sign in sign up
<<
Home , Purine analogue

Analysis of Tautomeric Equilibrium of the Analogue d(dinitro-tC(O))TP During Incorporation by the Klenow Fragment of DNA Polymerase I

Arielle Baker

Monday, April 7th, 2014: 4:00pm

Thesis Advisor Robert Kuchta, Ph.D. | Dept. of Chemistry and

Committee Members Robert Knight, Ph.D. | Dept. of Chemistry and Biochemistry Jennifer Martin, Ph.D. | Dept. of Molecular, Cellular and Developmental Biology

This thesis is submitted in partial fulfillment of the requirements for graduation with honors in the Department of Molecular, Cellular and Developmental Biology at the University of Colorado, Boulder.

Acknowledgements

My most heartfelt thanks goes to my advisor, mentor and friend Dr. Rob Kuchta, for his unending patience and willingness to share his knowledge with me, as well as his continued support for my pursuit of science. I would like to thank my friends in the Kuchta lab, and to some colleagues in particular. Thank you to Dr. Andrew Olsen and Dr. Gudrun Stengel for being the very first overseers of my work, and for not scaring me off. Thanks to Dr. Ashwani Vashishtha, Dr. Joshua Gosling, Sarah Dickerson, Clarinda Hougen and Taylor Minckley for the many laughs we’ve shared benchside, and for answering even the most insignificant of questions. Thank you to my committee members, Dr. Jennifer Martin and Dr. Rob Knight, for their assistance in navigating my thesis work. My work would not have been possible without generous funding from the National Institutes of Health. Thank you to my dear friend Makenna Morck for always being available to discuss biochemistry with me in the wee hours of the morning. I want to extend my profound thanks to my family: my parents, Jean and Michael, for being my first teachers, sparking a passion in me for learning; and my sisters, Sarah and Katelyn, for their unwavering support, and for pretending to be interested when I talk science. And, finally, to Grandma Baker- without you, I’d be an art history major.

2 Baker, Arielle Leigh (B.A., Molecular, Cellular and Developmental Biology) Analysis of Tautomeric Equilibrium of the Analogue d(dinitro-tC(O))TP During Incorporation by the Klenow Fragment of DNA Polymerase I Honors Thesis directed by Professor Robert Kuchta

The studies outlined in this thesis examines the kinetic parameters of incorporation of the novel analogue d(dinitro-tC(O))TP by the Klenow fragment of DNA polymerase I, accomplished through a series of polymerization assays. d(dinitro-tC(O))TP contains an additional pair of nitro groups at the top of the cyclic structure, expanding on the structures of previously studied synthetic tC and tC(O) (Stengel et al. 2009). Nitro groups have a large electron withdrawing effect due to the highly electron-withdrawing oxygens, resulting in the compound being recognized more as T than as C when compared to the parent tC and tC(O). KF incorporates d(dinitro- tC(O))TP as efficiently as dTTP opposite a templating base, and about twice as efficiently as dCTP opposite . My studies on d(dinitro-tC(O))TP support the existing hypothesis that selection for the correct nucleotide based on size is only true for analogues closely related to the canonical bases. KF appears to use both the size and hydrogen bonding patterns of incoming nucleotides to distinguish their correctness. The ability of d(dinitro-tC(O))TP to hydrogen bond in a manner similar to its parent compound, , allows the compound to be incorporated by KF while the bulky cyclic addition is not detrimental enough to sterically inhibit incorporation. The efficient incorporation opposite both A and G is consistent with it existing in roughly equal amounts of the two tautomers, amino (C- like) and imino (T-like). d(dinitro-tC(O))TP is an exciting compound because it retains traditional hydrogen bonding properties, thereby maintaining the identified prerequisite for selection of the correct incoming nucleotide, but expands upon the traditional cytosine structure by expanding into the major groove. Though not therapeutically viable, d(dinitro-tC(O))TP offers interesting insight into the effect of electron-withdrawing groups on tautomeric equilibrium when added to a nucleotide analogue.

3 TABLE OF CONTENTS

I. Introduction……………………………………………………………………..……..7 DNA REPLICATION………………………………..……………………………7 DNA POLYMERASE………………………..………………………….………..8 NUCLEOTIDE DISCRIMINATION BY DNA POLYMERASES…...……….…9 THERAPEUTICS…………..……………………………………………………13 THERAPEUTIC MECHANISMS OF ACTION….…………………….………13 II. Anticancer Nucleotide and Analogues………………………………...15 III. Antiviral Nucleotide and Nucleoside Analogues…………………………………20 IV. tC, tC(O), and dinitro-tC(O)…….. ………………………………………………….23 tC AND tC(O)………. ………………………………………………..…………..23 d(dinitro-tC(O))TP: A NOVEL NUCLEOTIDE ANALOGUE…………..………24 ASSAY METHODOLOGY………………..……………………..………..……25 V. Results………………………………………………………………………………..27 VI. Discussion and Conclusions………………………………………………………..30 VII. References………………………………………………………………………….35

4 LIST OF FIGURES

I. Introduction 1.1 Model for a replicative DNA polymerase 1.2 The chemical composition of DNA 1.3 Sample analogues lacking shape and hydrogen bond complementarity 1.4 analogues used to probe chemical features of natural purine II. Anticancer Nucleotide and Nucleoside Analogues 2.1 Chemical structure of 2.2 Chemical structures of and CNDAC 2.3 Chemical structure of 2.4 Chemical structure of troxacitabine 2.5 Chemical structure of ara-G III. Antiviral Nucleotide and Nucleoside Analogues 3.1 Chemical structure of FdC 3.2 Chemical structure of cyclopropavir IV. tC, tC(O), and dinitro-tC(O) 4.1 Hydrogen bonding of the amino and imino tautomers of tC 4.2 Chemical structure of d(dinitro-tC(O))TP V. Results 5.1 Primer elongation by Klenow fragment after incorporation of d(dinitro- tC(O))TP opposite G 5.2 Primer elongation by Klenow fragment after incorporation of d(dinitro- tC(O))TP opposite A VI. Discussion and Conclusions 6.1 Proposed hydrogen bonding of the imino tautomer of dinitro-tC(O) 6.2 Proposed hydrogen bonding of the amino tautomer of dinitro-tC(O) VII. References

5 LIST OF ABBREVIATIONS

CMV Cytomegalovirus CNDAC 1-(2-C-cyanodeoxybeta-D-arabino-pentofuranosyl) cytosine dATP triphosphate dCTP triphosphate dGTP triphosphate DNA Deoxyribonucleic acid dNMP Deoxynucleotide monophosphate dNTP Deoxynucleotide triphosphate dTTP Deoxythymidine triphosphate GW506U78 2-amino-9-beta-D-arabinosyl-6-methoxy-9H- HBV virus HIV Human immunodeficiency virus HSV Herpes simplex virus KF Klenow fragment NDP Nucleoside diphosphate RNA Ribonucleic acid ssDNA Single-stranded DNA

6 I. Introduction

DNA REPLICATION Cellular replication, proliferation and survival are contingent upon the process of DNA replication. In many domains of life, there are common features to the replication process, not only in its mechanisms but also in its machinery. This machinery includes DNA helicases, which topologically prepare the DNA helix by unraveling it; single- stranded DNA binding (SSB) proteins, which help to prevent reannealing of the DNA strands after unwinding by helicases as well as prevent destruction of the ssDNA by nucleases; , which synthesize a primer on the DNA template to which polymerases can adhere and begin elongation; topoisomerases, which relieve supercoiling stress in the DNA strand generated by helicase unwinding; and DNA polymerases, which synthesize daughter DNAs complementary to each single-stranded parent DNA in a semi-conservative fashion. Replication is an intricate process. It requires the perfect orchestration of a number of , and involves precisely copying genomes containing millions to billions of nucleotides. Despite these factors, replication of the genome is incredibly accurate; rarely do DNA polymerases incorporate the incorrect nucleotide. For A family polymerases, the frequency of error is approximately 10-3 to 10-6 errors per nucleotide inserted (Kunkel and Bebenek 1988). Many DNA polymerases also have exonuclease active sites that provide proofreading functions, which will repair many errors by excising incorrectly incorporated nucleotides, thus decreasing the misincorporation rate even further. Finally, repair enzymes that follow the replication machinery carry out a final examination of the replicated DNA, decreasing the frequency of errors to approximately 10-9 errors per nucleotide inserted (Roberts and Kunkel 1996). It is important to note that while erroneous replication can have deleterious consequences, evolution is founded in erroneous replication.

