Purinergic signaling is involved in immune regulation. Adenosine exerts an immunosuppressive effect through adenosine receptors (ARs) and is present in high concentration in the tumor microenvironment. Ecto-5'-nucleotidase (CD73) is part of the purinome and catalyzes the dephosphorylation of AMP into adenosine, acting as the major control point of extracellular adenosine levels. Moreover, CD73 is upregulated in the tumor microenvironment. The development of CD73 inhibitors might be beneficial for cancer immunotherapy.<br/>We have developed N4-[O-(4-benzyloxyimino)]-3-methylcytidine-5'-[(α,β)-methylene-diphosphonate derivatives as CD73 inhibitors, with different substitution at the benzyl group. Pyrimidine inhibitors, if hydrolyzed to the parent nucleoside, would not activate ARs. The in vitro CD73 inhibitory activity was assessed, and many of the analogues outperformed the potency of the lead compound bearing an unsubstituted benzyl group (Ki 10.6 nM). 4-Chloro (MRS4598, Ki 0.67 nM) and 4-iodo (MRS4620, Ki 0.44 nM) substitutions of the N4-benzyloxy group decreased Ki by ∼20-fold. The X-ray structures of hCD73 bound to two inhibitors (4-chloro (MRS4598) and 4-methoxycarbonyl (MRS4602, Ki 0.85 nM)) were obtained. The benzyloxyimino group binds the region between the C- and N-terminal domains of the enzyme, as previously reported for N6-benzyl groups in adenine inhibitors. Molecular dynamics (MD) followed by cluster analysis predicted conformational variability of the benzyloxy group, which appeared more stable in the simulations of the 4-iodo analogue. The pyrimidine scaffold showed low deviation from the crystallographic conformation and a persistent pattern of interactions with the enzyme. The ribose ring assumed a North conformation in the crystallographic structure of the complex, which was maintained during MD simulations. An analysis of CD73 structures in the Protein Data Bank (PDB) showed a preferred North puckering for nucleosides/tides bound to CD73. This suggested ribose ring substitution with a bicyclo[3.1.0]hexane, which constrains the five-membered ring in a North conformation. However, this substitution reduced potency, hypothetically because of the polar character of CD73 active site that favors a furanose ring.<br/>In conclusion, we have greatly enhanced the inhibitory potency of CD73 inhibitors by N4-benzyloxy substitution, and we have characterized their interaction with hCD73 through X-ray crystallography and molecular modeling.

Development and structural insights of N4-[O-(4-benzyloxyimino)]-3-methylcytidine-5'-[(α,β)-methylene-diphosphonate Inhibitors of hCD73

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