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

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 (-)-beta-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.

Figure 1

(A) Schematic of the d- and l-nucleosides (all in β-form): 3TC, (−)-l-2′,3′-dideoxy-3′-thiacytidine (Lamivudine, Epivir^®); TRO, (−)-l-2′,3′-dideoxy-3′-oxacytidine (Troxacitabine, Troxatyl™); d-dC, d-2′-deoxycytidine, and l-dC, l-2′-deoxycytidine. Experimental electron density map for the l-nucleoside analogs: (B) 3TC is shown in yellow and (C) TRO in magenta. The difference map (|F_obs| − |F_calc|) is contoured at 3σ (light blue) and 8σ (orange). Note the strong signal given by the sulfur atom in 3TC even at the high sigma cut-off. Figures were generated using Bobscript (26) and Molscript (27), and rendered with Raster3D (28).

Figure 2

Structures of dCK-3TC/TRO complexes. (A) Shown are four superimposed structures of human dCK: the C₄S variant in complex with 3TC/ADP (yellow), TRO/ADP (magenta) and d-dC/ADP (blue); the WT in complex with d-dC/ADP (gray; PDB ID 1P5Z). The structures in complex with d-dC/ADP of the dCK C₄S variant and the WT dCK are basically identical, apart from one loop involved in crystal contact. This validates the use of the C₄S mutant as a representative of WT dCK. Binding of l-nucleosides also does not elicit major structural perturbations. Non-carbon atom color coding: oxygen-red, nitrogen-blue, phosphorous-green and sulfur-orange. The N- and C-terminal residues visible in the experimental electron density map are labeled N₂₀ and C₂₆₀, respectively. (B) Stereo representation of the active site showing hydrogen bonding interactions (some water molecules omitted, for clarity). Curved arrow signifies the χ torsion angle around the glycosidic bond. (C) Stereo representation of the active site in an orientation rotated ∼90° relative to (B) depicting the cytosine base in the aromatic slot with Leu82 and Ile30 sandwiching the sugar. Structures have been deposited in the PDB with accession ID 2NOA for C₄S-3TC/ADP, 2NO9 for C₄S-TRO/ADP and 2NO1 for C₄S-d-dC/ADP.

Figure 3

Interactions activating the 5′-OH of the nucleosides for nucleophilic attack. (A) Stereoview depicting the interactions made by the 5′-OH groups of 3TC (yellow) and TRO (magenta). (B) Stereoview depicting the analogous interactions for d-dC (blue) and l-dC (cyan).

Figure 4

Enzyme-substrate interactions. Schematic representation of the (A) polar and (B) weakly polar and hydrophobic interactions made by 3TC and TRO with the residues in the dCK active site. Distances (Å) are color-coded in (A) with 3TC-yellow and TRO-magenta, while in (B) they are in black and correspond to the average closest contacts for both structures. Water molecules are represented as cyan balls.