ABSTRACT
ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease which is essential in multiple DNA repair pathways in mammalian cells. ERCC1-XPF (ERCC1-ERCC4) repairs cisplatin-DNA intrastrand adducts and interstrand crosslinks and its specific inhibition has been shown to enhance cisplatin cytotoxicity in cancer cells. In this study, we describe a high throughput screen (HTS) used to identify small molecules that inhibit the endonuclease activity of ERCC1-XPF. Primary screens identified two compounds that inhibit ERCC1-XPF activity in the nanomolar range. These compounds were validated in secondary screens against two other non-related endonucleases to ensure specificity. Results from these screens were validated using an in vitro gel-based nuclease assay. Electrophoretic mobility shift assays (EMSAs) further show that these compounds do not inhibit the binding of purified ERCC1-XPF to DNA. Next, in lung cancer cells these compounds potentiated cisplatin cytotoxicity and inhibited DNA repair. Structure activity relationship (SAR) studies identified related compounds for one of the original Hits, which also potentiated cisplatin cytotoxicity in cancer cells. Excitingly, dosing with NSC16168 compound potentiated cisplatin antitumor activity in a lung cancer xenograft model. Further development of ERCC1-XPF DNA repair inhibitors is expected to sensitize cancer cells to DNA damage-based chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , DNA Repair/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Endonucleases/antagonists & inhibitors , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Screening Assays, Antitumor , Drug Synergism , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Mice , Xenograft Model Antitumor AssaysABSTRACT
Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: DeltaF508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not DeltaF508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than DeltaF508. The ER exit code is necessary for DeltaF508 residual export and rescue. R555K, a mutation that rescues DeltaF508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in DeltaF508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling DeltaF508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving DeltaF508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of DeltaF508.
