Directed Evolution and Computational Design of Nucleoside Analog Kinases Open Access

Abstract

Directed Evolution and Computational Design of Nucleoside Analog Kinases
By Lingfeng Liu

Nucleoside analog (NA) prodrugs represent a promising group of viral and cancer therapeutics. Despite the relative ease of synthesizing diverse NAs, < 30 NAs have been FDA-approved. Difficulties arise since the prodrugs need to be phosphorylated by cellular nucleoside/nucleotide kinases to become activated triphophates. The NA-triphosphates then function as DNA replication terminators. Nevertheless, the human deoxynucleoside kinases generally catalyze NAs with poor efficiency, causing low drug potency and failure of many NAs in vivo.

As a potential solution, previous research found the co-administration of an exogenous kinase can accelerate the NA phosphorylation in vivo and increase drug potency. Kinase engineering by directed evolution provides an efficient strategy to evolve specific and efficient NA kinases from parental nucleoside kinases. Nevertheless, limited success has been achieved because of the lack of efficient selection/screening protocols that directly monitor NA phosphorylation. In this dissertation, I developed a fluorescence-activated cell sorting (FACS)-based screening that combines fluorescent nucleobases and modified 2'-deoxyriboses from NA prodrugs, and the phosphorylation was followed by monitoring the entrapment of the fluorescent NAs through FACS. Using this screening, an orthogonal ddT kinase was evolved with 20,000-fold higher specificity and 6-fold higher catalytic efficiency for ddT starting from Drosophila melanogaster deoxynucleoside kinase (DmdNK), a broad substrate-specificity kinase. To enhance the capability of searching through the sequence space for NA kinase activity, we used Rosetta program to switch the substrate specificity of DmdNK to ddT. Combining Rosetta design with a mutation learned from directed evolution also led to a specific ddT kinase with improved thermostability. Each method developed orthogonal kinases which we propose will be more effective at phosphorylating NAs while minimizing perturbation to cellular nucleoside metabolism.

We tested these ideas in both E. coli bacteria and three different human cancer cell lines, and the results support our hypothesis that specific NA kinases increase NA potency more efficiently than promiscuous kinases, suggesting an alternative perspective for clinical applications of evolving orthogonal enzymes with minimized perturbation to cellular environment.

Table of Contents

1 Introduction...1

1.1. Current HIV/AIDS and cancer therapeutic strategies Cellular...1

1.1.1. HIV/AIDS therapeutic strategies...1
1.1.2. Nucleoside analogs in cancer treatment...3

1.2. Nucleosides and nucleoside analogs design...4

1.2.1. Structure and working mechanism of NAs...4
1.2.2. Challenges in NA drug development...7

1.3. The salvage pathway and phosphorylation problems for NAs...8
1.4. Cellular dNKs, exogenous kinases and substrate specificity...11
1.5. Kinase engineering by directed evolution and the existing challenges...13

1.5.1. Directed evolution for enzyme engineering...13
1.5.2. Progress toward NA kinase engineering...15
1.5.3. Library generation strategies used for kinase engineering...17
1.5.4. Biases in auxotrophic complementation selection...17

1.6. Fluorescent nucleoside analogs...19
1.7. FACS-based screening for directed evolution...23
1.8. Combining computational design with directed evolution for enzyme engineering...25

2 Development of FACS-based screening for kinase phosphorylation...27

2.1. Introduction...27
2.2. Results and discussion...31

2.2.1. Fluorescence property of fT...31
2.2.2. Effect of furano-modification on thymine base on fT as substrates for DmdNK...32
2.2.3. Toxicity study for fT...33
2.2.4. Evaluation of different promoters for kinase expression in E. coli in the presence of fluorescent nucleosides...33
2.2.5. Method evaluation: enrichment experiments on fT phosphorylation...36

2.3. Concluding remarks...39
2.4. Materials and methods...41

2.4.1. Materials...41
2.4.2. Steady-state fluorescence spectroscopy...42
2.4.3. Toxicity assay for fT...42
2.4.4. FACS sorting of cell mixtures carrying DmdNK and hdCK...43
2.4.5. Over-expression and purification of DmdNK...43
2.4.6. Steady-state kinetic analysis by ITC...44

3 Directed evolution of orthogonal ddT kinases using FLUPS (FLUorescent nucleoside analog Phosphorylation Screen)...45

3.1. Introduction...45
3.2. Results and discussion...49

3.2.1. Effect of furano-modification on thymine base on fddT as substrates for DmdNK...49
3.2.2. Fluorescence property of fddT...50
3.2.3. Evolution of an orthogonal ddT kinase...51
3.2.4. Mutation study by Reverse engineering...54
3.2.5. Competition experiments of orthogonal kinases in E. coli...60
3.2.6. Variants performance in cancer cell lines...62

3.3. Concluding remarks...65
3.4. Materials and methods...66

3.4.1. Directed Evolution Library Construction...66
3.4.2. Library screening by fluorescence activated cell sorting...67
3.4.3. Protein over-expression and purification...68
3.4.4. Kinetic analysis...68
3.4.5. Cell culture experiments...69

4 Computational design of ddT kinase by Rosetta...71

4.1. Introduction...71
4.2. Results and discussion...73

4.2.1. Design ddT kinase from DmdNK by Rosetta...73
4.2.2. Kinetic analysis of Rosetta designed mutants...76
4.2.3. Modifying Rosetta designed DmdNK variants...77

4.3. Concluding remarks...85
4.4. Materials and methods...87

4.4.1. Materials...87
4.4.2. Protein expression and purification...88
4.4.3. Steady-state kinetic assays...88
4.4.4. Enzyme thermostability assay...89

5 Conclusions and perspectives...90

5.1. Summary...90
5.2. Comparison of our engineered kinases to previous evolution results...92
5.3. The advantages of using orthogonal enzymes for gene therapy...93
5.4. Extension of FACS-based screening to other NAs...94
5.5. Engineer human kinases...97
5.6. Combining computational design with directed evolution for kinase engineering...98

References...100

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Keyword	nucleoside analogs protein engineering suicide gene therapy nucleoside kinase protein design biocatalysis
Committee Chair / Thesis Advisor	Lutz, Stefan, Emory University
Committee Members	Menger, Fred M, Emory University Lynn, David, Emory University

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