Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin

© 20201,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980–995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions11Nsciescopu

Alkylation of nucleobases by 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) sensitizes PARP1-deficient tumors

Targeting BRCA1-and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1-and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi