High-Throughput Screening Assays for Cancer Immunotherapy Targets: Ectonucleotidases CD39 and CD73

Area of Science:

• Biochemistry
• Immunology
• Pharmacology

Background:

• Extracellular adenosine in tumors causes immunosuppression and promotes progression.
• Targeting adenosine-generating ectonucleotidases is a promising strategy to enhance antitumor immunity.
• Selective detection of ectonucleotidase activity for high-throughput screening (HTS) has been challenging due to similar reaction products.

Purpose of the Study:

• To develop HTS-compatible biochemical assays for measuring the activity of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) and ecto-5'-nucleotidase (CD73).
• To utilize these assays to identify novel inhibitors of CD39 and CD73, key enzymes in purinergic signaling within the tumor microenvironment.

Main Methods:

• Development of assays using the Transcreener HTS Assay platform for selective immunodetection of nucleotides.
• Utilized Transcreener AMP2 Assay for CD39 activity measurement and IC50 determination.
• Coupled Transcreener ADP2 Assay with adenosine kinase (AK) to detect CD73 activity by measuring adenosine production.

Main Results:

• The developed assays demonstrated reliable measurement of CD39 and CD73 activity with high sensitivity.
• Screening of compound libraries identified nine and eight candidate CD39 inhibitors from the LOPAC and ChemBridge libraries, respectively.
• Screening of the ChemBridge library identified 14 potential CD73 inhibitors.

Conclusions:

• HTS-compatible assays for ectonucleotidase activity have been successfully developed.
• These assays facilitate the identification of inhibitors targeting the purinergic signaling pathway.
• The identified inhibitors hold potential for stimulating antitumor immunity and treating cancer.