GW4869 br Experiment br Azacoccones C and E were obtained
Azacoccones C and E were obtained in our previous work . Anti-PHGDH antibody (14719-1-AP) was purchased from Proteintech Group Inc (China). And D-(-)-3-phosphoglyceric GW4869 disodium salt (sc-214793) was brought from Santa Cruz Blotechnology (U.S.). All of them were stored at −20 °C. MDA-MB-231, MDA-MB-468 and Hela cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences.
2.2. Molecular docking
The crystal structure of human PHGDH (PDB code: 2G76) was ob-tained from the Protein Data Bank (http://www.rcsb.org). Molecular docking was implemented in ICM-Pro 3.8.2 modeling software on an Intel i7 4960 processor (MolSoft LLC, San Diego, CA). Ligand binding pocket residue was selected by graphical tools in the ICM software to create the boundaries of the docking search. Chemical structures of compounds were input as mol2 files for docking. In the docking cal-culation, default parameters were applied to calculate the potential energy maps of the receptor. Compounds were imported into the ICM filed as an index project. Conformational sampling was based on the
Monte Carlo procedure, and finally the ligand with the lowest energy and the most favorable orientation was selected .
2.3. The expression and purification of PHGDH and PSAT1
2.4. Enzyme inhibition assays
To assay the inhibitory effects of azacoccones C and E on PHGDH, NADH which was generated through a coupled reaction with phos-phoserine amino transferase (PSAT1) was monitored by fluorescence (340/460 nm; Ex/Em) every minute for 20 min on a SpectraMax M5e plate reader. Briefly, 40 μL PHGDH protein (880 nM) was incubated with 10 μL compound solution at room temperature for 30 min. Then 50 μL assay mix containing 50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 2 μM PSAT1, 0.81 mM NAD+, 0.73 mM 3-phosphoglycerate (3-PG) and 30 mM glutamate was added. For IC50 assays, twofold serial dilutions of compound solution ranging from 100 μM to 0.3 μM were preincubated with enzyme for 30 min before initiating the enzyme reaction with substrate mix. For time-dependent IC50 assays, compound preincuba-tion time was 4, 1, or 0.5 h. For inhibition pattern measurements, compound (0, 5, 10, 20 μM) and enzyme were preincubated for 30 min. Initial rate plots were fit using Prism .
2.5. Microscale thermophoresis (MST) assay
The binding capacity of PHGDH to azacoccones C and E was de-termined on Monolith NT.115 (NanoTemper Technologies, München, Germany). The protein was diluted to 10 μM in standard buffer (20 mM HEPES pH 7.5, 0.05 (v/v) % Tween-20) and incubated for 30 min with an equal volume of 20 μM NT647 at room temperature. The labeled protein was separated from the excess dye by gravity flow column. The compounds were diluted into 12 standard dilutions with standard buffer in the appropriate concentration range. After incubation at 22 °C
Fig. 2. In vitro enzyme inhibition assay. (A) Dose-dependent inhibition of PHGDH by azacoccones C and E. (B) IC50 of azacoccone C against PHGDH and IDH1. (C) IC50 of azacoccone E against PHGDH and IDH1.
Inhibition activities of the compounds.
Compound Kd (μM)a IC50 for PHGDH activities (μM) IC50 for cancer cell activities (μM)
a The Kd value was automatically calculated by the curve fitting, and presented as means ± SD for three experiments.
Fig. 3. The inhibition activities of azacoccones C and E. (A and B) Measurement of affinity of azacoccones C and E to PHGDH by MST with Kd values of 61.30 ± 5.62 and 15.20 ± 2.94 μM, respectively. (C) Cellular thermal shift assay (CETSA) in Hela cells treated with 50 μM azacoccone E confirmed its binding capacity with PHGDH at the cellular level.
for 15 min, the samples were loaded into NT hydrophobic capillaries and measured with 20% laser power and 100% LED power. The dis-sociation constant Kd was fitted by NTA analysis software [13,14].
2.6. Cellular thermal shift assay (CETSA)
The cellular thermal shift assay (CETSA) was performed to confirm the interaction between PHGDH and azacoccone E in vitro by Western blot assay, and the technique was based on ligand-induced stabilization of target proteins. Briefly, Hela cells cultured with 90% confluent in 100 × 20 mm tissue culture dishes were treated with media containing DMSO (equal volume with compound) or azacoccone E (50 µM) for 8 h. After treatment, the cells were isolated with trypsin, collected by cen-trifugation, and then resuspended in PBS. The cell suspension was di-vided equally into 5 PCR tubes and heated at gradient temperature from 54 °C to 62 °C for 5 min. Subsequently, cells were analyzed by Western blot assay .
Cells were routinely grown in high Glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, 10% (v/