Finally, the GBSA binding energy of the last 2 ns and the dihedral angle of the last 5 ns were analyzed.
Akt1 and Akt2 inhibitory activity assay:
The Akt1 and Akt2 inhibitory activities were determined by Sundia MediTech Company using the mobility shift assay. Akt1 and Akt2 kinase were purchased from Carna Biosciences. The kinase reactions were carried out in a 384 OptiPlate (PerkinElmer) with 10 µL reaction volume per well containing Akt1or Akt2 kinase, Caliper substrate 6 (GL), test compound and ATP in assay buffer. After incubation for 1 h at room temperature, the reaction was stopped by the addition of 30 µL reaction stop buffer. The plate was sealed and incubated for 30 s at room temperature, and the resulting signal was measured on Caliper EZ Reader Ⅱ. The percent inhibitions were calculated as follows: Percent inhibition = (max-sample Ratio)/ (max-min)*100 (“Min” means the Ratio of no enzyme control and “max” means the Ratio of DMSO control). 
Anti-proliferative assay:
The cancer cells or HaCaT cells were treated with the compounds at various doses for 72h, and cell proliferation was tested by a sulforhodamine B (SRB) protein assay. Cells (5000/well) were incubated with 10% trichloroacetic acid (TCA) for 1 h (4 ℃) and then stained with SRB for 20 min. The SRB was washed away with 1% glacial acetic acid, and 100 μ l of 1% Tris-base was added to each well. The optical density (OD) was determined at 515 nm by a Multiskan Spectrum plate reader (Thermo Electron Corporation, Marietta, OH, USA).
Western blot assay:
Whole protein extracts from cultured cells or tissues were prepared and subjected to western blotting. Protein samples (40 μg) were loaded and run on 10% SDS-PAGE, then transferred to PVDF membranes (Merck Millipore, IPVH00010) and incubated with primary antibodies overnight at 4 °C. After that, membranes were washed three times by PBS with 0.1% Tween-20 (T-PBS), and incubated with secondary antibodies for 1 h at room temperature. After being washed three times with T-PBS, membranes were incubated with Western Lightning Plus-ECL reagent (PerkinElmer, NEL105001EA), and then were exposed using Amersham Imager 600 (General Electric Company, USA). The primary antibodies were listed as follows: Akt1 (Cell Signaling Technology, 75692s), Akt2 (Cell Signaling Technology, 2964S), Akt3 (Cell Signaling Technology, 14982S), pAKT-Ser473 (Santa Cruz Biotechnology, sc-514032), Cleaved Caspase 3 (HuaBio, ET1602-47), c-PARP (Cell Signaling Technology, 5625s), GAPDH (DiagBio, db106). Appropriate secondary antibodies (MultiSciences Biotech, GAM007 and GAR007) and ECL (PerkinElmer, The Netherlands) were used to visualize the protein signaling. 
hERG inhibition assay:
Currents were recorded from HEK-293 cells, using the whole-cell patch-clamp technique. The cells were transferred to a perfusion chamber and the perfusion was performed with extracellular fluid. The extracellular fluid (mM): KAspartate, 130; MgCl2, 5; EGTA, 5; HEPES, 10; Tris-ATP, 4; pH 7.2. Electrodes were pulled using a dual-stage glass micropipette puller (Narishige PC-10, Japan). Current traces of hERG channels were elicited by applying a pulse from -80 mV to +40 mV for 4 s followed by a step to -40 mV for 2 s. The procedure was repeated every 20 seconds. After the maximum current was stabilized, the tested compounds (3 μM) were perfused. The inhibition rate was calculated when the current was stable. 