7 DNA POLYMERASE There are several families of polymerases found in a variety of organisms, including A, B, C, X, Y and RT. DNA polymerases are classified according to a variety of structural homologies, both in sequence and in conformation. The studies I conducted utilized the Klenow fragment of DNA Polymerase I (Pol I) from Escherichia coli, an A family polymerase. Pol I was the first identified polymerase (Kornberg 1960). In general, A family polymerases replicate DNA with high fidelity, working with other accessory proteins and subunits to achieve a finished product. Pol I also functions in nucleotide excision repair (Rothwell and Waksman 2005). DNA polymerases have similar structures across the different families. One distinctive feature of polymerases is their hand-like shape (Figure 1.1). The hand contains subdomains that resemble a palm, fingers and a thumb (Wang et al. 1997). The palm, similar to a human hand, is situated between the fingers and the thumb subdomains; this contains the active site where dNTPs enter the complex to be catalytically incorporated (Figure 1.1). The palm domains Figure 1.1: Model for a replicative DNA polymerase. The of different polymerase differing domains are indicated, as well as the parent DNA strand (brown), the daughter DNA strand (yellow) and a dNTP families are largely (black) (DePamphilis 2006). superimposable (Li and Waksman 2001), highlighting the evolutionary adaptation of the . The fingers work to recognize incoming nucleotides, and help orient them to bind, while the thumb helps to bind the DNA substrate (Figure 1.1) (Klenow and Henningsen 1970; Wang et al. 1997). The Klenow fragment of DNA Polymerase I is an important catalytic protein. First discovered in 1970, it was the first solved polymerase crystal structure (Ollis et al.

8 1985). KF is formed from Pol I by removing its 5’—3’ exonuclease domain. The cleaved enzyme retains 5’—3’ polymerase activity and its 3’—5’ exonuclease domain (Klenow and Overgaard-Hansen 1970; Zhao et al. 2013; Zhao and Guan 2010). The 3’—5’ exonuclease domain can be knocked out, which is the case for the KF utilized in this study. Because of the importance of ensuring accurate genomic replication, many studies focus on the importance of polymerase fidelity, examining the various mechanisms by which polymerases distinguish between correct and incorrect nucleotides (Prindle et al. 2013; Donigan et al. 2014). Homologies in polymerase structure and function raise the question of why certain types of polymerases are significantly more efficient or accurate than other polymerases (Yao and Müller 2011; Nemec et al. 2014).

NUCLEOTIDE DISCRIMINATION BY DNA POLYMERASES Nucleotides are compounds naturally created within cells that are utilized in a variety of ways, ranging from traditional RNA and DNA synthesis to metabolism, signaling within the cell, and regulation of enzyme activity (Jordheim et al. 2013). There are four canonical bases found in DNA: guanine, cytosine, and . Specific nucleobases express pairing preferences based on the size and stereochemistry of their nucleotide partners; cytosine and guanine are pairs, and adenine and thymine are pairs (Figure Figure 1.2: The chemical composition of DNA. 1.2). Francis Crick and James Watson This model shows the four bases adenine, thymine, largely pioneered this research, finding cytosine and guanine, and the backbone. The backbone is composed of phosphorylated that alternate tautomeric forms of natural deoxyribose sugars (Klotsa et al. 2005). nucleotides could account for mutations in DNA (1953). They used a model to determine that cytosine and adenine had the ability to mutagenically pair with each other using amino and imino tautomeric forms, and that thymine and guanine could mutagenically

9 pair with each other using keto and enol tautomeric forms; because adenine and guanine are , and thymine and cytosine are , these tautomeric forms can exist without causing a large structural disturbance (Morgan 1993; Urban et al. 2010; Hirao et al. 2012). A foundational property of the discovery of DNA structure was the identification of the hydrogen bonding patterns between nucleobases. These distinctive patterns are thought to be a basis upon which polymerases discriminate incoming nucleotides (Moore et al. 2004; Ramirez-Aguilar et al. 2005; Wolfle and Washington 2005; Choi et al. 2009). It appears that A family polymerases use a mélange of positive and negative selectivity in nucleotide discrimination. Pol I, which is a high-fidelity polymerase, does not appear to utilize hydrogen bond pairing to achieve efficient nucleotide incorporation (Chiaramonte et al. 2003; Trostler et al. 2009); these mechanisms will be discussed in detail later. Nucleoside and nucleotide analogues are chemically altered agents that are made to simulate the actions of their physiological counterparts, but with key differences. Perhaps the most famous analogue alteration development came from researchers Gertrude Elion and George Hitchings, who developed, among other drugs, acycloguanosine for the treatment of herpes; their discoveries led to development of AZT, for the treatment of AIDS. Since their remarkable discovery, scientists continue to toy with these useful compounds, producing a variety of novel structures. These alterations can be made on different structural components of the nucleotide. These developments have been built on previous knowledge on how correct nucleotides are selected and incorporated by polymerases. Not only are analogues being tested clinically, but a large number are used clinically as chemotherapeutics for both cancer and viral infection.

10 The mechanism by which Pol I may in some cases choose its substrate appears to be through evaluation of the shape of the incoming nucleotide (Kool 2002). This mechanism has been explored using analogues of traditional nucleotides, with altered or removed hydrogen bonding capabilities. In addition to shape and hydrogen bond features, investigations have also included consideration of the hydrophobicity and polarity of nucleotides being incorporated. Studies by Urban et al. conclude that primer elongation rate by Pol I decreases significantly when the incoming dNTP is unable to hydrogen bond to its templating base in a manner consistent with canonical Watson-Crick base-pairing (2009; 2010). The acceptors Figure 1.3: Sample nucleobase analogues that of hydrogen bonds contained within the lack shape and hydrogen bonding complementarity yet are incorporated very minor groove appear to be of particular efficiently by A family polymerases importance to nucleotide incorporation (Chiaramonte et al. 2003). (Seeman et al. 1976; Hendrickson et al. 2004). Studies thus indicate that the ability of nucleobases to form canonical hydrogen- bonding interactions during incorporation is of paramount importance in determining the efficiency of A family polymerases. Chiaramonte et al., however, showed that Klenow fragment has the ability to incorporate nucleotides whose shapes differ significantly from those of canonical nucleotides (2003). The analogues studied, which are shown in Figure 1.3, have novel and distinct shapes, leading to alternative hydrogen bonding patterns. These studies help to conclude that polymerases of the A family do not discriminate between nucleobases based solely on shape or hydrogen bond patterns, allowing them to efficiently incorporate a variety of compounds. Knowing what features

11 A family polymerases do not use to discriminate, termed negative selection, a study by Trostler et al. set out to determine what aspects of the nucleotide is used for discrimination, termed positive selection. Using Pol I of Bacillus stearothermophilus, the group studied a number of purine analogue triphosphates (Figure 1.4) in which the N-1, N-3 and N6 of the purine ring were systematically replaced with carbons. This results in a decreased efficiency of correct incorporation opposite T, particularly with substitution of the nitrogen for a carbon in the heterocycle in the 1 or 3 position (2009). This indicates that Pol I does not merely evaluate the shape or the hydrogen bonding ability of a given nucleotide; the composition Figure 1.4: Purine analogues utilized to probe the roles of specific chemical features of the of the natural purine nucleobases (Trostler et al. 2009). heterocycle plays a key role in ensuring correct incorporation, steering the polymerase away from mutagenic base pairing. Thusly, one can see that it is of paramount importance to consider not only the addition of groups to the canonical compounds, but also the composition of the cyclic portion of the structure itself.

12 THERAPEUTICS The important role of nucleotides in so many cellular processes has prompted investigation into their clinical applications, and analogues are an excellent avenue for current and future therapeutics. Structural alterations allow researchers to manipulate the nuances of replication and nucleotide metabolism, thereby affecting a plethora of intracellular activities (Jordheim et al. 2013). Notably, analogues can inhibit DNA and RNA synthesis, thus halting cellular division and/or viral replication (De Clerq and Neyts 2004). Nucleotide and nucleoside analogues, then, represent powerful treatments for cancer tumor suppression and anti-viral therapy (Pockros 2013), as well as unconventional approaches to mechanisms involved in polymerase and phosphodiesterase inhibition, epigenetic modulation, immunosuppression, neuroprotection and cardioprotection (Moyle 2000; Squires 2001). In order for nucleoside analogues to be functional, they must undergo phosphorylation within the cell by nucleoside kinases, (d)NMP kinases and NDP kinase; triphosphorylation is necessary for nucleotides to become active in the body. Some nucleotide analogues already have this phosphorylation completed, and are thus already chemically active, but are unable to enter the cell because of the inability for charged phosphate groups to penetrate the hydrophobic cell membrane (Pockros 2013; Squires 2001). Thus, phosphorylation must be completed once inside the cell, which is a current challenge to clinical application.

