Presteady State Kinetic Investigation of the Incorporation of Anti

Article pubs.acs.org/crt

Presteady State Kinetic Investigation of the Incorporation of AntiHepatitis B Nucleotide Analogues Catalyzed by Noncanonical Human DNA Polymerases Jessica A. Brown,†,‡ Lindsey R. Pack,† Jason D. Fowler,† and Zucai Suo*,†,‡ †

Department of Biochemistry, ‡Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: Antiviral nucleoside analogues have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogues can be limited by drug toxicity because the 5′-triphosphates of these nucleoside analogues (nucleotide analogues) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogues are substrates for the recently discovered human X- and Y-family DNA polymerases. Using presteady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogues approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogues were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3′-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator, while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogues catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity.

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INTRODUCTION With more than two billion people infected worldwide, the hepatitis B virus (HBV) remains an important global health concern. Chronic HBV infection, which affects more than 350 million people, is a major cause of hepatocellular carcinoma and liver cirrhosis, two life-threatening disease states of the liver. Thus, HBV treatment is important to prevent or to slow the progression of these severe liver complications. Currently, seven antiviral agents are approved by the United States Food and Drug Administration (FDA) for the treatment of HBV: two immune modulators (interferon-alpha and pegylated interferon-alpha) and five nucleoside/nucleotide analogues [adefovir (PMEA), tenofovir (PMPA), lamivudine (L-3TC), telbivudine (L-TBV), and entecavir (ETV) (Figure 1)]. Following cellular uptake, these analogues undergo either two (PMEA and PMPA) or three (L-3TC, L-TBV, and ETV) phosphorylation events to be activated to their di (PMEA-DP and PMPA-DP)- or triphosphate (L-3TC-TP, L-TBV-TP, and ETV-TP) forms, respectively. These activated nucleotide analogues target the HBV DNA polymerase (Pol), which has enzymatic activity for a unique protein-priming event, RNAdependent and DNA-dependent DNA synthesis, and degradation of RNA in a RNA/DNA duplex (i.e., RNase H). Depending on the analogue, these drugs may function as © 2011 American Chemical Society

competitive inhibitors against natural dNTP substrates and/or as obligate or masked chain terminators that inhibit the priming and/or polymerization activities of the HBV Pol. Unfortunately, the usage of anti-HBV nucleoside analogues can be limited by drug resistance and adverse side effects.1,2 It has been postulated that cellular DNA polymerases, such as human DNA polymerase γ (Pol γ), may be potential drug targets and the cause of observed clinical toxicity since nucleoside analogues approved for human immunodeficiency virus type 1 (HIV-1) are associated with mitochondrial toxicity.3,4 However, mitochondrial toxicity induced by nucleotide analogue incorporation catalyzed by Pol γ does not account for all of the unwanted side effects.5 The human genome encodes at least 15 other DNA polymerases, which are members of the A-, B-, X-, or Y-family, that may be potential candidates for generating cellular toxicity via analogue incorporation into nuclear DNA.5−8 Using presteady state kinetic techniques, we determined the incorporation efficiency of five anti-HBV nucleotide analogues (Figure 1) with six noncanonical human DNA polymerases: Pols β, λ, η, ι, κ, and Rev1. Both Pols β and λ are X-family DNA Received: October 23, 2011 Published: December 1, 2011 225

dx.doi.org/10.1021/tx200458s | Chem. Res. Toxicol. 2012, 25, 225−233

Chemical Research in Toxicology

Article

Figure 1. Chemical structures of anti-HBV nucleoside/nucleotide analogues and their natural counterpart.

Table 1. Sequences of Oligonucleotidesa

polymerases. Pol β functions in base excision repair, while Pol λ is putatively involved in base excision repair, nonhomologous end-joining, and antibody generation.9 The human genome encodes four Y-family DNA polymerases (Pols η, ι, κ, and Rev1) that catalyze translesion DNA synthesis and may play a part in somatic hypermutation.10 Therefore, inhibition of these selected X- and Y-family pols could lead to unwanted toxicity including apoptosis, genetic instability, and immunodeficiency. Our kinetic data showed that most of the analogues were substrates for the noncanonical pols and that the kinetic basis of incorporation varied for each analogue. These results suggested that human X- and Y-family enzymes are capable of inserting nucleotide analogues in vivo and established structure−function relationships that are important for future anti-HBV drug design.

