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.

Domainex Expertise

Protein ScienceVirtual Screening via LeadBuilder Biochemistry  • Structural Biology 

MicroScale Thermophoresis (MST) Services • BiophysicsX-ray Crystallography


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