THERAPEUTIC MECHANISMS OF ACTION Nucleotide and nucleoside analogues often behave as anti-metabolites in the process of metabolism. They can utilize the exact metabolic pathways that are used by natural and nucleotides (Jordheim et al. 2013). Classical nucleotides are hydrophilic in nature, and are derived from two pathways: the de novo pathway and the salvage pathway. The de novo pathway converts various small molecules into nucleoside triphosphates; this pathway, then, is essential in replication of cells. The salvage pathway, on the other hand, recycles leftover nucleosides from DNA and RNA catabolism (Kufe et al. 1984). Exogenous nucleotides use kinases of the salvage pathway. These analogues are ingested by cells through compound-specific

13 membrane transport carriers or via passive diffusion across the plasma membrane (Minuesa et al. 2011; Cano-Soldado and Pastor-Anglada 2012). Ongoing research indicates that some antiviral analogues can be taken up by organic anion and cation transporters, in addition to peptide transporters (Roizman et al. 2001), but this data is presently inconclusive. Once ingested, the analogues undergo a series of phosphorylation events, allowing di- and tri-phosphorylated analogues to collect in the target cells. Without these phosphorylation steps, the drugs remain inactive (Jordheim et al. 2013). The active analogues are then readily available for incorporation into DNA and RNA strands being synthesized, or for inhibition of enzymes other than polymerases, or oftentimes for both. Many analogues, due to the structural and chemical changes they possess, cause elongation to halt, as the polymerase enzyme cannot extend the strand past such a compound (Pockros 2013). Typically in replication of the viral genome, incorporation of analogues causes the cell to recognize the strand as severely mutated, causing replication termination of the viral DNA or RNA, called error catastrophe (De Clerq and Neyts 2004; Leyssen et al. 2008;). Researchers postulate that analogues results in a chain- termination event that prevents progression of the cell cycle S phase (Ewald et al. 2008), but further research in this area is needed in order to draw more decisive conclusions. It is important to understand the structural and mechanistic basis for nucleotide and nucleoside analogues in order to develop new compounds. Such considerations have fostered the creation of agents that interact more efficiently with membrane transporters, or that are less liable to degradation (Jordheim et al. 2013). New compounds can be developed with enhanced properties that increase the efficiency and rate of success of nucleotide and nucleoside analogues as therapeutics (Pockros 2013). Research in the nucleotide and field has seen the development of many new nucleoside drugs, several of which are presently in early-phase clinical trials, and a significant number that have become regularly prescribed drugs (Jordheim et al. 2013). I will briefly review the history and present state of such therapeutics, to set the stage for the investigation of d(dinitro-tC(O))TP. Nucleoside and nucleotide analogues fall into two primary therapeutic categories: anticancer compounds and antiviral compounds.

14 II. Anticancer Nucleotide and Nucleoside Analogues

The first anticancer nucleoside analogue that targeted DNA polymerases was cytarabine (Figure 2.1) for acute myeloid ; approval of this compound by the US Food and Drug Administration (FDA) in 1969 initiated a cascade of compound development for anticancer therapeutics (USA National Institutes of Health 2011). In terms of anticancer nucleoside analogues, as of 2007, nine are currently approved and on the market: cytarabine, , , gemcitabine, , , , , and deoxycoformycin (Nekhai et al. 2007). These drugs treat specific types of cancer, and are largely derivatives of deoxycytidine, deoxyadenosine or deoxyguanosine (Van Rompay et al. 2003; Galmarini et al. 2010; Senanayake et al. 2011). Two additional compounds, Figure 2.1: Chemical (Kaminskas et al. 2005) and (Stewart et structure of cytarabine (USA al. 2009; Jordheim et al. 2013) are known as demethylating National Institutes of Health 2011). agents and act through a different mechanism to prevent proliferation in cancer cells. Presently, many consider existing anticancer agents to not be impactful enough to merit continued clinical treatment. Cytarabine, the most powerful clinically used compound, provides cures of acute myeloid leukemia in fewer than 30% of all adult patients (Kantarijian et al. 2012). A couple of other cytotoxic nucleoside analogues have been developed (Jordheim et al. 2013), but do not impact the patient long-term, simply prolonging survival, but not ultimately curing the cancer. A few of the newly developed nucleoside analogues have the same cytotoxic mechanisms as those of older nucleoside analogues. One such compound is 1-(2-C- cyanodeoxybeta-D-arabino-pentofuranosyl) cytosine (Figure 4), also referred to as CNDAC (Jordheim et al. 2013), from which an orally administered drug has been created: sapacitabine (Figure 2.2) (Chaio et al 2013).

15 CNDAC is a cytarabine analogue that functions by cleaving the DNA strand through nucleophilic attack of the cyano groups. Human

Figure 2.2: Chemical structures of sapacitabine (left) and CNDAC cancer xenografts (right) (Chaio et al. 2013). responded well in vivo to both CNDAC and sapacitabine. This has resulted in the initiation of sapacitabine’s clinical testing, as well as in combination with other anticancer agents, and appears to have a beneficial effect in patients being treated for chronic lymphocytic leukemia, acute myeloid leukemia, as well as some small tumors, largely by inhibiting the polymerases that copy genetic information. By disallowing these processes to occur, sapacitabine prevents the growth of tumors (Kantarjian et al. 2012). Another realm of anticancer analogue research is focused on inhibiting RNA synthesis (Krett et al. 2004). Two such compounds utilized in this research are 8-chloro adenosine and 8-amino-adenosine. 8-Chloro-adenosine decreases RNA synthesis while 8- amino-adenosine decreases both RNA and DNA synthesis (Gandhi et al. 2001). This results in a decrease in the production of a variety of proteins. The cytotoxic effects of 8- chloro-adenosine are manifested in decreased expression of MET. MET is the for hepatocyte growth factor in myeloma cells (Stellrecht et al. 2007). 8-Chloro- adenosine is also implicated in decreased cyclin-E expression in breast cancer cells. Due to these properties, this compound is presently being clinically investigated in a Phase I clinical trial (Stellrecht et al. 2010; Jordheim et al. 2013). And yet, these compounds display inconsistency in terms of impact, largely due to patient variability. For example, cytarabine, as well as fludarabine, are functional in a few hematological malignancies, but not in solid tumors. This inconsistency may rise from a matter of patient variability or a mechanism of action. The compounds are specific for reasons not yet understood. The effects of gemcitabine (Figure 2.3) are presently the most consistently predictable in the field of anticancer nucleoside analogues; it displays activity in both hematological malignancies and several solid tumors (Song et al. 2013).

16 These studies have shaped the direction of current cancer therapeutics research—research has evolved from the notion of an overlying “cancer treatment”, due to tumor specificity; rather, current efforts aim to treat each tumor type as a separate disease. 4’-Thioaracytidine is a nucleoside in which the sugar has been modified, and demonstrates antitumor activity in several tumor subtypes. 4’-thioaracytidine exhibits a unique ability to retain a tri-phosphorylated state, unlike cytarabine and fludarabine. A tri- phosphorylated state is more difficult to degrade intracellularly, thus making it a more stable compound. 4’-Thioaracytidine is currently being evaluated in Phase I clinical trials (Someya et al. 2006). Figure 2.3: Chemical structure of gemcitabine Many anticancer compounds can also target (Song et al. 2013). viruses, the most prominent of which is cytarabine (Figure 2.1). Cytarabine can target herpesvirus infection (Renis 1973; Hryniuk et al. 1972). The compounds toxacitabine and 2-amino-9-beta-D-arabinosyl-6-methoxy-9H- guanine, both of which are anticancer compounds, display promising attributes in their pre-clinical trials (Jordheim et al. 2013). Troxacitabine (Figure 2.4) is a synthetic nucleotide analogue (Galmarini et al. 2001) that, in pre-clinical trials, appeared to behave as an antitumor agent (Grove et al. 1995; Kadhim et al. 1997; Parker 2013). It has a nontraditional stereochemical configuration, and is the first of its kind to be developed clinically. Being a nucleoside analogue, troxacitabine requires phosphorylation in vitro by cellular kinases, after which its incorporation into the DNA strand causes inhibition of the polymerase. This compound does not have inhibitory effects on reductase nor mitochondrial DNA Figure 2.4: Chemical structure of synthesis; in the case of gemcitabine, the inhibition of troxacitabine (Galmarini et al. 2001). ribonucleotide reductase may enhance its efficacy.

