Identification of allosteric inhibitors of the ecto-5'-nucleotidase (CD73) targeting the dimer interface

Abstract

The ecto-5’-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues (D366, H456, K471, Y484 and E543 for polar interactions and G453-454, I455, H456, L475, V542 and G544 for hydrophobic contacts) located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response.

CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer

“…One caveat of the in vitro study was that APCP is a competitive inhibitor. Its efficiency and metabolic stability are lower when the concentration of substrate is high compared to allosteric inhibitors 42 . However, we demonstrated that blocking of CD73 by APCP had significant effect on cellular behavior and EMT progression in TNBC cells.…”

Be Aware of Aggregators in the Search for Potential Human ecto-5′-Nucleotidase Inhibitors

“…Due to its key role in purinergic signaling pathways regulation, ecto -5′-NT has been recognized as a promising biological target for multiple diseases and pathophysiological events, including cancer, autoimmune diseases, inflammation, infections and ischemia-reperfusion injury. The remarkable efforts that have been made by scientific community towards discovery of novel ecto -5′-NT inhibitors can be attested by the numerous studies that account for potential bioactive compounds and/or drug candidates targeting this enzyme [ 42 , 43 , 44 , 45 , 46 , 47 , 49 , 59 ]. Despite the encouraging results obtained by most of them, controls for inhibitors aggregation and/or precipitation have not been systematically reported so far.…”

“…Considering its importance for therapy, the screening for ecto -5′-NT inhibitors has become urgent. Although numerous studies describing ecto -5′-NT inhibitors have been published in the literature [ 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 ], the corresponding procedures and controls concerning compound aggregation have not been systematically described so far for this target enzyme.…”

Anti‐Inflammatory Effects of Quercetin on High‐Glucose and Pro‐Inflammatory Cytokine Challenged Vascular Endothelial Cell Metabolism

“…[51] 5′-Nucleotidases can be found either membrane bound (ecto-5′−-nucleotidase/CD73) or in soluble form in the cells. [57] However in the present study we have tested our polyphenols only on CD73 (ecto-5′-nucleotidase) activity since high cellular ATP concentrations deactivates soluble 5′-nucleotidase function. [58] The only flavonol that inhibited CD73 activity in intact HUVECs was quercetin, and the inhibition was only partial and requiring relatively high quercetin concentrations (50 m).…”