用于筛选癌症中选择性整合素抑制剂的高通量测定平台的开发

Integrins are critical mediators of cancer progression, facilitating cancer cell invasion, survival in circulation, and metastatic outgrowth. Integrins result in cancer progression by activating pro-survival signaling pathways and remodeling the cancer microenvironment, and thus represent promising targets for anticancer treatments. To accelerate the discovery of novel integrin-targeted therapeutics, we developed an integrated platform of high-throughput screening assays for the evaluating integrin inhibitors of diverse modalities, including small molecules, peptides, antibodies, and antibody-drug conjugates. In this study, we developed a panel of cell-free fluorescence polarization (FP) assays in 384-well plate format using a Cy3B-RGD probe to screen compounds against multiple RGD-binding integrins (alphavbeta1, alphavbeta3, alphavbeta5, alphavbeta6, alphavbeta8 and alpha8beta1). In addition, we generated HEK293 cell lines stably overexpressing integrins such as alpha8beta1, and developed ELISA as well as IncuCyte based alpha8beta1-Mfge8 competition binding assays for compounds screening in cell-based assays. The assay results were further validated using cancer cell lines endogenously expressing high levels of integrins such as alpha8beta1, confirming compound binding and activity in a more physiologically relevant model. In summary, we have created a robust high-throughput screening platform that combines cell-free and cell-based assays. This integrated integrin assay platform enables the efficient evaluation of selective integrin inhibitors, accelerating the development of novel integrin inhibitors for target-based cancer treatment.