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a

The 21-mer strand was 5′-radiolabeled. For single-nucleotide gap DNA substrates, the downstream 19-mer strand was 5′-phosphorylated. The identity of important base changes is highlighted in bold.

5′-radiolabeled with [γ-32P]ATP and OptiKinase according to the manufacturer’s protocol, and the unreacted [γ-32 P]ATP was subsequently removed via a Bio-Spin 6 column. The 21−41-mer primer-template DNA substrates23 and 21−19−41-mer singlenucleotide gapped DNA substrates4,26,27 were annealed as described previously. Single-Turnover Kinetic Assays to Measure the kp and Kd. Kinetic assays were completed using buffer B (50 mM Tris-HCl, pH 7.8, at 37 °C, 5 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, and 0.1 mg/mL of bovine serum albumin (BSA)) for Pol β, buffer L (50 mM Tris-HCl, pH 8.4, at 37 °C, 5 mM MgCl2, 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, and 0.1 mg/mL of BSA) for Pol λ,14 and buffer Y (50 mM HEPES, pH 7.5, at 37 °C, 5 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 10% glycerol, and 0.1 mg/mL of BSA) for Pols η, ι, κ, and Rev1. The primer-template DNA substrates were used in assays with Pols η, ι, κ, and Rev1, while the gap-filling Pols β and λ were provided 21−19−41mer gap DNA. All kinetic experiments described herein were performed at 37 °C, and the reported concentrations are final after mixing all the components. A preincubated solution of the Pol (120 or 300 nM) and 5′-[32P]-radiolabeled DNA substrate (30 nM) was mixed with increasing concentrations (0.01−900 μM) of a single nucleotide or nucleotide analogue in the appropriate buffer at 37 °C. The enzyme

EXPERIMENTAL PROCEDURES

Materials. These chemicals were purchased from the following companies: [γ-32P]ATP, MP Biomedicals; ATP, GE Healthcare; BioSpin 6 columns, Bio-Rad Laboratories; deoxyribonucleotide 5′triphosphates, GE Healthcare; β-L-2′-deoxythymidine 5′-triphosphate (L-TBV-TP), TriLink Biotechnologies; OptiKinase, USB Corporation; synthetic oligodeoxyribonucleotides 21-mer, 5′-phosphorylated 19mer, and 41-mers, Integrated DNA Technologies. The diphosphate of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA-DP) and 9-[2(phosphonomethoxy)propyl]adenine (PMPA-DP), β-L-2′,3′-dideoxy3′-thiacytidine 5′-triphosphate (L-3TC-TP), and entecavir 5′-triphosphate (ETV-TP) were kind gifts from Gilead Sciences, Inc. Preparation of Human DNA Polymerases and DNA Substrates. The plasmids, expression, and purification of human DNA polymerases β,11−13 λ,14−16 η,17 truncated ι (1−420),17,18 truncated κ (9−518),17 and truncated Rev1 (341−829)19−21 were described previously, and the purity of each enzyme is greater than 95% based on a Coomassie-stained polyacrylamide gel. Purified human Δ235 DNA ligase I was a kind gift of Dr. Tom Ellenberger.22 Commercially synthesized oligonucleotides in Table 1 were purified using polyacrylamide gel electrophoresis.23−25 The 21-mer primer was 226



