Methods and tools for studying ecto-5'-nucleotidase (CD73)

Abstract

Ecto-5’-nucleotidase (CD73), a glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mainly in the membranes of immune and endothelial cells and released in exosomes or as a soluble protein, has emerged as a potential target in cancer immunotherapy. CD73 is the main extracellular protein that catalyzes the hydrolysis of AMP to adenosine. CD73 expression is upregulated in several types of cancer, resulting in a surge in extracellular adenosine concentration in the tumor milieu. Adenosine activates adenosine receptors, thereby exerting immunosuppressive effects and enhancing tumor proliferation and metastasis. Small molecule inhibitors of CD73 and monoclonal antibodies are currently being evaluated in clinical trials as monotherapy or in combination for cancer therapy. <br /> Research on small molecule inhibitors of CD73 has already identified potent a,ß-methylene-ADP analogs (AMPCP derivatives) with Ki values in the subnanomolar range. In this study, we aimed to explore the structure-activity relationships (SARs) of AMPCP derivatives and to identify new scaffolds with potentially more favorable pharmacokinetic profiles as inhibitors of CD73. We identified several highly potent inhibitors of CD73 based on N6-substitued AMPCP showing picomolar potency (for example, PSB-19294 and PSB-19301). Additionally, potent derivatives with reduced molecular weight were obtained (e.g. N6-alkynyl-AMPCP and 2-substituted AMPCP derivatives). An adenine-based a-carboxy-phosphononucleoside derivative (Cork82) was identified as a dual CD73/CD39 inhibitor with moderate potency. Several hit compounds that target allosteric sites on CD73 have been identified. For example, indole scaffolds have been thoroughly studied, leading to the identification of the hit compound PZB17718005A. An anthraquinone-based inhibitor, PZB17718022A, has been identified displaying submicromolar potency. Interestingly, these inhibitors selectively inhibited recombinant soluble human CD73, but not membrane-anchored CD73 natively expressed in MDA-MB-231 cells. Moreover, a xanthine derivative (GR178) was identified as a potent activator of human CD73. Such a compound has potential for the treatment of inflammatory and cardiovascular diseases. <br /> Biological results obtained for several scaffolds showed discrepancies between rat and human CD73 or between soluble and membrane-bound CD73. This observed selectivity prompted us to express various soluble human and mouse CD73 proteins and to additionally study a phospholipase C-cleaved soluble CD73 preparation. Modulators that were inactive at membrane-bound CD73 were also inactive at the cleaved soluble CD73, suggesting that variations in the glycosylation pattern of CD73 might affect the inhibitors’ activity determined on MDA-MB-231 tumor cells. The obtained results indicated that a C-terminal His-tag did not interfere with the activity. <br /> A further aim was to establish new binding assays for CD73 to enable studies of the binding kinetics and residence times of inhibitors. Radioligand binding assays and nano-luciferase bioluminescence resonance energy transfer (NanoBRET) assays were successfully developed and applied to characterize a series of CD73 inhibitors. Moreover, through nano-luciferase tagging of the soluble and membrane-bound forms of CD73, we provide structural insights, namely which sites and methods are tolerated for conjugation of CD73 without affecting enzyme functionality. <br /> This study provides valuable insights into the biochemistry and pharmacology of the promising drug target CD73, a key player in purinergic signaling.