Extracellular adenosine (ADO) is present in high concentrations within the tumor micro-environment (TME). ADO is a potent inhibitor of T cell and NK cell activation, resulting in suppression of immune function. The generation of ADO is dependent upon the ecto-nucleotidases CD39 (transforms ATP to AMP) and CD73 (transforms AMP to ADO). Inhibition of CD73 represents a promising therapeutic approach for preventing ADO-mediated immunosuppression in the TME. Here we present the discovery of a novel class of small molecules that are capable of inhibiting CD73, starting from a high throughput screening hit. Several potent small-molecule CD73 inhibitors have been co-crystallized with recombinant human CD73. They have been found to bind to the closed form of CD73 and to occupy the adenosine binding pocket, maintaining the same π-π stacking interaction observed with the CD73 inhibitor AMPCP. This class of molecules also demonstrates a key hydrogen bond with CD73 residue ASP506 to provide a drastic improvement in CD73 inhibition. Compounds exhibit potent CD73 inhibition in multiple biochemical and cell-based assays, providing IC<sub>50</sub> values of less than 20 nM against both soluble and membrane-bound CD73. Inhibitors have also been screened against various NTPDases as well as the adenosine receptors and were found to be highly selective for CD73. Select molecules do not show significant inhibition of the major CYP450 isoforms. Pharmacokinetic properties in rodents indicate the potential for oral bioavailability with low to moderate clearance. Structure-activity relationships, X-ray co-crystal structural characterization, and preclinical pharmacokinetic parameters for this series of inhibitors will be presented.

Progress towards orally bioavailable, potent, and selective small-molecule inhibitors of CD73 for immunooncology

2019 Spring National Meeting

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