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Figure 2. Concentration dependence on the presteady state rate constant of PMEA-DP incorporation catalyzed by Pol λ. A preincubated solution of Pol λ (120 nM) and 21−19C-41-merTG DNA (30 nM) was rapidly mixed with increasing concentrations of PMEA-DP·Mg2+ (0.2 μM, ●; 0.5 μM, ○; 1 μM, ■; 2 μM, □; 5 μM, ▲; 10 μM, Δ; and 25 μM, ⧫) for various time intervals. (A) A representative gel image for PMEA-DP incorporation at 25 μM is shown. The length of the DNA primer is indicated in the right margin. (B) The concentration of the DNA product is plotted as a function of time. The solid lines are the best fits to a single-exponential equation which determined the observed rate constant, kobs. (C) The kobs values were plotted as a function of PMEA-DP concentration. The data (●) were then fit to a hyperbolic equation, yielding a kp of 0.175 ± 0.004 s−1 and a Kd of 4.5 ± 0.3 μM. was in molar excess over DNA, whereby the enzyme to DNA ratio was 4:1 for Pols λ, η, ι, and Rev1 and 10:1 for Pols β and κ. Aliquots of the reaction mixtures were quenched at various times using 0.37 M EDTA. A rapid chemical-quench flow apparatus (KinTek) was utilized for fast nucleotide incorporations. Reaction products were resolved using sequencing gel electrophoresis (17% acrylamide, 8 M urea) and quantitated with a Typhoon TRIO (GE Healthcare). The time course of product formation at each nucleotide concentration was fit to a single-exponential equation (eq 1) using a nonlinear regression program, KaleidaGraph (Synergy Software), to yield an observed rate constant of nucleotide incorporation (kobs). The kobs values were then plotted as a function of nucleotide concentration and fit using the hyperbolic equation (eq 2), which resolved the maximum rate of incorporation (kp) and the equilibrium dissociation constant (Kd) of an incoming nucleotide for each Pol·DNA complex.

[Product] = A[1 − exp( − kobst )]

(1)

kobs = k p[dNTP]/{[dNTP] + Kd}

(2)

DNA ligase I (30 nM) and 1 mM ATP to the polymerization mixture. Aliquots of the reaction mixtures were quenched at various times by adding them to 0.37 M EDTA and immediate heat denaturation for 2 min at 95 °C. The DNA products were resolved using sequencing gel electrophoresis (20% acrylamide, 8 M urea).

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RESULTS Determination of Selection Factors. The mechanism of anti-HBV nucleotide analogue incorporation catalyzed by Pols β, λ, η, ι, κ, and Rev1 was determined by utilizing singleturnover kinetic methodology. When the enzyme is in molar excess over DNA, the conversion of the DNA substrate into product is observed directly in a single pass through the enzymatic pathway so that the nucleotide concentration dependence on the observed rate constant (kobs) can resolve the equilibrium dissociation constant (Kd) and maximum rate of incorporation (kp) for an incoming nucleotide.28−30 As a representative example, a preincubated solution of Pol λ and 5′[32P]-radiolabeled 21−19C-41-merTG (Table 1) was mixed with increasing concentrations of PMEA-DP (see Experimental Procedures and Figure 2A). After plotting and fitting the data to eqs 1 and 2 (Figures 2B and C), the single-turnover kinetic parameters were resolved: a kp of 0.175 ± 0.004 s−1 and a Kd of 4.5 ± 0.3 μM. Using similar single-turnover kinetic assays, the kinetic parameters were measured for each enzyme incorporating a natural nucleotide or one of the analogues, which were subsequently used to calculate the substrate specificity constants (kp/Kd) and selection factors ((kp/Kd)dNTP/(kp/ Kd)analog) in Tables 2−5. Incorporation of Acyclic Adenine Analogues. Tenofovir (PMPA) and adefovir (PMEA) both have an acyclic moiety and a phosphonate group, but PMPA has an additional methyl group (Figure 1). In general, the selection factors were lower

Extension Assay for Nonchain Terminators. A preincubated solution of Pol (240 or 600 nM) and 5′-[32P]-radiolabeled 21−41-mer DNA (60 nM) in the appropriate buffer was mixed with either ETVTP·Mg2+ (1−50 μM) or L-TBV-TP·Mg2+ (100−500 μM) to allow sufficient extension (at least 8 half-lives) of the primer before adding the four natural dNTPs (200 μM each) for various reaction times. The enzyme to DNA ratio was 4:1 for Pols λ, η, ι, and Rev1 and 10:1 for Pols β and κ. Aliquots of the reaction mixtures were quenched using 0.37 M EDTA, and the reaction products were resolved using sequencing gel electrophoresis (20% acrylamide, 8 M urea). DNA Ligation Assay for Nonchain Terminators. A preincubated solution of Pol β (120 nM) and 5′-[32P]-radiolabeled 21−19− 41-mer DNA (30 nM) in buffer B was mixed in independent reactions with dGTP·Mg2+ (1 μM), dTTP·Mg2+ (1 μM), ETV-TP·Mg2+ (1 μM), or L-TBV-TP·Mg2+ (500 μM) to allow sufficient extension (at least 8 half-lives; 30 s for dGTP and dTTP, 5 min for ETV-TP, and 120 min for L-TBV-TP) of the primer before adding human Δ235 227



