In battling existing and newly emerging viral infections and cancer, nucleoside analogues (NAs) represent a prominent and highly potent weapon in the medicinal arsenal. Administered as membrane-penetrating nucleosidic prodrugs, the compounds require intracellular activation by deoxynucleoside kinases (dNKs) and deoxynucleotide kinases (dNPKs) into their bioactive triphosphate anabolites. In recent years, dNKs and dNPKs have taken on a key role in the quest for new and more potent antiviral and anticancer drugs. Emerging resistance to NAs upon long-term exposure, accumulation of cytotoxic intermediates, and failure of a large percentage of new prodrugs in vivo has been linked to problems associated with the phosphorylation cascade. The proposed research program focuses on the engineering of mammalian and bacterial dNKs and dNPKs, tailoring catalysts for optimal specificity toward NAs to maximize prodrug efficiency upon codelivery in cancer and antiviral therapy. Employing a novel, homology- independent combinatorial technique named SCRATCHY enables us to produce vast numbers of hybrid constructs from parents with high sequence diversity as found in the dNK/dNPK family. Functional hybrid candidates are subsequently selected by genetic selection and high-throughput screening. We will 1) Demonstrate that protein fragment swapping between structure-homologous proteins can generate hybrid enzymes that exhibit desired function, 2) Validate the performance of hybrid kinases through co-administration with prodrugs in cell-based assays, and 3) Develop a framework to study the structure-function relationship of dNKs and dNPKs.
The specific aims of this work are: 1) To identify engineered dNKs with improved NA specificity, 2) To generate novel catalysts that perform multistep phosphorylation, 3) To improve the versatility of structure-based protein engineering, and 4) To evaluate functional hybrid enzymes on established and novel NAs in vitro and in vivo. The results from these studies will have far-reaching implications on viral disease and cancer treatment, affecting existing prodrugs as well as potential drug candidates by facilitating their phosphorylation independent of cellular dNK activation. Finally, the structural diversity of the hybrid proteins will be a rich source for fundamental structure-function studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069958-04
Application #
7449749
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ikeda, Richard A
Project Start
2005-07-01
Project End
2010-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
4
Fiscal Year
2008
Total Cost
$228,489
Indirect Cost
Name
Emory University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Lutz, Stefan (2010) Beyond directed evolution--semi-rational protein engineering and design. Curr Opin Biotechnol 21:734-43
Lutz, Stefan (2010) Biochemistry. Reengineering enzymes. Science 329:285-7
Li, Yongfeng; Soni, Priti B; Liu, Lingfeng et al. (2010) Synthesis of fluorescent nucleoside analogs as probes for 2'-deoxyribonucleoside kinases. Bioorg Med Chem Lett 20:841-3
Liu, Lingfeng; Murphy, Paul; Baker, David et al. (2010) Computational design of orthogonal nucleoside kinases. Chem Commun (Camb) 46:8803-5
Liu, Lingfeng; Li, Yongfeng; Liotta, Dennis et al. (2009) Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting. Nucleic Acids Res 37:4472-81
Lutz, Stefan; Liu, Lingfeng; Liu, Yichen (2009) Engineering Kinases to Phosphorylate Nucleoside Analogs for Antiviral and Cancer Therapy. Chimia (Aarau) 63:737-744
Kazlauskas, Romas; Lutz, Stefan (2009) Engineering enzymes by 'intelligent' design. Curr Opin Chem Biol 13:1-2
Iyidogan, Pinar; Lutz, Stefan (2008) Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity. Biochemistry 47:4711-20
Segura-Pena, Dario; Lichter, Joseph; Trani, Manuela et al. (2007) Quaternary structure change as a mechanism for the regulation of thymidine kinase 1-like enzymes. Structure 15:1555-66
Lutz, Stefan; Lichter, Joseph; Liu, Lingfeng (2007) Exploiting temperature-dependent substrate promiscuity for nucleoside analogue activation by thymidine kinase from Thermotoga maritima. J Am Chem Soc 129:8714-5

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