Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase

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Abstract
L -nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the l -nucleosides (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC)—an approved anti-human immunodeficiency virus (HIV) agent—and troxacitabine (TRO)—an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural d -nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both d - and l -nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the l - and d -enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of l -nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.