To analyze BrdU incorporation, cells were labeled with 10 M BrdU after which cells were collected, washed and, fixed in 70% EtOH

To analyze BrdU incorporation, cells were labeled with 10 M BrdU after which cells were collected, washed and, fixed in 70% EtOH. are sustained by the numerous oligosaccharide/oligonucleotide binding (OB)-folds present on each of the three subunits (2;3). OB-folds in DNA binding domains A and B (DBD-A and DBD-B) in the central region of the p70 subunit contribute most of the binding energy for RPA-ssDNA interactions (2). Individual OB-folds are compact modular domains populated with hydrophobic and basic amino acids. These structural features make the OB-folds an attractive target for development of small molecule inhibitors (SMIs) of DNA binding activity. Inhibiting RPA-DNA interactions has the potential to impact numerous DNA metabolic pathways that are differentially active in cancer cells. In DNA replication, RPA inhibition can be used D-glutamine to exploit the highly proliferative nature of cancer cells which is characterized by a large population of cells in S-phase. Consistent with this, a recent clinical trial correlated disease stage and metastasis in colon cancer with increased RPA p70 and p34 expression (4). RPA is also essential for several DNA repair pathways in the cell including nucleotide excision repair (NER). Cisplatin, a common chemotherapeutic used in the treatment of various cancers, induces its cytotoxic effect by forming intrastrand covalent DNA adducts that are repaired primarily by the NER pathway (5). Consistent with the role of NER in the repair of cisplatin-induced DNA damage, resistance to this treatment has been observed to be influenced by DNA repair capacity (6;7). Consequently, cisplatin treatment, in conjunction with decreased RPA ssDNA binding activity, would be expected to result in decreased efficiency of cellular repair of cisplatin-DNA adducts and increased cytotoxicity. Thus, targeting RPA has the potential not only for single agent activity but also to sensitize cancer cells to therapies that induce DNA damage and genetic instability, such as cisplatin, etoposide and ionizing radiation (IR). We present the identification and development of the first small molecule that inhibits the ssDNA binding activity of RPA. Cellular RPA inhibition results in the inability to enter S phase, D-glutamine cytotoxicity and synergistic activity with the chemotherapeutic agents cisplatin and etoposide. This is the first characterization of a small molecule that is able to inhibit the ssDNA binding activity of RPA and presents a novel chemotherapeutic target both as a single agent and in conjunction with commonly used chemotherapeutics. Materials and Methods protein analysis Small molecule inhibitors were obtained from ChemDiv and resuspended in DMSO. Compound 505 was independently synthesized and structure confirmed by mass spectrometry analysis. Human RPA was purified as previously described (8). Fluorescence Anisotropy based DNA binding assays were performed with 40 nM RPA and 20 nM 5fluoroscein-labeled ss-dT12 DNA as previously described (9). EMSAs were performed in 20 L reactions containing 25 nM RPA, 25 nM 5[32P]-labeled 34-base pair DNA as previously described (8). Molecular Modeling Molecular modeling of compound 505 with the central DNA binding domain of RPA p70 (1FGU) was performed using Autodock 4.2 (10). Three independent grids established 60? in each dimension to encompass either the interdomain region, DBD-A or DBD-B. Semi-flexible automated ligand docking was performed using the Monte Carlo based simulated annealing and locality search algorithms. The most stable complexes were selected based on binding energies from multiple analyses. Coordinates of the final docked complexes were displayed with PyMOL. Flow Cytometry H460 cells were analyzed for apoptosis using an Annexin V-FITC/Propidium iodide (PI) Vybrant Apoptosis Assay Kit (Invitrogen), according to manufacturers instructions. Cells were plated Rabbit Polyclonal to Cofilin at a density of 1 1 104 cells/cm2 for 24 hours and then treated with compound 505 for 48 hours. Following plating and treatment of H460 cells, adherent and non-adherent cells were collected, D-glutamine processed, and analyzed on a BD FACScan flow cytometer. Data was analyzed using WinMDI software (The Scripps Research Institute, San Diego, CA). Cell cycle analysis was performed by PI staining. Briefly, cells were plated and treated with compound, collected and then washed.

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