In vitro activity: HAMNO (formerly known as NSC-111847) is a novel, potent and selective protein interaction inhibitor of replication protein A (RPA). RPA is thought to interact with proteins involved in the replication stress response. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions that 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. Therefore, 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.

Kinase Assay: RPA was purified using a published protocol as described. DBD-F fused to maltose binding protein was generated and purified as described. Quality of both proteins were assessed by SDS-PAGE, followed by coomassie staining. For ssDNA binding studies, 7 nM RPA was added to 10 nM labeled polyT 30mer in EMSA buffer (10 mM Tris, pH 7.5, 10 mM KCl, 10% glycerol) for 10 min at 25 °C. Samples were run on 1% agarose gels in 40 mM Tris-Acetate buffer, pH 7.5, and then scanned on an infrared scanner. For DNA unwinding assays, 14 nM RPA was added to 10 nM PAGE purified annealed polyA:polyT 30mer oligonucleotides.

Cell Assay: The squamous cell carcinoma cell lines UMSCC38 and UMSCC11B (kindly obtained from Dr. Thomas G. Carey, University of Michigan, Ann Arbor, MI) were propagated in DMEM with 10% fetal bovine serum (FBS). The immortalized primary oral keratinocyte cell line, OKF4 (obtained from Dr. James G. Rheinwald, Harvard Institutes of Medicine, Boston, MA) was propagated in fortified KBM-2 media (Lonza) with 10% fetal bovine serum (Hyclone). For clonogenic assays, cells were trypsinized and diluted in media to 1000 cells/mL, then dispersed into 60 mm dishes (3 mL) overnight. After addition of HAMNO, cells were grown for 9 d, then fixed in PBS containing 6% glutaraldehyde for 30 min, and then dyed in 0.5% crystal violet for 30 min and rinsed. Colonies containing over 50 cells were counted. For studies requiring etoposide, HAMNO was added one h before addition of 2 μM etoposide. After 2 h of etoposide exposure, media was removed and rinsed with PBS, before adding back media containing HAMNO. Data were analyzed using an unpaired 2-tailed Student t test to determine statistical significance.