Quinazoline Derivative kzl052 Suppresses Prostate Cancer by Targeting WRN Helicase to Stabilize DNA Replication Forks

Abstract

WRN helicases play a key role in DNA replication, repair, and other processes in a variety of tumors. It has become one of the hot targets of genotoxic drugs, but the effect and mechanism of targeting WRN against prostate cancer is still unclear. In our previous study, we found a quinazoline compound kzl052, which has a WRN-dependent inhibitory effect on prostate cancer cells, but its molecular mechanism needs to be further explored. In this study, kzl052 significantly inhibited the growth of PC3 (IC₅₀ = 0.39 ± 0.01 μM) and LNCaP (IC₅₀ = 0.11 ± 0.01 μM) cells in vitro and showed a good inhibition effect on PCa in vivo. It inhibits PC3 cell growth by binding to WRN proteins and affecting its non-enzymatic function. Then the mechanism of kzl052 against prostate cancer progression was revealed to be by regulating the stability of DNA replication forks and the RB pathway. This study will provide a theoretical basis and treatment strategy for targeting WRN helicase in the treatment of prostate cancer.

Keywords: DNA replication fork; WRN helicase; prostate cancer; quinazoline derivative.

Figure 1

kzl052 inhibits the cell growth of PCa. (A) The structure of compound kzl052. (B,C) MTT results of PC3 and LNCaP cells treated with different concentrations of kzl052 after 24 h. (D) Bright field and Hoechst staining results of PC3 and LNCaP cells treated with kzl052 at different concentrations after 24 h. * p < 0.05, ** p < 0.01, and all experiments were repeated three times.

Figure 2

kzl052 inhibited the PCa cell growth by targeting WRN. (A) Visual results of molecular docking between kzl052 and WRN (PDB:6YHR). (B) CETSA results of kzl052 combined with WRN protein. (C) Western blot results of WRN-silenced PC3 cells. (D) Cell growth curve of kzl052 in WRN-silenced cells. (E) The WRN protein levels under kzl052 treatment for 24 h. * p < 0.05, ** p < 0.01, and all experiments were repeated three times.

Figure 3

kzl052 promotes DNA damage in PC3 and LNCaP cells. γ-H2A.X immunofluorescence staining and its statistical results in PC3 (A,B) and LNCaP (C,D) cells. ** p < 0.01, and all experiments were repeated three times.

Figure 4

Regulatory signal pathway of kzl052 against PCa. (A) Venny analysis of kzl052 target genes and CRPC key genes. (B) Results of protein–protein interactions (PPI) of key genes. (C–E) Gene ontology function annotation results of key genes. (F) The pathway analysis results of target genes.

Figure 5

kzl052 regulates the expression of the replication fork, key protein components, and the RB signaling pathway. (A) kzl052 regulates the expression of key factors of the cell cycle and cell apoptosis. (B) kzl052 regulates RB signals. (C) kzl052 regulates the expression of key factors of the replication fork complexes. * p < 0.05, ** p < 0.01, *** p < 0.001, and all experiments were repeated three times.

Figure 6

kzl052 inhibited PCa cell growth by regulating DNA replication fork stability in vivo. (A) Tumor volume changes after treatment with kzl052. (B) Tumor quality. (C) Tumor tissue. (D) H&E staining results. (E) Mouse body weight. (F) Internal organ morphology in mice. (G) The immunohistochemistry and its statistical results of γ-H2A.X, PARP1, RB1, PCNA, RPA, and Mre11 in cancer tissues. * p < 0.05, ** p < 0.01, and all experiments were repeated three times. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; the number of mice in the animals was eight.

Figure 7

kzl052 against PCa by regulating WRN—DNA replication fork stability.