17 Trozacitabine is hence in phase II testing due to its breadth: it is cytotoxic against both leukemic and epithelial malignancies, and may have effects on acute leukemia (Giles et al. 1999; Jordheim et al. 2013). The compound 9-beta-arabinofuranosylguanine, also referred to as ara-G (Figure 2.5), is a distinctive anticancer nucleoside analogue (Galmarini et al. 2001) that exhibits resistance to cleavage by purine nucleoside phosphorylase (PNP) (Mahmoudian et al. 1999). Pre-clinical trials revealed that ara-G was toxic to T-lymphocytes, and therefore represented a powerful antitumor candidate, but development of this compound was halted due to problems with insolubility in water (Aguayo et al. 1999). This led researchers to the discovery of the compound 2-amino-9-beta-D-arabinosyl-6- methoxy-9H-guanine, also referred to as GW506U78, which is a derivative of ara-G, but with improved

Figure 2.5: Chemical structure solubility, allowing the compound the access the cell of ara-G (Mahmoudian et al. 1999). (Aguayo et al. 1999). After accessing the cell, GW506U78 can then be converted to ara-G via plasma adenosine deaminase (Kurtzberg et al. 1999; Jordheim et al. 2013). Troxacitabine and GW506U78 are excellent examples of the progression of anticancer nucleoside and nucleotides analogue research. Developmental research on troxacitabine has helped researchers understand the selectivity of its stereochemistry in regards to specific types of DNA. This enables researchers to develop selective drug targets. The logical development of GW506U78 from ara-G demonstrates the ability of researchers to examine analogues structurally, and make appropriate changes based on specific properties of that compound. This is the direction of current nucleoside and nucleotide analogue research— trial and error, slowly molding the ideal compound for particular cancer or viral infection types. This will yield new agents with enhanced solubility and bioavailability, as well as the ability to overcome resistance mechanisms (Jordheim et al. 2013).

18 Research on anticancer nucleoside and nucleotide analogues has brought about the development of many successful candidates for therapeutics. The breakthroughs with cytarabine and other analogues brought about a profound interest in nucleoside and nucleotide analogues for anticancer therapeutics. Studies on the chemical structure of novel compounds, however, have brought about candidates for specificity and retention. Thusly, there is significant effort being made to improve their utility. These novel analogues exhibit the potential to transform the position of nucleoside and nucleotide analogues in cancer therapy.

19 III. Antiviral Nucleotide and Nucleoside Analogues

Antiviral nucleoside analogues emerged in 1969, the first of which was . Since the development of edoxudine, 24 additional antiviral nucleoside and nucleotide analogues have been created and approved by the FDA. Antiviral analogues can target several viruses, including HIV, HBV, CMV and HSV infections (De Clercq and Holy 2005). HIV, HBV and HCV infections are life threatening and chronic, and represent the largest class of antiviral research; these infections affect about 600 million people throughout the world (Hurwitz and Schinazi 2013). Another classification is acute infections: independent viruses that cycle through the population with minor impact for the majority of people, but dangerous to some individuals, such as pregnant and feeding women, or the elderly (De Clerq and Holy 2005). The final classification of viral infections is those that are common, such as a cold (caused by rhinoviruses), but do pose an economic impact in the form of sick employees. The ability of nucleoside and nucleotide analogues to treat a variety of viral diseases is both a benefit and a potential roadblock It is beneficial because it allows one simple mechanism to be applied across the board to many diseases without extensive time used and research to be conducted. It can be problematic, however, because each virus is specific, variable and irregular, making it difficult to predict the outcome of clinical application of a given antiviral agent (Jordheim et al. 2013). There are four main antiviral targets: HBV, HCV nucleosides, HIV nucleosides, and herpes. Anti-HCV nucleoside analogues largely revolve around the compound 2’-deoxy-2’fluorocytidine (FdC) (Figure 3.1), and chemically altered derivatives (Stuyver et al. 2004). One such derivative is valopicitabine, which was the first clinically used anti-HCV nucleoside analogue. Valopicitabine can function by itself, but is often combined with pegylated or , to decrease chance of Figure 3.1: Chemical structure of FdC (Stuyver et al. 2004). resistance. The success of this compound prompted the

20 development of more anti-HCV compounds, notably mericitabine. Mericitabine is another FdC derivative that is currently in Phase II clinical trials, and is presently on the forefront of anti-HCV nucleoside development (Le Pogam et al. 2010; Wedemeyer et al. 2013). Anti-HIV nucleosides are dideoxyribose derivatives. One particularly potent derivative is apricitabine, which has elevated genetic resistance, making it an attractive agent. Apricitabine functions best against HIV-1 strains and HIV strains with mutations in reverse transcriptase. In 2005, tested over a 48-week treatment period, apricitabine was highly effective and did not show signs of resistance (Bethall et al. 2005). Despite anticipation of its arrival on the market in 2010, however, apricitabine was suddenly withdrawn, seemingly due to legal and commercial complications. One year later, it was reinstituted as an anti-HIV candidate, and is currently in Phase III clinical trials (Cahn and Wainberg 2010). Apricitabine represents the progression of anti-HIV analogues, as well as that which needs to be addressed in future compounds: apricitabine is a worthy candidate due to its resistance to mutation; this effect ought to be taken into consideration upon further development of more anti-HIV nucleosides. Studies since the development of apricitabine have led to the generation of the compound festinavir. Festinavir, which is a derivative of an early anti-HIV nucleoside analogue names stavudine but with an additional 4’ position ethynyl group on the ring, has been found have reduced toxicity in patients, as well as increased potencies. Festinavir, then, expresses properties better than its forefathers, and is presently being evaluated in Phase II clinical trials. It has a prolonged antiviral effect, and is an attractive model for development of differing resistance profiles. Anti-herpes nucleosides are acyclic sugars. The most famous anti-herpes nucleoside on the market is acyclovir, from which stemmed several other compounds: , , and most recently, Figure 3.2: Chemical structure cyclopropavir. Cyclopropavir (Figure 3.2) is a derivative of cyclopropavir (Chou et al. 2012). of the traditional nucleoside guanosine, but with a

21 methylenecyclopropane group as a side chain (Chou et al. 2012). Cyclopropavir is interesting because it combats multiple strains of herpesvirus, including human herpesviruses 6A, 6B and 8, and human cytomegalovirus (Price and Prichard 2011). It exhibits many unique properties not present in currently approved anti-herpes agents, including the ability to inhibit replication of human cytomegalovirus, and the ability to remain continuously active against ganciclovir-resistant strains (Chou and Bowlin 2011). Antiviral nucleosides and nucleotides have seen an influx of development over the past ten years, making them attractive subjects for future studies. Future research, however, needs to focus on ensuring the analogues’ incorporation into the viral genome, and not the genomic and mitochondrial DNA of the host cells. In addition, it is important that a variety of compounds continue to be developed, to stave off resistance to particular compounds by viruses being treated solely by a few compounds (Jordheim et al. 2013). Effective treatment will most likely require the use of many compounds, for which research ought to be continued. The success and failures of current therapeutics, as well as the number of studies elucidating DNA polymerase mechanisms, have helped develop a greater understanding of the way in which polymerases accurately and successfully incorporate a given nucleotide. This research has lead to the amelioration of current nucleotide analogues, and still leave quite a bit of room for development of further systems.

