Identification of Novel CD39 Inhibitors Based on Virtual Screening and Enzymatic Assays

The accumulation of adenosine in the tumor microenvironment mediates immunosuppression and promotes tumor growth and proliferation. Intervention of the adenosine pathway is an important direction of antitumor immunity research. CD39 is an important ecto-nucleotidases for adenosine generation, therefore targeting the CD39-adenosine pathway is an emerging immune checkpoint for anticancer treatment. However, currently no CD39 inhibitor has been approved by the U.S. Food and Drug Administration. The development of CD39 drugs is urgent for clinical application. In this study, we combined homology modeling, virtual screening, and in vitro enzymatic activity to characterize the structural features of the CD39 protein and identify a triazinoindole-based compound as a CD39 inhibitor. The identified inhibitor and one of its analogues could effectively prevent the enzymatic activity of CD39 with IC₅₀ values of 27.42 ± 5.52 and 79.24 ± 12.21 μM, respectively. At the same time, the inhibitor significantly inhibited the adenosine monophosphate production in colorectal cancer cell lines (HT29 and MC38) and thereafter prevented cell proliferation. Molecular docking studies, mutagenesis, and microscale thermophoresis indicated that residues such as R85 could be the main contributor in binding triazinoindole compounds. The binding mode can potentially be utilized for hit-to-lead optimization, and the identified inhibitor can be further tested for its anticancer activity in vivo or may serve as a chemical agent to study CD39-related functions.