Discovery of Protein–Protein Interaction Inhibitors of Replication Protein A

Patrone, James D.; Jp, Kennedy; Frank, Albin; Feldkamp,; Vangamudi, Bhavatarini; Nf, Pelz; Rossanese, Olivia W.; Ag, Waterson; Wj, Chazin; Sw, Fesik

doi:10.1021/ml400032y

Cited by 27 publications

(42 citation statements)

References 21 publications

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“…ATR pathway genes are mutated or deleted in up to 15–25% of samples from some cancer types suggesting that reduced ATR pathway functionality may be a useful criteria for selecting patients for ATR targeted drugs(33). Furthermore, the screening strategy is also a useful approach to identify novel ATR-pathway proteins, some of which may prove to be druggable targets, such as the recently reported Replication Protein A inhibitors(43, 44). …”

Section: Discussionmentioning

confidence: 99%

ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency

2014

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The DNA damage response kinase ATR and its effector kinase CHEK1 are required for cancer cells to survive oncogene-induced replication stress. ATR inhibitors exhibit synthetic lethal interactions with deficiencies in the DNA damage response enzymes ATM and XRCC1 and with overexpression of the cell cycle kinase Cyclin E. Here we report a systematic screen to identify synthetic lethal interactions with ATR-pathway targeted drugs, rationalized by their predicted therapeutic utility in the oncology clinic. We found that reduced function in the ATR pathway itself provided the strongest synthetic lethal interaction. In addition, we found that loss of the structure specific-endonuclease ERCC1-XPF (ERCC4) is synthetic lethal with ATR pathway inhibitors. ERCC1-deficient cells exhibited elevated levels of DNA damage, which was increased further by ATR inhibition. When treated with ATR or CHEK1 inhibitors, ERCC1-deficient cells arrested in S phase and failed to complete cell cycle transit even after drug removal. Notably, triple-negative breast cancer cells and non-small cell lung cancer cells depleted of ERCC1 exhibited increased sensitivity to ATR-pathway targeted drugs. Overall, we concluded that ATR pathway-targeted drugs may offer particular utility in cancers with reduced ATR pathway function or reduced levels of ERCC4 activity.

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Section: Discussionmentioning

confidence: 99%

ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency

2014

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“…FPA inhibited the DBD-F with high affinity and selectivity, but had a preponderance of high negative charge that restricted the compound from crossing cell membranes (22, 25). Fesik, et al also utilized both HTS and a fragment-based NMR spectroscopy method to identify in vitro inhibitors of RPA with affinities similar to FPA (48, 49). HAMNO, with its neutral charge and hydrophobic ring structures allow passage of the compound through the cell membrane, where it can bind to DBD-F, an improvement over FPA.…”

Section: Discussionmentioning

confidence: 99%

RPA Inhibition Increases Replication Stress and Suppresses Tumor Growth

et al. 2014

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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.

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“…22–24 Using these methods, our group has previously reported the discovery of compounds that bind to RPA70N with affinities as low as 11 µM and X-ray crystal structures that reveal how they bind to the protein. 25 …”

Section: Introductionmentioning

confidence: 99%

Discovery of a Potent Inhibitor of Replication Protein A Protein–Protein Interactions Using a Fragment-Linking Approach

et al. 2013

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Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is involved in nearly all cellular DNA transactions. The RPA N-terminal domain (RPA70N) is a recruitment site for proteins involved in DNA damage response and repair. Selective inhibition of these protein-protein interactions has the potential to inhibit the DNA damage response and sensitize cancer cells to DNA-damaging agents without affecting other functions of RPA. To discover a potent, selective inhibitor of the RPA70N protein-protein interactions to test this hypothesis, we used NMR spectroscopy to identify fragment hits that bind to two adjacent sites in the basic cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N-ligand complexes revealed how these fragments bind to RPA and guided the design of linked compounds that simultaneously occupy both sites. We have synthesized linked molecules that bind to RPA70N with submicromolar affinity and minimal disruption of RPA’s interaction with ssDNA.

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Discovery of Protein–Protein Interaction Inhibitors of Replication Protein A

Cited by 27 publications

References 21 publications

ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency

ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency

RPA Inhibition Increases Replication Stress and Suppresses Tumor Growth

Discovery of a Potent Inhibitor of Replication Protein A Protein–Protein Interactions Using a Fragment-Linking Approach

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