22 IV. tC, tC(O), and dinitro-tC(O) tC AND tC(O) A study conducted in 2009 investigated of the compound 1,3-diaza-2-oxo- phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tC(O)) (Figure 4.1) (Stengel et al.). These compounds are unique cytosine analogues with fluorescent properties. Unlike traditional cytosine, tC contains substantial extra mass in the major groove, but Figure 4.1: Hydrogen bonding of the amino and imino tautomers of tC remains similar to with G and A, respectively (X = S for tC; X = O for tC(O)) (Stengel et al. 2009). cytosine in its hydrogen bonding abilities (Stengel et al. 2009). Extensive studies characterizing these compounds in relation to all four natural nucleobases using the KF of DNA Pol I helped elucidate their incorporation properties. Stengel et al. discovered many unique characteristics of these compounds. KF inserts dtCTP and dtC(O)TP efficiently opposite both G and A. KF inserts both analogues opposite G with the same efficiency (defined by Vmax/KM) with which dCTP is inserted. KF incorporated the analogues approximately 4- to 11-fold less efficiently opposite template A than incorporation of dTTP. The KF is able to distinguish the structure of the template, thereby not generating dtC(O)TP- mismatches. KF polymerizes dtCTP and dtC(O)TP 200- and 1000-fold less effectively opposite T, respectively, than it does dATP (Stengel et al. 2009). Many N4-substituted cytosine analogues prefer to exist in the imino tautomer (Harris et al. 2003). The analogues tC and tC(O), however, have an unclear tautomeric equilibrium- and one cannot determine the tautomeric equilibrium base solely on structure. The compounds likely with G in the amino tautomer, and in the imino tautomer they base pair with A (Figure 4.1). Polymerization favored the amino tautomer

23 10:1 because the amino tautomer is 10-fold more stable; this helps to explain why multiple polymerases are able to insert the compound more efficiently opposite G than A (Table 4.1).

The studies by Stengel et al. investigated incorporation of dtCTP and dtC(O)TP by both KF and pol α, as well KM as some studies using dNTP DNA Vmax Vmax/KM Discrimination [µM] BF pol and HSV pol, dTTP DNA 4.1 0.6 6.5 1 A and found that despite dtCTP DNAA 1.6 2.7 0.6 11 differing substrate (O) dtC TP DNAA 2.2 2.4 0.9 7 specificities, they dCTP DNAG 3.63 0.23 15.8 1 polymerize the dtCTP DNAG 4.5 0.18 25 0.6 analogues with (O) dtC TP DNAG 3.4 0.13 26.2 0.6 strikingly similar Table 4. 1: Kinetic parameters for incorporation of dtCTP and dtC(O)TP efficiency. The analogues into DNAA and DNAG by KF (Stengel et al. 2009). formation of base pairs occurs nearly identically regardless of placement of the analogue in the primer or the template strand, thereby showing that the imino tautomer of tC(O) is the main proponent

of mutagenic pairing opposite A. Calculation of Vmax/KM found that the tautomeric equilibrium of tC and tC(O) is relatively the same; this demonstrates that the tautomeric equilibrium plays a significant role in defining incorporation ability (Stengel et al. 2009).

d(dinitro-tC(O))TP: A NOVEL NUCLEOTIDE ANALOGUE The nucleotide analogue d(dinitro-tC(O))TP (Figure 4.2) was synthesized in

O2N NO2 December 2011 by Brittney Rodgers and Byron Purse of the Purse Laboratory at the University of San Diego.

HN O Due to the structural similarities of dinitro-tC(O) to tC and tC(O) , I founded my N assays on the studies of these compounds. N (O) O O O Whereas tC and tC are derived from O O

O P O P O P OH

OH OH O-

OH 24 Figure 4. 2: Chemical structure of d(dinitro- tC(O))TP (amino tautomer). cytosine via an expansion of the carbon ring at the N4 and C5 positions (Stengel et al. 2009), d(dinitro-tC(O))TP has an additional two nitro groups at the top of the tricyclic portion of the structure. I examined the incorporation of a triphosphorylated dinitro-tC(O) opposite natural bases, characterizing the structural and electronic properties of the compound to understand its incorporation preferences using the Klenow Fragment of DNA polymerase I. Nitro groups are extremely electron withdrawing. My studies aim to address how the two nitro groups affected the tautomeric equilibrium between imino and amino forms.

ASSAY METHODOLOGY All reagents were of highest quality commercially available. Unlabeled dNTPs were purchased from Invitrogen. d(dinitro-tC(O))TP was synthesized in the Purse Laboratory at University of San Diego. E. coli DNA Polymerase I Klenow fragment (exo-) was purchased from New England Biolabs.

5’-labeling of primer strands DNA primers were 5’-32P-labeled using T4 kinase (purchased from New England Biolabs) and [ ! - 32P]ATP. The labeled primer was annealed to the appropriate template strands. The Chart 1: Sequences of Primer-Templates. The letter after "DNA" designates the template base being replicated. sequences of the used primer-templates are given in Chart 1.

Polymerization assays All kinetic data were determined under steady-state conditions. 32 The reactions with KF contained 2 µM 5’- P-primer-template, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, and varying concentrations of natural or analogue dNTPs in a total volume of 10 µl. Reactions that served to derive kinetic parameters contained 0.5 nM KF

25 (-exo). Polymerization was initiated by mixing equal volumes of reaction mixture and enzyme followed by incubation at 37 ºC. The reactions were stopped by addition of two volumes gel loading buffer (90% formamide with 50 mM EDTA) after 1—10 minutes. Extension products were separated by denaturing gel electrophoresis (20% polyacrylamide, 8 M urea) and analyzed by phosphor imaging (Typhoon scanner,

Molecular Dynamics). The parameters KM and Vmax were obtained by nonlinear curve fitting.

26 V. Results KF inserts d(dinitro-tC(O))TP efficiently opposite G and A The single nucleotide insertion assays evaluated the incorporation of d(dinitro- tC(O))TP opposite guanosine and adenosine; G and A were positioned on synthetic oligonucleotides (Chart 1); these assays allowed for the determination of the KM and

Vmax. Preliminary experiments indicated poor to nonexistent incorporation of d(dinitro- tC(O))TP opposite T and C. Knowing that d(dinitro-tC(O))TP is derived from the canonical pyrimidine dCTP, I wanted to investigate its ability to be incorporated across from a templating guanosine. Figure 5.1 shows the result.

Figure 5.1: Primer elongation by KF after incorporation of d(dinitro-tC(O))TP. The primer/template sequence is shown above the figure. Assay contained: 2 µM DNAG/standard primer; 2, 5, 10, 20, 40 and 500 µM dCTP. The enzyme concentration was 0.5 nM KF. The time points recorded were 2.5 and 10 minutes.

Knowing the ambivalence with which dtCTP and dtC(O)TP were incorporated opposite both a templating guanosine and adenosine, I investigated the incorporation of d(dinitro-tC(O))TP across from a templating adenosine. Figure 5.2 shows the result.

27

Figure 5.2: Primer elongation by KF after incorporation of d(dinitro-tC(O))TP. The primer/template sequence is shown above the figure. Assay contained: 2 µM (O) DNAA/standard primer; 2, 5, 10, 20, 40 and 500 µM d(dinitro-tC )TP; 0.5, 0.75, 1, 2, 4 and 6 µM dTTP. The enzyme concentration was 0.5 nM KF. The time points recorded were 2.5 and 10 minutes.

Graph 5.1 visually depicts the Michaelis-Menten kinetics with which d(dinitro-tC(O))TP is incorporated opposite a templating adenosine.

Incorporation of d(dinitro-tC(O))TP opposite A

1.5

1

Measured 0.5 V(initial) Calculated 0 0 1 2 3 4 5 [S]

Graph 5.1: Michaelis-Menten parameters for incorporation of d(dinitro-tC(O))TP opposite template adenosine.

28

Graph 5.2 visually depicts the Michaelis-Menten kinetics with which dTTP, the correct nucleotide, is incorporated opposite a templating adenosine.

Incorporation of dTTP opposite A

1.4 1.2 1 0.8 0.6 Measured

V(initial) 0.4 Calculated 0.2 0 0 1 2 3 4 5 [S]

Graph 5.2: Michaelis-Menten parameters for incorporation of dTTP opposite template adenosine.

Table 5.1 shows the quantitative data of how the rates of incorporation compared.

dNTP DNA Vmax KM [µM] Vmax/KM Discrimination

dTTP DNAA 2.47 0.32 7.70 1 (O) d(dinitro-tC )TP DNAA 2.38 0.25 9.48 1.23

dCTP DNAG 0.69 0.19 3.59 1 (O) d(dinitro-tC )TP DNAG 2.93 0.38 7.70 2.15

Table 5. 1: Kinetic parameters for incorporation of d(dinitro-tC(O))TP analogues and their correct counterparts into DNAN by KF.

KF polymerizes d(dinitro-tC(O))TP opposite A with approximately the same efficiency, Vmax/KM, with which it inserts the correct nucleotide, dTTP. KF polymerizes d(dinitro-tC(O))TP opposite G with approximately 2-fold more efficiency than it does the correct nucleotide, dCTP (Table 5.1).

