A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA)

Abstract

Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box–mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box–mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.

Structure of proliferating cell nuclear antigen (PCNA) bound to an APIM peptide reveals the universality of PCNA interaction

“…Based on our structure, we further explored structural features of APIM by comparison with PIPM and revealed details of the mechanism underlying the PCNA-APIM interaction. Recently, the PCNA interaction has attracted much interest as a drug target for cancer therapy (Kontopidis et al, 2005;Punchihewa et al, 2012;Mü ller et al, 2013;Evison et al, 2014;Olaisen et al, 2015). Our results could provide a structural basis for the development of chemical compounds or peptides to inhibit PCNA interactions for cancer therapy.…”

Modulation of human melanoma cell proliferation and apoptosis by hydatid cyst fluid of <em>Echinococcus granulosus</em>

“… 11 , 12 Proliferating cell nuclear antigen (PCNA) is responsible for its indispensable role in cellular DNA replication and repair by the organization of numerous protein components of ERK/MAPK signaling pathway. 13 HCF has been observed to activate the MAPK signaling pathway in rat liver cells, thereby achieving phosphorylation and a high expression of ERK signaling pathway. 14 Besides, alveolar echinococcosis is a severe chronic helminthic disease that mimics slow-growing liver cancer, 15 and Aghayan et al found that melanoma could transfer into the liver, 16 which indicated that HCF and melanoma have the common action site.…”

The Rev1-Polζ translesion synthesis mutasome: Structure, interactions and inhibition

“…For instance, inhibitors of the PPI between Rev1-UBM and ubiquitin have been developed, and also demonstrated an anti-TLS activity in cells [176]. The interaction between PCNA and the PIP box is similarly mediated by a pair of aromatic (F or Y) side chains and which inserts into a pocket on PCNA surface (Figure 13C), and small molecule inhibitions of this PPI have been reported [177][178][179][180].…”