CD73: A structural biology case study

Expression and structural determination of CD73 to facilitate drug discovery

CD73 (also known as ecto-50-nucleotidase, e5NT) is a eukaryotic extracellular glycoprotein with potential applications in the treatment of cancer and inflammation.1-3 

CD73 catalyses the hydrolysis of extracellular AMP to adenosine and plays a pivotal role in switching on adenosine signalling via the P1 receptors of the purinergic signalling pathway.


Domainex was tasked with establishing a CD73 biochemical assay and a ligand-binding assay using Microscale Thermophoresis (MST) to support a small-molecule drug discovery programme. Commercially available CD73 was found to be of insufficient quality for the MST studies, so Domainex recommended that it produce high-quality CD73 protein in-house to support these assays.

Recombinant CD73 was successfully isolated from E. coli inclusion bodies, refolded and purified to homogeneity in multi-milligram quantities. A Design-of-Experiments approach was used to find the optimum conditions for the protein refolding step, resulting in a significant improvement over the initial conditions, and a reproducible outcome. The resulting protein was not only used to screen the output from a LeadBuilder virtual screen by both biochemical and MST assays, but was of sufficiently high yield and quality to enable additional STD-NMR and X-ray crystallography studies at Domainex (an example structure is shown in Figure 1).

CD73 Case Study
Figure 1: X-ray crystal structure of CD73 bound to adenosine 5′-(α,β-methylene)diphosphate (AMPCP), a CD73 inhibitor (dataset resolution 1.7Å). The structure overlays well with the Apo open conformation structure which was also solved.


1. Targeting adenosine for cancer immunotherapy. Robert D. Leone and Leisha A. Emens. j. immunotherapy cancer, 2018, 6, 57

2. Anti-CD73 in Cancer Immunotherapy: Awakening New Opportunities. Luca Antonioli, Gennady G. Yegutkin, Pál Pacher, Corrado Blandizzi and György Haskó. Trends in Cancer2016, 2, 2, 95-109

3. CD73 as a potential opportunity for cancer immunotherapy. Ghasem Ghalamfarsa, Mohammad Hossein Kazemi, Sahar Raoofi Mohseni, Ali Masjedi, Mohammad Hojjat-Farsangi, Gholamreza Azizi, Mehdi Yousefi and Farhad Jadidi-Niaragh. Expert Opinion on Therapeutic Targets2019, 23, 2, 127-142