br PHGDH microscale thermophoresis MST
PHGDH, microscale thermophoresis (MST) method which had pre-viously been used to investigate protein-protein, small organic mole-cule-protein and antibody-protein interactions was performed. Measuring the thermophoretic behavior of a protein in the presence of different ligand concentrations by MST allows quantitative analysis of molecular interactions in solution. In this study, MST was utilized for the confirmation of the dissociation constant (Kd) of azacoccones C and E to PHGDH. As shown in Fig. 3A and B, the Kd values of azacoccones C and E to PHGDH were 61.30 ± 5.62 and 15.20 ± 2.94 μM, respec-tively. Therefore, the results further confirmed the specific binding of 3.3. Azacoccones C and E selectively inhibited the proliferation of high PHGDH-expressing cell lines
Human breast cancer cell lines MDA-MB-231 and MDA-MB-468, and cervical cancer cell line Hela were initially selected to examine the PHGDH expression level by Western blot assay. PHGDH protein content in these samples was normalized by the signal intensity of structure protein β-actin. As is shown in Fig. 4A, MDA-MB-468 22862-76-6 and Hela cells exhibited higher expression level of PHGDH than that in MDA-MB-231 cells by analyzing the quantitation of band intensity. After ex-posure to azacoccones C and E for 3 days, MDA-MB-468 and Hela cells
Fig. 7. Low-energy binding conformations of azacoccone E bound to PHGDH generated by molecular docking. (A) Detailed view of azacoccone E binding in the allosteric site of the enzyme. (B) Ligand interaction diagram of azacoccone E with PHGDH.
with higher expression of PHGDH showed more sensitive to aza-coccones C and E than MDA-MB-231 cells (Fig. 4B and C), indicating that azacoccones C and E induced specific cancer cells to death through a PHGDH-dependent manner. To test whether tumor cells were more sensitive to azacoccone compounds in a serine-deplete media, low PHGDH-expressing lines MDA-MB-231 and high PHGDH-expressing lines MDA-MB-468 were cultured respectively in serine-replete or serine-deplete media. The results showed that azacoccone E (50 μM) inhibited the proliferation of MDA-MB-468 cells by 37.57% in serine-replete medium. When the serine in the medium was withdrawn, the inhibition effect of compound on MDA-MB-468 proliferation can be improved by 70.13%. However, the azacoccone compounds had no effect on MDA-MB-231, futher confirming that azacoccones C and E were selectively toxic to cells with high serine synthesis activity (Fig. 4D and E).
3.4. Azacoccone E-induced apoptotic effect detected by flow cytometry analysis and Hoechst 33258 staining
The apoptosis effects of azacoccone E were monitored by flow cy-tometry analysis. Followed by an exposure to 75 and 150 μM of aza-coccone E for 72 h, the apoptotic cells positive for annexin-V and PI increased from 11.5% (vehicle) to 15.8% and 27.4% at 75 and 150 μM, respectively (Fig. 5A), which disclosed that azacoccone E showed a
dose-dependent proapoptotic activity.
To determine the morphological changes induced by azacoccone E in Hela cells, Hoechst 33258 staining was carried out. Hela cells were treated with a variety of concentrations (0, 50, 100 µM) of azacoccone E for 72 h. Then the cells were stained with Hoechst 33258 and the ty-pical morphological features of later stage apoptosis increased con-densation of chromatin material and fragmentation of the nuclei were observed. The number of apoptotic nuclei significantly increased with the increase of the concentration of azacoccone E to 100 µM (Fig. 5B).
3.5. Manner of azacoccone E inhibition
We sought to more deeply characterize the inhibitory mechanism of azacoccone E on PHGDH. As displayed in Fig. 6A, B and Table 2, in the presence of azacoccone E, the maximal reaction rate (Vmax) values of 3-PG was decreased in a concentration-dependent manner while the ki-netic constants (Km) value of 3-PG was not obviously affected, sug-gesting a non-competitive inhibition manner of azacoccone E against PHGDH. Furthermore, in order to assess whether the inhibition was time-dependent, azacoccone E and PHGDH were preincubated before initiating the enzymatic reaction. As expected, azacoccone E was pro-gressively more potent with increasing preincubation time (Fig. 6C).
3.6. Molecular docking revealed the possible binding mode of azacoccone E with PHGDH
In order to further elucidate the binding mode of azacoccone E with PHGDH, molecular docking was performed by using ICM-Pro 3.8.2 modeling software (MolSoft LLC, San Diego, CA). The lowest-energy binding conformation of azacoccone E was shown as Fig. 7A. From the generated docking model, azacoccone E was well fitted an allosteric site of the enzyme, where it was away from the active site. This ligand binding pocket was relatively hydrophilic in the entrance, with two arginines protruded from the shallow pocket. Hydrogen binding was predicted between Arg118 with two phenol group, Arg134 with lactam carbonyl (Fig. 7B). The predicted binding of azacoccone E in the al-losteric site of the enzyme was consistent with observed enzyme in-hibition kinetics.