186–192 Nucleic Acids Research, 2007, Vol. 35, No. 1 doi:10.1093/nar/gkl1038 Published online 7 December 2006 Structural basis for activation of the therapeutic L-nucleoside analogs 3TC and troxacitabine by human deoxycytidine kinase Elisabetta Sabini, Saugata Hazra, Manfred Konrad 1 , Stephen K. Burley2 and Arnon Lavie* Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago 900 S. Ashland (M/C 669), Chicago, IL 60607, USA, 1Max Planck Institute for Biophysical Chemistry, Am Fassberg 11 D-37077, Go¨ ttingen, Germany and 2SGX Pharmaceuticals Incorporation, 10505 Roselle Street, San Diego, CA 92121, USA Received September 20, 2006; Revised October 26, 2006; Accepted November 7, 2006 ABSTRACT L-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmaco- logical activity only after enzyme-catalyzed conver- sion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycyti- dine kinase (dCK) in complex with the L-nucleosides (-)-b-20,30-dideoxy-30-thiacytidine (3TC)—an appro- ved 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 phosphory- lation 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 virtu- ally 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. INTRODUCTION Chirality is a hallmark of biological systems: proteins are composed of L-amino acids and DNA and RNA are com- posed of b-D-nucleotides. It has, therefore, been widely accepted that enzymes that must correctly bind ligands and catalyze chemistry on their respective substrates, will invari- ably show high chiral selectivity. Quite unexpectedly, however, it was demonstrated that it is the L-component of the racemic mixture (±)-BCH-189 [(±)-b-20,30-dideoxy- 30-thiacytidine], which possesses signiﬁcantly better anti- human immunodeﬁciency virus (HIV) properties than its D-enantiomeric counterpart (1,2). Since this discovery, two non-physiologic L-nucleoside analogs [(-)-b-20,30-dideoxy- 30-thiacytidine (3TC), the L-enantiomer of BCH-189, and its 5-ﬂuorinated analog FTC] have been approved for treat- ment of human acquired immune deﬁciency syndrome (AIDS). An additional six D-nucleoside analogs have been approved as AIDS therapeutics, and it is customary for patients to receive both L- and D-enantiomer anti-HIV agents simultaneously. The pharmacological potential of L-nucleoside analogs has been recently reviewed (3–5). The dioxolane analog of 3TC, troxacitabine [TRO, (-)-L-b-20, 30-dideoxy-30-oxacytidine; Figure 1A] is currently in a Phase II/III clinical trial for third-line treatment of acute myeloge- nous leukemia (AML), a blood cancer. The L-nucleoside analogs 3TC and FTC are preferred as anti-virals over their D-analogs [(+)-BCH-189 and (+)-FTC, respectively], primar- ily because these HIV reverse transcriptase inhibitors do not affect cellular DNA polymerases (4), resulting in minimal toxicity. In contrast, TRO, the ﬁrst L-nucleoside analog to be used as a cytotoxic anti-cancer agent (6), which differs from 3TC by only one atom (S versus O in the ﬁve membered ring, Figure 1A), has potent anti-viral activity yet displays substantial cellular toxicity due to inhibition of DNA poly- merases a, b and g (7). Unlike the D-nucleoside analog *To whom correspondence should be addressed. Tel: +1 312 355 5029; Fax: +1 312 355 4535; Email: lavie@uic.edu 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.