Novel ATR/PARP1 Dual Inhibitors Demonstrate Synergistic Antitumor Efficacy in Triple-Negative Breast Cancer Models

Abstract

Concomitant inhibition of ataxia telangiectasia and Rad3-related protein (ATR) and poly ADP-ribose Polymerase (PARP) pathways is a promising strategy in cancer therapy, potentially expanding the clinical utility of ATR inhibitor (ATRi) and PARP inhibitor (PARPi). A novel series of ATR/PARP1 dual inhibitors is developed through the pharmacophore fusion of AZD6738 and Olaparib. Among them, B8 emerges as the most promising candidate, exhibiting potent ATR (IC₅₀: 17.3 nM) and PARP1 (IC₅₀: 0.38 nM) inhibition. B8 effectively reduced cell viability, induced apoptosis, and caused G2/M cell cycle arrest in TNBC cells. Additionally, B8 significantly impaired TNBC colony formation, migration, and invasion. Mechanistically, B8 induces DNA damage, evidenced by increased γH2AX levels. In in vivo studies, B8 suppressed tumor growth more effectively than the combination in MDA-MB-468 xenografted mice, with no significant body weight loss. B8 also enhanced γH2AX expression in tumor tissues. These findings confirm the synergistic effects of ATR/PARP1 co-inhibition and highlight the potential of this novel inhibitor class for TNBC therapy.

Keywords: ATR; Anticancer; Dual Inhibitor; PARP1; TNBC.

Figure 1

Biological functions of PARP1 and ATR.

Figure 2

Chemical structures of PARPi and ATRi. A) PARPi approval drugs (1‐6); B) representative ATRi clinical candidates (7‐13).

Figure 3

Bioinformatics analysis of the relationship between ATR and PARP1. A) The workflow of bioinformatics analyses of ATR‐PARP1 and cluster analysis network diagram of ATR and PARP1 interaction proteins related to cell cycle and apoptosis; B) Predicted ATR‐ and PARP1‐related proteins involved in the regulation of cell cycle and apoptosis, respectively; C) Correlation between the mRNA expression of ATR and PARP1 in breast cancer samples (n = 1089).

Figure 4

A) Binding mode of AZD6738 docked with rationally designed PI3Kα mutant that mimics ATR (PDB code: 5UL1). B) Co‐crystal structure of Olaparib in complex with PARP1 (PDB code: 5DS3). Green dashed lines indicate hydrogen bond interactions. The carbons of small molecules are in black. The oxygen atoms of small molecules are in red. The nitrogen atoms are in purple. The sulphur atom is in yellow, while the fluorine atom is in pale blue.

Figure 5

Design strategy of ATR/PARP1 dual inhibitors based on the pharmacophores of AZD6738 and Olaparib.

Figure 6

Head‐to‐head docking experiment of 8 (AZD6738) A), C1 B), and C2 C) with the designed PI3Kα mutant that mimics ATR (PDB: 5UL1) using Schrodinger 2024. Yellow dashed lines indicate hydrogen bond interactions.

Scheme 1

Synthesis of key intermediate 23. ^a Reagents and conditions: a) TEA, EtOH, rt, 16 h, 66.2%; b) NaHMDS, Toluene, 0 °C ∼ rt, 2.5 h, 56.4%; c) KOH, H₂O₂ (30%), MeOH/DCM (1:1), 40 °C, overnight, 56.3%; d) NaOH, NaClO·5 H₂O, H₂O, rt, 16 h, 74.6%; e) Boc₂O, DMAP, TEA, DCM, rt, overnight, 55%; f) K₂CO₃, Pd(dppf)Cl₂, 1,4‐Dioxane/H₂O, 100 °C, 4 h, 57.3%; g) HCl in 1,4‐Dioxane (4 M), MeOH, rt, 2 h, 93%; (h) aqueous NaOH, THF, 1,4‐Dioxane, 75 °C, overnight, 51.9%.

Scheme 2

Synthesis of key intermediate 28. Reagents and conditions: a) TEA, DCM, rt, 2 h, 79.7%; b) K₂CO₃, Pd(dppf)Cl₂, 1,4‐Dioxane/H₂O, 100 °C, 4 h, 87.1%; c) H₂, Raney Nickel, NH₃/MeOH, rt, 16 h, 39.6%; d) aqueous NaOH, THF, 1,4‐Dioxane, 75 °C, overnight, 73.4%.

Scheme 3

Synthesis of compounds A1–A5 and B1–B6. Reagents and conditions: a) DIPEA, EDCI, HOBT, DMF, rt, 4 h, 64% ∼ 89%; b) NaOH, H₂O, 40 °C, 2 h, 67% ∼ 86%; c) DIPEA, EDCI, HOBT, DMF, rt, 6 h, 61% ∼ 78%; d) Et₃N, DCM, 0 °C ∼ rt; 3 h, 73%; e) HCl in 1,4‐Dioxane (4 M), MeOH, rt, 3 h, 88%; f) 29, DIPEA, EDCI, HOBT, DMF, rt, 6 h, 70%.