Liver microsome stability assay:
Microsomes from different species were purchased from Ruide Research Institute for Liver Diseases (Shanghai) Co. Ltd. (Human: SUNK, Monkey: CYJC, Beagle Dog: DMXD, Rat: JPXY, Mouse: STOM-2). 1 mg/mL microsome solution was mixed with 20 mL of 50 mM NADPH (Roche) solution to prepare a microsome-NADPH solution. 500 µL of the microsome-NADPH solution was pre-warmed at 37 ℃ for 5 minutes. Hu7691 solution was then added to a final concentration of 50 μM to initiate the reaction. The incubation mixture was kept at 37 ℃ and 200 µL aliquots were taken at 0, 10, 30 and 60 minutes. In each aliquot, the reaction was quenched using 400 µL of acetonitrile containing 5 ng/mL internal standard compound (Verapamil, Aladdin). After quenching, the mixtures were vortexed and centrifuged at 4 ℃. The supernatant was transferred and 10 µL was injected into a XEVO TQ-S LC-MS/MS system. The peak area ratio of a test article versus the internal standard was used in the calculation of the rate of disappearance of a test article.
Plasma protein binding rate assay:
The plasma from different species such as Beagle dog, monkey and rat were provided by Center for Drug Safety Evaluation and Research, College of Pharmaceutical Sciences, Zhejiang University. The human plasma was provided by the Second Affiliated Hospital Zhejiang University School of Medicine. A 300 μL plasma containing 50 ng/mL or 2500 ng/mL Hu7691 was added to the sample chamber of Thermo Equilibrium Dialysis Device (Red Device Insert), and 500 μL PBS dialysate was added to the buffer chamber. After sealing, the mixture was incubated at 37 ℃ for 8h. An aliquot of 50 µL of each mixture was quenched using 500 µL of acetonitrile containing 5 ng/mL internal standard compound (Loratadine, Aladdin). After quenching, the mixture was vortexed and centrifuged, the resulting solution was injected into a Waters XEVO TQ-S system. The PPB % = (1-concentration in PBS/concentration in plasma) ×100. 
Pharmacokinetic and metabolites studies:
SD rats or Beagle dogs were administered Hu7691 by oral gavage in saline. Venous blood (100 µL) samples of rats were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 24, 30, 48, 54 and 72 h for oral gavage or 0, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 24 h for intravenous injection. Venous blood (100 µL) samples of dogs were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 10, 24, 30, 48, 72 and 96 h. Plasma was separated from whole blood by centrifugation and stored at -20 ℃ until analysis. The rat plasma (from 0.5, 1, 2, 4, 6 h), urine (from 4~7, 24~48, 48~72 h), excrement (from 0~4, 4~7, 7~24 h) and bile (from 0~6, 6~12, 12~24 h) were collected for metabolites study. For pharmacokinetic studies, an aliquot of 50 µL plasma was quenched using 300 µL of acetonitrile containing 5 ng/mL internal standard compound (Loratadine, Aladdin). For metabolites study, the samples were quenched using 300 µL of acetonitrile. After quenching, the mixture was vortexed and centrifuged, the resulting solution was injected into an AB Sciex Qtrap 5500 LC-MS/MS system (pharmacokinetic study) or Waters XEVO G2-XS Q-TOF LC-MS/MS system (metabolites study). The Cmax, Tmax, T1/2 and AUC were evaluated using Winnolin 8.0. 
Toxicology evaluation:
The rats were randomly divided into four groups and received Hu7691 (25, 50, 75 mg/kg, i.g.) or vehicle (saline, i.g.) once daily for 28 days. The body weight of mice was recorded every 7 days. During the experiment, the scab clinical symptom on rat back skin was recorded. At the end of the experiment, mice were sacrificed, the serum hematological parameters and serum biochemistry parameters were recorded on Sysmex XT-2000i and Roche Cobas c 311, respectively.
In vivo evaluation of tumor inhibition:
BALB/c (nu/nu) mice were used for the experiments. When 786-O or KHOS xenograft tumors reached 100~300 mm3, mice were randomly divided into five groups and received Hu7691 (12.5, 25, 50 mg/kg, i.g.), AZD5363 (50 or 100 mg/kg, i.g.) or vehicle (saline, i.g.) once daily for 22 days. The body weight of mice and tumor volume was recorded every 3 days. At the end of the experiment, mice were sacrificed, the tumors were weighed and photographed.