Discovery and characterization of potent And‐1 inhibitors for cancer treatment

Abstract Acidic nucleoplasmic DNA‐binding protein 1 (And‐1), an important factor for deoxyribonucleic acid (DNA) replication and repair, is overexpressed in many types of cancer but not in normal tissues. Although multiple independent studies have elucidated And‐1 as a promising target gene for cancer therapy, an And‐1 inhibitor has yet to be identified. Using an And‐1 luciferase reporter assay to screen the Library of Pharmacologically Active Compounds (LOPAC) in a high throughput screening (HTS) platform, and then further screen the compound analog collection, we identified two potent And‐1 inhibitors, bazedoxifene acetate (BZA) and an uncharacterized compound [(E)‐5‐(3,4‐dichlorostyryl)benzo[c][1,2]oxaborol‐1(3H)‐ol] (CH3), which specifically inhibit And‐1 by promoting its degradation. Specifically, through direct interaction with And‐1 WD40 domain, CH3 interrupts the polymerization of And‐1. Depolymerization of And‐1 promotes its interaction with E3 ligase Cullin 4B (CUL4B), resulting in its ubiquitination and subsequent degradation. Furthermore, CH3 suppresses the growth of a broad range of cancers. Moreover, And‐1 inhibitors re‐sensitize platinum‐resistant ovarian cancer cells to platinum drugs in vitro and in vivo. Since BZA is an FDA approved drug, we expect a clinical trial of BZA‐mediated cancer therapy in the near future. Taken together, our findings suggest that targeting And‐1 by its inhibitors is a potential broad‐spectrum anti‐cancer chemotherapy regimen.


Animal experiments
Mouse housing and all procedures were followed regulation accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) of The George Washington University. 5-6week old female BALB/c athymic nude mice (CByJ.Cg-Foxn1nu/J, weighting 20-25g, from the Jackson Laboratory) were inoculated subcutaneously by injecting the OC cells, cisplatin resistant OC cells or breast cancer cells suspended in 100 µl ice-cold Matrigel/PBS (1:1, V:V; 5 x 10 6 /mouse) into the dorsal flank of each mouse. When the average tumor volume reached 100-150 mm 3 , the mice were randomized into subsequent experiment groups. For tumors treated with CH3 only, CH3 was given at 0 mg/kg, 20 mg/kg and 40 mg/kg twice a week for three weeks. For combinational treatment, cisplatin, CH3 and BZA were then given intraperitoneally at 8 mg/kg, 20 mg/kg and 2 mg/kg respectively twice a week for three weeks. The relative tumor volumes were calculated using the formula: a × (b 2 ) / 2, for which a and b represent the longest and shortest diameters, respectively. During the drug treatment, the tumor volume and body weight were measured. Tumor samples were collected 4 days after final administration of the drug.

Ovarian cancer patients
The specimens of chemosensitive and matched recurrent or chemoresistant tumor tissues from OC patients were kept as formalin-fixed paraffin-embedded (FFPE) samples in the Department of Pathology at The University of Hong Kong. Studies using human tissues were approved by the local institutional ethics committee (institutional review board reference No: UW 05-143 T/806 and UW 11-298). Written informed consent was received from patients prior to their inclusion in the study. The histological types, disease stages, and cancer cell contents in each FFPE section were examined by the three independent pathologists. Platinum-sensitive was defined as patients who have a total response to platinum-based therapy and no recurrence within 6 months. Platinum resistance means patients who had the recurrence occur within 6 months following the completion of platinum-based therapy.

Identification of And-1-ATR signature gene and analyses
The detailed approach to identify And-1-ATR signature gene (23 genes were identified) was described as below. The Kaplan Meier plotter is capable to assess the effect of 54k genes (mRNA, miRNA, protein) on survival in 21 cancer types including breast (n=7,830), ovarian (n=2,190), lung (n=3,452), and gastric (n=1,440) cancer. Sources for the databases include GEO, EGA, and TCGA. Primary purpose of the tool is a meta-analysis based discovery and validation of survival biomarkers.
By performing the steps described in Example 4, the following intermediates were prepared:

(E)-4-(3,4-dichlorostyryl)-2-formylphenyl trifluoromethanesulfonate (6b)
B 2 (Pin) 2 (0.870 g, 3.43 mmol) and potassium acetate (0.510 g, 5.19 mmol) were added to dry 1,4-dioxane (18 mL) under nitrogen atmosphere. (E)-4-(3,5-dichlorostyryl)-2-formylphenyl trifluoromethanesulfonate (0.725 g, 1.71 mmol) and Pd(dppf)Cl 2 (0.140 g, 0.17 mmol) were then added. The mixture was stirred at 80℃ for two hours. After the dioxane was removed under reduced pressure, the residue was purified by flash column chromatography to obtain a crude product, which was directly used in the next reaction. The crude product was dissolved in a mixed solution of 4 mL methanol and 3 mL tetrahydrofuran, and NaBH 4 (0.280 g, 7.41 mmol) was added in portions at 0℃. After the mixture was stirred for one hour, part of the solvent was removed under reduced pressure, 15 mL of water was added, and the pH was adjusted to 3 with dilute hydrochloric acid. After extraction with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was first purified by column chromatography and then recrystallized from toluene to obtain 0.250 g of white solid.
By performing the steps described in above, the following intermediates were prepared: