Carbonic Anhdrase IX Inhibitors Screening And Their Proliferation Inhibition Activity Against Hypoxic Tumor Cells

Carbonic Anhydrase IX related with the tumor hypoxia is one of the most important members inα-CAs family. It is not only the marker of tumor poor prognosis, but also the significant drug target for hypoxic tumor cure.Fistly,51 candidate compounds were selected as CA IX inhibitors from the SPECS commercial database, by using of the structure-based virtual screening method. Then, an effective CA IX inhibitors screening platform was established based on the stopped-flow instrument, which was verified by two CA IX standard inhibitors Acetazolamide (AZA) and Ethoxzolamide (EZA). Furthermore, these 51 compounds were evaluated on this platform. As a result,15 compounds showed the excellent inhibitory activity toward to CA IX. Among them, the inhibitory activity of compounds 1,2,3 and 4 was as good as two positive compounds AZA and EZA. We also tested these 15 compounds'IC50 against CAⅠand CAⅡ, which were irrelevant with tumor. The results demonstrated that compounds 1,2,3 and 10 could selectively suppress CA IX over CAⅠor/and CAⅡ. Finally, MTT assay were performed to estimate whether these compounds could inhibit the proliferation of hypoxic tumor cells or not, and compounds 7 and 11 showed the proliferation inhibition against hypoxic MDA-MB-231 breast tumor cells.In addition, we constructed another method to test IC50 values of CA I and CA II by multi-detection microplate reader, while 4-Nitrophenylacetat was used as the substrate of the enzymatic reaction. This method was pretty more efficient, convenient and inexpensive.