Scheme 4

Synthesis of compounds A6–A9 and B7–B11. Reagents and conditions: a) 23 or 28, DIPEA, CDI, rt, 5 h, 85% ∼ 93%; b) DIPEA, EDCI, HOBt, DCM, rt, 6 h, 71% ∼ 84%; c) NaOH, H₂O, MeOH, rt, 2 h, 87% ∼ 91%; d) Et₃N, DCM, 0 °C ∼ rt; 3 h, 53% ∼ 65%; e) 34, DIPEA, EDCI, HOBt, DCM, rt, 6 h, 71%.

Scheme 5

Synthesis of compounds A10–A12 and B12–B14. Reagents and conditions: a) DIPEA, DMF, 60 °C, 6 h, 74%; b) NaOH, H₂O, MeOH, rt, 2 h, 78% ∼ 85%; c) 23 or 28, DIPEA, EDCI, HOBt, DMF, rt, 6 h, 63% ∼ 77%; d) DIPEA, EDCI, HOBt, DCM, rt, 6 h, 81% ∼ 88%.

Figure 7

Diagram summary of structural optimization of ATR/PARP1 dual inhibitors. The processes were divided into Stages I–V.

Figure 8

Cell cycle profile and distribution of MDA‐MB‐231 cells a) and MDA‐MB‐468 cells b) under 48 h treatment with different concentrations of compounds as indicated. Quantitative data of cell cycle distribution were calculated as the mean ± SD of three sets of experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = no significance.

Figure 9

A,B) Annexin V‐FITC/PI dual staining assay to determine the apoptosis of MDA‐MB‐231 cells and MDA‐MB‐468 cells after 72 h of various treatments as indicated. C,D) Quantitative data of flow cytometry were calculated as the mean ± SD of three sets of experiments (n = 3). E) Western blotting of BCL‐2, BAX, Cleaved‐caspase‐3, and caspase‐3 in MDA‐MB‐468 cells exposed to different compounds for 48 h. F) Relative densitometric values of BCL‐2, BAX, Cleaved‐caspase‐3, and caspase‐3. Quantitative data were calculated as the mean ± SD of three sets of experiments (n = 3). Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 10

B8 exerts anti‐tumor activity on TNBC cells in vitro. A,B) Colony formation capacity of MDA‐MB‐231 and MDA‐MB‐468 cells after treatment with compounds for 10 days. C) Representative images of wound‐healing assay and the percentage of 48 h wound‐healed distance in MDA‐MB‐231 cells. Scale bar = 500 µm. D,E) Representative images of the transwell migration assay and the percentage of 24 h areas of migrated cells per field of cells in MDA‐MB‐231 cells and MDA‐MB‐468 cells. Scale bar = 100 µm. F,G) Representative images of the transwell invasion assay and the percentage of 24 h areas of invasion cells per field of cells in MDA‐MB‐231 cells and MDA‐MB‐468 cells. H) Western blotting and relative densitometric values of Vimentin and E‐cadherin in MDA‐MB‐468 cells exposed to different compounds as indicated for 48 h. Quantitative data were calculated as the mean ± SD of three sets of experiments (n = 3). Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 11

B8 can inhibit DSB repair. A) Representative images of the comet assay for the measurement of DNA damage in MDA‐MB‐231 cells under treatment with the indicated compounds for 24 h. Scale bar = 50 µm. B,C) Representative images of immunofluorescence staining of γH2AX foci in MDA‐MB‐231 B) and MDA‐MB‐468 C) cells treated with compounds as indicated for 48 h. Cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). D) Western blotting of ATR, PARP1, γH2AX, p‐CHK1/CHK1, and p‐CDK1/CDK1 in MDA‐MB‐468 cells exposed to different compounds as indicated for 48 h. E,F) Statistical analysis of ATR E) and PARP1 F) protein levels. G) Mechanism of action of B8 in the treatment of TNBC. The data are shown as the mean ± SD of three independent experiments. Scale bar = 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 12

Molecular docking and binding modes of compound B8 with ATR and PARP1, respectively. A) Compound B8 docking using PI3Kα mutant (a mimetic ATR protein, PDB ID: 5UL1). The close view of the key residues from the protein with B8 was presented in the right diagram. B) compound B8 docking with PARP1 co‐crystal structures (PDB ID: 5DS3). The close view of the key residues from the protein with B8 was presented in the right diagram. Only the surrounding amino acid residues with close proximity to B8 are shown for clarity. See Supporting Information for the 2D view of the interactions.

Figure 13

Compound B8 exerts anti‐tumor efficacy in the MDA‐MB‐468 xenografted mice model. A) Schematic procedure for the in vivo animal experiment. B) Tumor volumes of each group of mice were measured at the indicated time after treatments. C) Animal weights of each group of mice were measured at the indicated time after treatments. D) Tumor weights in each group mice following treatment with different compounds for 28 days. E) Representative image of tumors. F) IHC staining of Ki67, γH2AX, CD8, and cleaved‐caspase 3 levels in MDA‐MB‐468 xenograft tumors (scale bar = 100 µm). G) H&E staining of heart, liver, kidney, lung, and spleen tissues of each group xenograft mice (scale bar = 50 µm). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.