(A) Binding sites of the 6 small molecules on CD73. The diagram displays the superposition of all 6 small molecules illustrating that they essentially target 2 distinct sites in the vicinity of the region V275–D311 of CD73 (in indigo) which makes interfacial contacts OTUD4 in the CD73/OTUD4 complex. (B and C) MDA-MB231(B) and MDA-MB468 (C) were treated with 0.5 μM screened compounds and CD73 expression levels were determined. (D) MDA-MB231 were treated with 0.5 μM ST80 or Z22 and CD73 protein turnover rate was determined by pulse-chase analysis. (E) MDA-MB231-CD73^WT and MDA-MB231-CD73^V300P/I301Q cells were treated with 0.5 μM ST80 and Z22, CD73 immune complexes were immunoprecipitated, OTUD4, CD73, and CD73 ubiquitylation were determined by immunoblotting. (F) Coordination of ST80 and Z22. ST80 (marine) and Z22 (blue) are located in and around residues V275–D311 (indigo) of CD73. The yellow spheres correspond to the labelled CD73 residues that predominantly interact with Z22. (G) MDA-MB231 cells were treated with different doses of ST80 or Z22 for 24 hours and CD73 protein levels were determined. (H) MDA-MB231 cells were treated with 0.5 μM ST80 or Z22, cell lysates were collected at different time points,and CD73 protein levels were determined. (I) MDA-MB231 cells were cocultured with human PBMCs at different Effector (E) to Target (T) ratios (2:1 or 4:1) and treated with ST80 and Z22 (0.5 μM), and cell proliferation rate was determined. (J and K) MDA-MB231 cells were treated with 1 μM, 0.5 μM, and 0.1 μM ST80 (J) and Z22 (K), adenosine productions were determined. (L and M) MDA-MB231 cells were cocultured with human PBMCs and treated with 0.5 μM ST80 or Z22. Percentage of IFN-γ⁺ in CD8⁺ T cells (L) and percentage of GzmB⁺ in CD8⁺ T cells (M) were measured and quantified using flow cytometry. Data (mean ± SEM) are representative of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple comparisons test.