29 VI. Discussion and Conclusions

d(dinitro-C(O))TP was derived in part from the studies conducted by Stengel et al. using tC and tC(O). Whereas tC and tC(O) are derived from cytosine via an expansion of the carbon ring at the N4 and C5 positions (Stengel et al. 2009), dinitro-tC(O) expands on the structure of tC(O) through the addition of two nitro groups at the top of the tricyclic portion of the structure. Studies of dtCTP and dtC(O)TP found that these compounds hydrogen bond similarly to their parental structure cytosine, but extend their bulky tricyclic rings into the major groove of the DNA; despite this addition, KF is able to polymerize the compounds equally as efficiently as a dCTP analogue, and only 4-11 times less efficiently as a dTTP analogue (Table 1). Thus, KF ambivalently incorporates dtCTP and dtC(O)TP opposite both A and G, which appears remarkable in light of the natural ability of KF to recognize mismatches (Stengel et al. 2009). KF incorporates d(dinitro-tC(O))TP efficiently opposite both A and G (Table 5.1), but with varying degrees. KF incorporates d(dinitro-tC(O))TP as efficiently as dTTP opposite A, and about twice as efficiently as dCTP opposite G (Table 5.1). A higher

Vmax/Km indicates a higher affinity for the substrate; this would indicate that KF has the greatest affinity for d(dinitro-tC(O))TP as a T analogue. KF appears to have an equal affinity for dTTP upon incorporation opposite A as it does d(dinitro-tC(O))TP upon incorporation opposite G. Of all the substrates investigated, KF has the least affinity for incorporation of dCTP opposite G. Acting as a T and C analogue, this indicates that d(dinitro-tC(O))TP behaves as a . Stengel et al. found that a dtC(O)TP-G base pair has a higher melting temperature than the canonical C-G base pair, leading to the conclusion that the novel compound lends a stabilizing effect to the duplex. This effect may be derived from π-π interactions between the aromatic tricycle and the heterocycles of the traditional bases. This is most likely true of d(dinitro-C(O))TP.

30 I propose that d(dinitro-tC(O))TP O2N NO2 incorporates opposite A in the imino tautomer (Figure 6.1); in the same manner, d(dinitro-tC(O))TP most likely incorporates H opposite G via the amino tautomer (Figure

N N H N O 6.2). This is largely because the hydrogen bonding ability of d(dinitro-tC(O))TP N (O) N should not differ from that of tC and tC ; C1 H N N the nitro groups do not influence the N compounds ability to pair with other bases. O C1 The cytosine analogue dinitro-tC(O) Figure 6.1: Proposed hydrogen bonding of the imino tautomer of dinitro-tC(O) (right) with A (left). and its sister compounds tC and tC(O) are a largely uncharted category of traditional nucleotide analogues; usually, nucleotide analogues are not quite as sizable as these compounds, but the size difference appears to have minimal effect on incorporation, particularly because hydrogen bonding patterns remain unaffected. A number of studies examine compounds in which the hydrogen bond donors and acceptors are substituted with differing chemical groups comparable in size to that which is being replaced, leading to the conclusion that hydrogen bonding is not the O2N NO2 key component in correct polymerization- rather, concluded that size appears to

be the deciding factor. (Kool N O H N O 2002; Henry and Romesberg N 2003). N C1 H N My studies on d(dinitro- N N tC(O))TP further supports the N H O C1 idea that selection for the H correct nucleotide based on Figure 6.2: Proposed hydrogen bonding of the amino (O) size is only true for analogues tautomer of dinitro-tC (right) with G (left).

31 closely related to the canonical bases. The size of d(dinitro-tC(O))TP is radically different from traditional nucleotides, and polymerization assays show that it can be incorporated efficiently but unselectively via KF, much like dtCTP and dtC(O)TP (Stengel et al. 2009). Studies of these compounds indicate that KF uses different mechanisms to select for the incoming nucleotide- and that both the size and hydrogen bonding patterns of the incoming nucleotide are equally important. With d(dinitro-tC(O))TP, then, it would appear that its ability to hydrogen bond in the same pattern as its parent compound, cytosine, is sufficient to allow KF to identify it as correct. The function of the bulky cyclic addition that juts into the major groove is less clear. Because it is readily incorporated into the DNA strand, it does not appear to be detrimental enough to disqualify the compound as incorrect. d(dinitro-tC(O))TP is an exciting compound because it retains traditional hydrogen bonding properties, thereby maintaining the identified prerequisite for selection of the correct incoming nucleotide, but expands upon the traditional cytosine structure by expanding into the major groove. d(dinitro-tC(O))TP represents an expansion of the studies conducted by Stengel et al. by the addition of the nitro groups. tC and tC(O) express tenfold preference for C, whereas dinitro-tC(O) appears to exist in closer tautomeric equilibrium. Thus, the addition of nitro groups causes d(dinitro-tC(O))TP to be recognized more like T. Electron-withdrawing groups therefore has an undesirable effect, causing the strand to be less mutagenic, and future compounds to be explored should evaluate the effect of adding electron-donating groups to the structure. The development of nucleotide and nucleoside analogues over the past decade has resulted in the creation of a powerful tool against a variety of viruses and cancers. These compounds are structurally and mechanistically diverse, leading to a plethora of applications. As seen above, nucleotide and nucleoside analogues have progressed significantly and have a dramatic impact on cancer and viral therapeutics; the applications of these compounds clinically have led to the recognition of that which is beneficial and detrimental to the compounds therapeutically. Background research has worked to widen knowledge about the individual mechanisms of resistance to nucleotide and nucleoside analogues, such as cellular drug uptake, interaction with cellular targets, drug metabolism, and apoptosis. Researchers have taken this knowledge and applied it to

32 more compounds, expanding the library of agents available to patients (Jordheim et al. 2013). Researchers are currently developing many additional agents that are presently in clinical trials. However, viruses continue to mutate and emerge, presenting a need for constant development of new compounds. Several viral infections, both life-threatening and not, do not have viable therapeutics, and could be addressed by new or repositioned nucleoside and nucleotide analogues. There are still fundamental flaws in the nucleoside and nucleotide analogue therapeutic system. Viral genomes demonstrate strong variation, and viruses have heterogenous resistance mechanisms (Hurwitz and Schinazi 2013). Patients have diverse genetic variability. Nucleoside and nucleotide analogues can attack host nucleic acid, leading to extremely dangerous circumstances for the patient. Moreover, due to the novelty of the system, the cost of such therapeutics is not yet a viable, low-cost option for diseases in low-income countries, which is one of the places that needs it the most (Veltkamp et al. 2008). Nucleosides with ambivalent base pairing properties, like tC, tC(O) and dinitro- tC(O), have the potential to have huge impacts on cancer therapeutics. This is because they can be introduced to the genome and incorporated extensively as mutagens, allowing the cell to recognize the sequence as error catastrophe. Currently, however, d(dinitro- tC(O))TP is therapeutically unviable; it is unable to be phosphorylated in the cell, potentially due to inhibition of the kinases by the bulky cyclic structure. Further studies on d(dinitro-tC(O))TP could need to elucidate its mutagenic capabilities as the templating base, as the studies by Stengel et al. determined for tC and tC(O). If d(dinitro-tC(O))TP were to exhibit mutagenic capabilities in the templating strand, this would support multiple rounds of cell division, thereby making it a suitable candidate that would survive past the first few rounds of replication. Thus, it appears that the merit of d(dinitro- tC(O))TP is founded in kinetic studies, not actual therapeutic application. Pursuit of a different compound, with electron-donating groups rather than electron-withdrawing groups, would be an interesting avenue for future research. It appears as though the addition of nitro groups shifts the tautomeric equilibrium from the amino preference of tC(O) to a greater preference for the imino tautomer. Perhaps the addition of electron- donating groups would push the tC(O) compound significantly more towards the amino

33 tautomer. This would help to confirm the effects of adding electron-affective groups to nucleotide analogues.

34 VII. References

Bethell, R. C., Y. S. Lie, and N. T. Parkin. "In Vitro Activity of SPD754, a New Deoxycytidine Nucleoside Reverse Transcriptase Inhibitor (NRTI), against 215 HIV-1 Isolates Resistant to Other NRTIs." Antiviral Chemistry & Chemotherapy 16 (2005): 295–302.

Cahn, P., and M. A. Wainberg. "Resistance Profile of the New Nucleoside Reverse Transcriptase Inhibitor Apricitabine." Journal of Antimicrobial Chemotherapy 65 (2010): 213–17.

