Redesigning Human 2′-Deoxycytidine Kinase Enantioselectivity for l-Nucleoside Analogues as Reporters in Positron Emission Tomography

Recent advances in nuclear medicine have allowed for positron emission tomography (PET) to track transgenes in cell-based therapies using PET reporter gene/probe pairs. A promising example for such reporter gene/probe pairs are engineered nucleoside kinases that effectively phosphorylate isotopically labeled nucleoside analogues. Upon expression in target cells, the kinase facilitates the intracellular accumulation of radionuclide monophosphate, which can be detected by PET imaging. We have employed computational design for the semi-rational engineering of human 2'-deoxycytidine kinase to create a reporter gene with selectivity for L-nucleosides including L-thymidine and 1-(2'-fluoro-5-methyl-β-L-arabinofuranosyl) uracil. Our design strategy relied on a combination of preexisting data from kinetic and structural studies of native kinases, as well as two small, focused libraries of kinase variants to generate an in silico model for assessing the effects of single amino acid changes on favorable activation of L-nucleosides over their corresponding D-enantiomers. The approach identified multiple amino acid positions distal to the active site that conferred desired L-enantioselectivity. Recombination of individual amino acid substitutions yielded orthogonal kinase variants with significantly improved catalytic performance for unnatural L-nucleosides but reduced activity for natural D-nucleosides.