PD-L1 dimerisation induced by biphenyl derivatives mediates anti-breast cancer activity via the non-immune PD-L1–AKT–mTOR/Bcl2 pathway

Figures & data

Scheme 1. Synthesis of compounds 13h-1–3, 13b-4–6, 7b-2, 7b-4, 12j-3–4. Reagents and conditions: (a) K₂CO₃, NaI, DMF, 60 °C, 2 h, 35%−55%; (b) Cs₂CO₃, DMF, 60 °C, 2 h, 60%−80%; (c) appropriate amine, HOAc, NaBH₃CN, DMF, rt, overnight, 10%−40%.

Table 1. The inhibitory activities of compounds 13h-1–3, 13b-4–6, 7b-2, 7b-4, and 12j-3–4 on the PD-1/PD-L1 interaction.

Figure 1. Detailed interactions of compound 12j-4 (the green stick) with the PD-L1 dimer. The crystal structure of the dimeric PD-L1 protein was taken from PDB ID: 5J89. Chain A is a solid cyan ribbon, while chain B is a solid magenta ribbon. The green dashed lines represent H-Bond interactions. The magenta dashed line represents hydrophobic interactions. The orange dashed lines represent electrostatic interactions. The cyan-dashed line represented halogen interactions.

Table 2. IC₅₀ values (μM) for the MDA-MB-231, MCF-7, A549, HCC827, and MRC-5 cell lines treated with 12j-4, 13h-1, and 13h-3.

IC50(μM)	12j-4	13h-1	13h-3
MCF-7	4.71 ± 0.44	4.81 ± 0.45	5.52 ± 0.57
MDA-MB-231	2.68 ± 0.27	4.67 ± 0.79	4.96 ± 0.82
A549	7.79 ± 0.69	7.27 ± 0.71	13.47 ± 0.86
HCC827	3.24 ± 0.09	6.16 ± 0.76	5.65 ± 0.56
MRC-5	10.28 ± 0.12	9.31 ± 0.16	11.14 ± 0.20

Figure 2. Viability of MDA-MB-231 cells treated with 4 μM of compounds (A) 13h-1–3, 13b-4–6, 7b-2, 7b-4, and 12j-3–4, and (B) MDA-MB-231, (C) A549, (D) HCC-827, (E) MCF-7, and (F) MRC-5 treated with compounds 12j-4, 13h-1, and 13h-3 with 0, 1, 2, 4, 8, 16, and 32 μM for 60 h, respectively. Cell viability was determined using the Cell Counting Kit-8 assay. Positive control: paclitaxel and BMS202. Negative control: 1% DMSO. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Inhibition of the proliferation and migration of MDA-MB-231 cells with compound 12j-4. (A) The clone formation rates of MDA-MB-231 cells were treated with 12j-4 for 24 h at a concentration of 1 μM. (B) Migration ability of MDA-MB-231 cells treated with 12j-4 (1 μM) for 24 h and 48 h. Positive control: paclitaxel and BMS202. Negative control: 1% DMSO. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. 12j-4 induced apoptosis in MDA-MB-231 cells. (A) The apoptosis of MDA-MB-231 cells was evaluated by flow cytometry after they were treated with 12j-4 for 48 h at a concentration of 4 μM. (B) The expression of Bcl-2, Caspase 3, cleaved-Caspase 3, PARP, and cleaved-PARP proteins was detected by Western blot in MDA-MB-231 cells treated with compound 12j-4 for 48 h at a concentration of 1, 2, and 4 μM, respectively. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. The expression level of PD-L1 is regulated by compound 12j-4. MDA-MB-231 cells were treated with 12j-4 for 48 h at a concentration of 1, 2, and 4 μM, respectively. BMS202 was used as a positive drug. (A) The expression of the PD-L1 protein and (B) PD-L1 mRNA was detected by Western blot and qPCR, respectively. The expression of PD-L1 protein was detected by (C) Western blot after MDA-MB-231 cells were treated with or without 12j-4 (4 μM) combined with GSK-3β inhibitor (40 nM) for 48 h. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6. Representative immunofluorescence images of PD-L1 (red) and nucleic (blue) foci in MDA-MB-231 cells treated with or without 12j-4 (4 μM) combined with GSK-3β inhibitor (40 nM) for 48 h. (A) PD-L1 protein expressed in organelle membranes with cell membrane ruptured; (B) PD-L1 protein expressed in organelle membranes with cell membrane non-ruptured.

Figure 7. The location of 12j-4/PD-L1 and the interaction between both molecules. (A) The structure of 12j-4-FITC. (B) PD-L1 protein expressed in organelle membranes with cell membrane ruptured; (C) PD-L1 protein expressed in organelle membranes with cell membrane non-ruptured. Representative immunofluorescence images of PD-L1 (red), nucleic (blue), and 12j-4 (green) foci in MDA-MB-231 cells treated with 12j-4-FITC (4 μM) for 48 h.

Figure 8. Blocking of the AKT/mTOR pathway by 12j-4 in MDA-MB-231 cells. (A) Representative immunofluorescence images of PD-L1 (red), p-AKT (green), and nucleic (blue) foci in MDA-MB-231 cells treated with 12j-4 (4 μM) for 24 h. (B) Detection of the expression of AKT, p-AKT, mTOR, and p-mTOR proteins by Western blot in MDA-MB-231 cells treated with compound 12j-4 for 48 h at a concentration of 1, 2, and 4 μM, respectively. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9. Anti-tumour effect of 12j-4 in nude mice. (A) 12j-4 inhibited tumour growth as indicated by the tumour volume. (B) Body weight curve of mice during treatment. (C) Tumour weight. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 10. The mechanism of 12j-4 inducing the tumour cells’ apoptosis and downregulating the expression of PD-L1.