Cano-Soldado, P., and M. Pastor-Anglada. "Transporters That Translocate Nucleosides and Structural Similar Drugs: Structural Requirements for Substrate Recognition." Medicinal Research Reviews 32 (2012): 428–57.

Chiao, Judy, David Blake, Daniella Zheleva, Susan Davis, Simon Green, and Geoffrey Shapiro. Dosage Regimen for Sapacitabine and . Cyclacel Limited, assignee. Patent 2013171473 A1. 21 Nov. 2013. Print.

Chiaramonte, M., C.L. Moore, K. Kincaid, and R.D. Kuchta. “Facile polymerization of dNTPs bearing unnatural base analogues by DNA polymerase alpha and Klenow fragment (DNA polymerase I).” Biochemistry 42 (2003): 10472–81.

Choi, J.Y., S. Lim, R.L. Eoff, and F.P. Guengerich. “Kinetic analysis of base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase iota.” Journal of Molecular Biology 389 (2009) 264–74.

Chou, S., and T. L. Bowlin. "Cytomegalovirus UL97 Mutations Affecting Cyclopropavir and Ganciclovir Susceptibility." Antimicrobial Agents and Chemotherapy 55 (2011): 382–84.

Chou, S., G. Marousek, and T. L. Bowlin. "Cyclopropavir Susceptibility of Cytomegalovirus DNA Polymerase Mutants Selected after Antiviral Drug Exposure." Antimicrobial Agents and Chemotherapy 56 (2012): 197–201.

De Clercq, Erik, and Antonin Holy. "Acyclic Nucleoside Phosphonates: A Key Class of Antiviral Drugs." Nature 4 (2005): 928–40. Nature Reviews: Drug Discovery. Nature.

De Clerq, E. and J. Neyts. “Therapeutic potential of nucleoside/nucleotide analogues against poxvirus infections.” Reviews in Medical Virology 14 (2004): 289—300.

DePamphilis, Melvin L. DNA Replication and Human Disease. Cold Spring Harbor: Cold Spring Harbor Laboratory, 2006. Print.

35 Donigan, K.A., M.P. McLenigan, W. Yang, M.F. Goodman, and R. Woodgate. “The Steric Gate of DNA Polymerase I Regulates Ribonucleotide Incorporation and Fidelity.” The Journal of Biological Chemistry 289 (2014): 9136–45

Ewald, B., D. Sampath, and W. Plunkett. "Nucleoside Analogs: Molecular Mechanisms Signaling Cell Death." Oncogene 27 (2008): 6522–37.

Fazakerley, G.V., Z. Gdaniec, and L.C. Sowers. “Base-Pair Induced Shifts in the Tautomeric Equilibrium of a Modified DNA-Base.” Journal of Molecular Biology 230 (1993): 6—10.

Galmarini, C.M., G. Warren, M.T. Senanayake, and S.V. Vinogradov. “Efficient overcoming of drug resistance to anticancer nucleoside analogs by nanodelivery of active phosphorylated drugs.” International Journal of Pharmaceutics 16 (2010): 281—9.

Gandhi, V., M. Ayres, R.G. Halgren, N.L. Krett, R.A. Newman, and S.T. Rosen. “8 chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells.” Cancer Research 61 (2001): 5474-9.

Harris, V.H., C.L. Smith, C.W. Jonathan, A.L. Hamilton, H. Adams, M. Dickman, D.P. Hornby, and D.M. Williams. “The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis.” The Journal of Molecular Biology 326 (2003): 1389–401

Hendrickson, C.L., K.G. Devine, and S. Benner. “Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2’- deoxyadenosine.” Nucleic Acids Research 32 (2004): 2241–50.

Henry, A.A., and F.E. Romesberg. “Beyond A, C, G and T: augmenting nature’s alphabet.” Current Opinion in Chemical Biology 7 (2003): 727–33.

Hirao, I., M. Kimoto, and R. Yamashige. “Natural versus artificial creation of base pairs in DNA: origin of nucleobases from the perspectives of unnatural base pair studies.” Accounts of Chemical Research 45 (2012): 2055–65.

Hryniuk, W., J. Foerster, M. Shojania, and A. Chow. “Cytarabine for herpesvirus infections.” The Journal of the American Medical Association 219 (1972): 715— 8

Hurwitz, S. J., and R. F. Schinazi. "Prodrug Strategies for Improved Efficacy of Nucleoside Antiviral Inhibitors." Current Opinion in HIV and AIDS 8 (2013): 556–64.

36 Jordheim, Lars P., David Durantel, Fabien Zoulim, and Charles Dumontet. "Advances in the Development of Nucleoside and Nucleotide Analogues for Cancer and Viral Diseases." Nature Reviews Drug Discovery 12 (2013): 447–64.

Kaminskas, E., A.T. Farrell, Y.C. Wang, R. Sridhara, and R. Pazdur. “FDA drug approval summary: azacitidine (5-azacytidine, Vidaza) for injectable suspension.” Oncologist 10 (2005): 176—82.

Kincaid, K., J. Beckman, A. Zivkovic, R.L. Halcomb, J.W. Engels, and R.D. Kuchta. “Exploration of factors driving incorporation of unnatural dNTPs into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha.” Nucleic Acids Research 33 (2005): 2620–28.

Klenow, H. and I Henningsen. “Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis.” Proceedings of the National Academy of Sciences 65 (1970): 168– 75.

Klenow, H. and K. Overgaard-Hansen. “Proteolytic cleavage of DNA polymerase from Escherichia Coli B into an exonuclease unit and a polymerase unit.” FEBS Letters 6 (1970): 25–7.

Klotsa, Daphne, Rudolf A. Romer, and Matthew S. Turner. “Electronic Transport in DNA.” Biophysical Journal 89 (2005): 2187–98.

Kool, E.T. “Active site tightness and substrate fit in DNA replication.” Annual Review of Biochemistry 71 (2002) 191–219.

Kornberg, A. “Biological synthesis of deoxyribonucleic acid.” Science 131 (1960): 1503 –8.

Kufe, D. W., R. Weichselbaum, E. M. Egan, W. Dahlberg, and R. J. Fram. "Lethal Effects of 1-beta-D-arabinofuranosylcytosine Incorporation into Deoxyribonucleic Acid during Ultraviolet Repair." Molecular Pharmacology 25 (1984): 322–26.

Kunkel, T.A., and K. Bebenek. “Recent studies of the fidelity of DNA synthesis.” Biochimica et Biophysica Acta 951 (1988): 1–15.

Kunkel, T. A., and K. Bebenek. "DNA Replication Fidelity." Annual Review of Biochemistry 69 (2000): 497–529.

Le Pogam, S., A. Seshaadri, A. Ewing, H. Kang, A. Kosaka, J. M. Yan, M. Berrey, B. Symonds, A. De La Rosa, N. Cammack, and I. Najera. "RG7128 Alone or in Combination with Pegylated Interferon-α2a and Ribavirin Prevents Hepatitis C

37 Virus (HCV) Replication and Selection of Resistant Variants in HCV-infected Patients." The Journal of Infectious Diseases 202 (2010): 1510–19.

Leyssen, P., E. De Clercq, and J. Neyts. "Molecular Strategies to Inhibit the Replication of RNA Viruses." Antiviral Research 78 (2008): 9–25.

Li, Y., and G. Waksman. “Crystal structures of a ddATP-, ddTTP- ddCTP-, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity.” Protein Science 10 (2001): 1225–33.

Mahmoudian, Mahmoud, John Eaddy, and Mike Dawson. "Enzymic Acylation of 506U78 (2-amino-9---arabinofuranosyl-6-methoxy-9H-purine),a Powerful New Anti-leukaemic Agent." Biotechnology and Applied Biochemistry 29 (1999): 229– 33.

Minuesa, G., I. Huber-Ruano, M. Pastor-Anglada, H. Koepsell, B. Clotet, and J. Martinez-Picado. "Drug Uptake Transporters in Antiretroviral Therapy." Pharmacology & Therapeutics 132 (2011): 268–79.

Moore, C.L., A. Zivkovic, J.W. Engels, and R.D. Kuchta. “Human DNA uses Watson-Crick hydrogen bonds to distinguish between correct and incorrect nucleoside triphosphates.” Biochemistry 43 (2004): 12367–74.

Morgan, A. R. "Base Mismatches and Mutagenesis: How Important Is Tautomerism?" Trends in Biochemical Sciences 18 (1993): 160–3.

Moyle, G. “Toxicity of antiretroviral nucleoside and nucleotide analogues: is mitochondrial toxicity the only mechanism?” Drug Safety 23 (2000):467—81.

