CD73 is a crucial regulator of adenosine production in the tumor microenvironment and, therefore, represents a valuable target for cancer immunotherapy. While different inhibitors of CD73 have been studied, the progress remains hindered by a lack of high-throughput assays that would allow the screening of large chemical libraries. Establishing a sensitive assay for the detection of CD73 activity could enable additions to the CD73 inhibitor chemical space as well as help facilitate a better understanding of the CD73 reaction mechanism. In this study, we focused on the development and adaptation of DIANA for CD73 high-throughput screening and showed that we can detect enzyme inhibition with high sensitivity. We then used this assay to screen an IOCB library, a proprietary set of chemical compounds with a special focus on nucleotide analogues. We identified several scaffolds that inhibit CD73 and in an SAR study demonstrated fine-tuning of the inhibition properties of monophosphonate analogues. Moreover, using a breast cancer cell line as a model with endogenous CD73 expression, we demonstrated the inhibition of CD73 directly on cells. The establishment of a sensitive assay for the detection of CD73 activity allowed us to develop potent inhibitors of the enzyme with low nanomolar inhibition constants. Our findings further promote the importance of CD73 inhibitors in cancer therapy.

• Keywords
• CD73, DNA-linked probe, ecto-5′-nucleotidase, high-throughput screening, monophosphonate, tumor microenvironment,
• Publication type
• Journal Article MeSH