Adenosine (ADO) is a potent inhibitor of T cell and NK cell activation and is present in high concentrations in the tumor micro-environment (TME), resulting in an immunosuppressed phenotype. In the TME, generation of ADO relies on the sequential hydrolysis of ATP by two ecto-nucleotidases, CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major pathway of ADO production and can reverse ADO-mediated immune suppression. We have developed a novel series of potent and selective CD73 inhibitors via interrogation of structure activity relationships (SAR) and structure-based drug design. Key molecular interactions were identified from high resolution X-ray structure data of select inhibitors bound to human CD73. These inhibitors were found to occupy the adenosine pocket and form an array of hydrogen bonds with CD73 residues D506, R354, and N390. Furthermore, a strong hydrophobic π-stacking interaction between two phenylalanine residues (F417 and F500) and coordination of the di-zinc catalytic center was integral to retain high potency. A001202, a representative member of this series, potently inhibits soluble and membrane-bound CD73 (IC<sub>50</sub> = 0.86 and 2.6 nM, respectively). Similar potency was measured in cell lines (IC<sub>50</sub> = 0.55 nM, SKOV-3). A001202 exhibits a favorable preclinical profile. A001202 is &gt;10,000-fold selective against related ecto-nucleotidases (NTPDase 2,3,8 and CD39), does not inhibit the major CYP450 isoforms, and its pharmacokinetic properties in rodents are characterized by low clearance and a long half-life. These efforts have expanded the understanding of the structural underpinnings of small-molecule CD73 inhibition.

Discovery and characterization of potent and selective small-molecule inhibitors of ecto-nucleotidase CD73 for cancer immunotherapy

2019 Spring National Meeting

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