Nedderman, A.N.R., M.J. Stone, P.K.T. Lin, D.M. Brown, and D.H. Williams. “Base pairing of Cytosine Analogs with Adenine and Guanine in Oligonucleotide Duplexes— Evidence for Exchange between Watson-Crick and Wobble Base- Pairs Using H-1-NMR Spectroscopy.” Journal of Molecular Biology 222 (1991): 711—23

Nedderman, A.N.R., M.J. Stone, D.H. Williams, P.K.T. Lin, and D.M. Brown. “Molecular-Basis for Methoxyamine-Initiated Mutagenesis— H-1 Nuclear- Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Base- Modified Cytosine Residues.” Journal of Molecular Biology 230 (1993): 1068— 76

Nekhai, S., U.G. Bhat, T. Ammosova, S.K. Radhakrishnan, M. Jerebtsova, X. Niu, A. Foster, T.J. Layden, and A.L. Gartel. “A novel anticancer agent ARC antagonizes HIV-1 and HCV.” Oncogene 26 (2007): 3899—903.

Nemec, A.A., D.L. Murphy, K.A. Donigan, and J.B. Sweasy. “The S229L Colon Tumor

38 Associated Variant of DNA Polymerase Beta Induces Cellular Transformation As A Result Of Decreased Polymerization Efficiency.” Journal of Biological Chemistry (2014).

Ogawa, A.K., Y.Q. Wu, M. Berger, P.G. Schultz, and F.E. Romesberg. “Rational design of an unnatural lbase pair with increased kinetic selectivity.” Journal of the American Chemical Society 122 (2000): 8803–4.

Ollis, D.L., P. Brick, R. Hamlin, N.G. Xuong, and T.A. Steitz. “Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP.” Nature 313 (1985): 762–6.

Parker, William B. "Enzymology of Purine and Pyrimidine Used in the Treatment of Cancer." Chemical Reviews 109 (2009): 2880–93.

Pockros, P.J. “Nucleoside/nucleotide analogue polymerase inhibitors in development.” Clinical Liver Disease 17 (2013): 105—10.

Price, N. B., and M. N. Prichard. "Progress in the Development of New Therapies for Herpesvirus Infections." Current Opinion in Virology 1 (2011): 548–54.

Prindle, M.J., M.W. Schmitt, F. Parmeggiani, and L.A. Loeb. “A substitution in the fingers domain of DNA polymerase reduces fidelity by altering nucleotide discrimination in the catalytic site.” Journal of Biological Chemistry 288 (2013): 5572—80.

Ramirez-Aguilar, K., C.L. Moore, and R.D. Kuchta. Herpes simplex virus 1 primase employs Watson-Crick hydrogen bonding to identify cognate nucleoside triphosphates.” Biochemistry 44 (2005): 15585–93.

Renis, Harold E. “Antiviral Activity of Cytarabine in Herpesvirus-Infected Rats.” Antimicrobial Agents and Chemotherapy 4(1973): 439—44.

Roberts, J. and T. Kunkel. “The Fidelity of Eukaryotic DNA Replication. DNA Replication in Eukaryotic Cells: Concepts, Enzymes, and Systems. DePamphilis, M., Ed., pp. 217–47. Cold Spring Harbor Press, Cold Spring Harbor, NY.

Roizman, B. & Knipe, D. M. in Fundamental Virology (eds Knipe, D. M. & Howley, P. M.)1123–84 (Lippincott William and Wilkins, 2001).

Rothwell, P. and G. Waksman. “Structure and mechanism of DNA polymerases.” Advances in Protein Chemistry 71 (2005)) 401–40.

Seeman, N.C., J.M. Rosenberg, and A. Rich. “Sequence-specific recognition of double helical nucleic acids by proteins.” Proceedings of the National Academy of Sciences of the United States of America 73 (1976): 804–8.

39

Senanayake, T.H., G. Warren, and S.V. Vinogradov. “Novel anticancer polymeric conjugates of activated nucleoside analogues.” Bioconjugate Chemistry 22 (2011) 1983—93.

Song, Bing, X. S. Liu, Steven Rice, Shihuan Kuang, Bennett D. Elzey, Stephen F. Konieczny, Timothy L. Ratliff, Tony Hazbun, Elena G. Chiorean, and Xiaoqi Liu. "Plk1 Phosphorylation of Orc2 and Hbo1 Contributes to Gemcitabine Resistance in Pancreatic Cancer." Molecular Cancer Therapeutics 12 (2013): 58–68.

Squires, K.E. “An introduction to nucleoside and nucleotide analogues.” Antiviral Therapy 6 (2001): 1—14

Stengel, Gudrun, Byron W. Purse, L. Marcus Wilhelmsson, Milan Urban, and Robert D. Kuchta. “Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase a and Klenow Fragment.” Biochemistry 48 (2009): 7547–55.

Stewart, D.J., J.P. Issa, R. Kurzrock, M.I. Nunez, J. Jelinek, D. Hong, Y. Oki, Z. Guo, S. Gupta, I. Wistuba. “Decitabine effect on tumor global DNA methylation and other parameters in a phase I trial in refractory solid tumors and lymphomas.” Clinical Cancer Research 15 (2009): 3881-8.

Stuyver, L. J., T. R. McBrayer, T. Whitaker, P. M. Tharnish, M. Ramesh, S. Lostia, L. Cartee, J. Shi, A. Hobbs, R. F. Schinazi, K. A. Watanabe, and M. J. Otto. "Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2'- deoxy-2'-fluorocytidine." Antimicrobial Agents and Chemotherapy 48 (2004): 651–4.

Trostler, M., A. Delier, J. Beckman, and M. Urban. “Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.” Biochemistry 48 (2009): 10866–81.

Urban, M., N. Joubert, M. Hocek, R.E. Alexander, and R.D. Kuchta. “Herpes simplex virus-1 DNA primase: a remarkably inaccurate yet selective polymerase.” Biochemistry 48 (2009): 10806–81.

Urban, M., N. Joubert, B.W. Purse, M. Hocek, and R.D. Kuchta. “Mechanisms by which human DNA primase chooses to polymerize a .” Biochemistry 49 (2010): 727–35.

United States of America National Institutes of Health. Health and Human Services. Cytarabine Injection. U.S. National Library of Medicine, May 2011. Web.

Van Rompay, A.R., M. Johansson, and A. Karlsson. “Substrate specificity and

40 phosphorylation of antiviral and anticancer nucleoside analogues by human kinases and kinases.” Pharmacology & Therapeutics 100 (2003): 119—39.

Veltcamp, S. A., R. S. Jansen, S. Callies, D. Pluim, C. M. Visseren-Grul, H. Rosing, S. Kloeker-Rhoades, V. A. Andre, J. H. Beijnen, C. A. Slapak, and J. H. Schellens. "Oral Administration of Gemcitabine in Patients with Refractory Tumors: A Clinical and Pharmacologic Study." Clinical Cancer Research 14 (2008): 3477– 86.

Wang, J., a K. Sattar, C. C. Wang, J.D. Karam, W.H. Konigsberg, and T. Steitz. “Crystal structure of a pol alpha family replication DNA polymerase from bacteriophage RB69.” Cell 89 (1997): 1087–99.

Watson, J. D., and F. H. Crick. "Genetical Implications of the Structure of Deoxyribonucleic Acid." Nature 171 (1953): 964–7.

Wedemeyer, Heiner, Donald Jensen, Robert Herring, Jr., Peter Ferenci, Mang-Ming Ma, Stefan Seusem, Maribel Rodriguez-Torres, Natalie Bzowej, Paul Packros, John Vierling, David Ipe, Marie L. Munson, Ya-Chi Chen, Isabel Najera, and James Thommes. "PROPEL: A Randomized Trial of Mericitabine plus Peginterferon Alpha-2a/ribavirin Therapy in Treatment-naïve HCV Genotype 1/4 Patients."Hepatology 58 (2013): 524–37.

Wolfle, W., and M. Washington. “Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa.” Molecular and Cellular Biology 25 (2005): 7137–43.

Yao, C. and U.F. Müller. “Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides.” RNA 17 (2011): 1274–81.

Zhao, G. and Y. Guan. “Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions.” Acta Biochimica et Biophysica Sinica 42 (2010): 722–8

Zhao, G., H. Wei, and Y. Guan. “Identification of a premature termination of DNA polymerization in vitro by Klenow fragment mutants.” Journal of Biosciences 38 (2013): 276–89.

41