RPA Inhibition Increases Replication Stress and Suppresses Tumor Growth

Abstract

The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions which rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress.

Dissociation of gemcitabine chemosensitization by CHK1 inhibition from cell cycle checkpoint abrogation and aberrant mitotic entry

“…Indeed, CHK1 inhibitors have played a critical role in establishing the potential of the general strategy of targeting DNA damage response pathways for cancer therapy and newer CHK1 inhibitors are in development. 16 This field has grown considerably in the last decade with the introduction of many agents designed to target other proteins in the DNA replication, DNA repair, and cell cycle checkpoint pathways such as ATR, 40 RPA 41 and WEE1. 32,42 Given the lack of effective strategies to treat pancreatic cancer, and the demonstrated efficacy of CHK1 inhibitors in sensitizing pancreatic cancers to gemcitabine or gemcitabine-based chemoradiation, 13,20,27,28 second generation CHK1 inhibitors or other agents which target these pathways should continue to be explored as potential therapeutic options.…”

Human single-stranded DNA binding proteins: guardians of genome stability

“…As a promising target, several inhibitors have been developed for obstructing RPA-ssDNA-binding activity [110][111][112][113]. TDRL-505, the first small molecule shown to be able to block the ssDNA-binding activity of RPA, can prevent cell cycle progression, induce cytotoxicity, and enhance chemosensitivity in cancer cells [110].…”

“…Additionally, another molecule that also targets the RPA N-terminus, named HAMNO, inhibits ATR phosphorylation and creates DNA replication stress in cancer cells that are already experiencing replication stress but not in normal cells. Moreover, it synergistically acts with etoposide to kill cancer cells both in vitro and in vivo [113]. Therefore, small molecules such as these could be used to investigate the role of RPA in cancer biology, which may provide a viable foundation for a cancer drug discovery program that targets the N-terminus of RPA1.…”

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

“…In this assay, UMSCC-38 cells are well characterized and will mimic highly replicative normal cells in humans that would be particularly susceptible to compounds that inhibit DNA replication and repair. 29 Of the five compounds tested, compounds 2 (NSC35676: 2,3,4,5-tetrahydroxybenzo [7]annulen-6-one), more commonly known as purpurogallin, and 6 [NSC9037: 9-hydroxyphenylfluoron (9-HPF)] showed nominal toxicity at the highest dose (128 mg/L) as shown in Figure 5(a). From the bacterial and cell toxicity data, an SI was calculated (Table 2).…”