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Table 2. Kinetic Parameters for Nucleotide Incorporation Opposite Template dT at 37 °C DNA polymerase Pol β

Pol λ

Pol η

Pol κ

Pol ι

Rev1

Pol γ T7 exo a

dNTP b

dATP PMPA-DPb PMEA-DP dATPc PMPA-DPb PMEA-DP dATPb PMPA-DPb PMEA-DP dATPb PMPA-DPb PMEA-DP dATPb PMPA-DPb PMEA-DP dATPb PMPA-DPb PMEA-DP dATPd PMPA-DPe dATPf PMPA-DPf

kp (s−1)

Kd (μM)

32 ± 1 4.7 ± 0.5 1.31 ± 0.04 1.5 ± 0.1 0.095 ± 0.008 0.175 ± 0.004 35 ± 3 0.0134 ± 0.0007 0.069 ± 0.008 2.49 ± 0.08 0.010 ± 0.005 0.039 ± 0.001 0.015 ± 0.001 no observed incorporation 0.00047 ± 0.00004 0.00152 ± 0.00005 no observed incorporation 0.00037 ± 0.00002 45 ± 1 0.21 ± 0.01 156 ± 8 0.096 ± 0.009

9.2 ± 1.0 50 ± 10 65 ± 6 0.9 ± 0.3 3.7 ± 0.9 4.5 ± 0.3 130 ± 30 90 ± 10 55 ± 18 7.0 ± 1.0 3000 ± 2000 23 ± 3 260 ± 40

3.5 9.4 2.0 1.7 2.6 3.9 2.7 1.5 1.3 3.6 3.3 1.7 5.8

180 ± 40 2.0 ± 0.3

2.6 × 10−6 7.6 × 10−4

120 ± 20 0.8 ± 0.1 40.3 ± 5.7 8±2 268 ± 39

3.1 × 10−6 56 5.2 × 10−3 19.5 3.6 × 10−4

b

c

kp/Kd (μM−1s−1) × 10−2 × 10−2 × × × × × × × × ×

10−2 10−2 10−1 10−4 10−3 10−1 10−6 10−3 10−5

selection factora 40 170 65 44 1,800 210 110,000 210 High 48 High 250 10,800 54,400

d

Calculated as (kp/Kd)dATP/(kp/Kd)Analogue. Kinetic parameters are from ref 31. Kinetic parameters are from ref 14. Kinetic parameters are from ref 49. eKinetic parameters are from ref 38. fKinetic parameters are from ref 50.

ground-state binding affinity (1/Kd) was 3-fold weaker, unchanged, and 60-fold weaker, respectively. As noted previously, PMPA-DP is a poor substrate for Pols κ, ι, and Rev1.31 The kinetic parameters could only be measured for Pol κ because PMPA-DP incorporation was too inefficient to be observed with Pol ι and Rev1. Incorporation of Nucleotides with L-Stereochemistry. Previously,31 the selection factors were determined for Pols β, λ, η, ι, κ, and Rev1 inserting L-3TC-TP, and the following general trend emerged: the kp drops significantly (6,100-fold on average), while the Kd decreases (6-fold on average) compared to that of dCTP (Table 3). Telbivudine (LTBV) is the L-isomer of thymidine (Figure 1). No observable L-TBV-TP incorporation was detected for Rev1, likely because it is a deoxycytidyl transferase and prefers dCTP regardless of the template base.21 For the remaining DNA polymerases, relatively large selection factors of greater than 8,000 were determined (Table 4 and Figure 3). Unlike L-3TC-TP, the binding of L-TBV-TP to the Pol·DNA complex was weakened by 10-fold on average, but the rate of incorporation remained slow compared to dTTP. These kinetic results suggested that (i) the oxathiolane ring of L-3TC-TP is important for the tight ground-state binding affinity of L-3TC-TP and (ii) that the polymerase active sites of these Y-family polymerases could accommodate L-dNTPs, albeit inefficiently. Incorporation of Entecavir 5′-Triphosphate. Entecavir (ETV) is a deoxyguanosine analogue with a cyclopentyl sugar ring (Figure 1). Interestingly, Pols β, λ, η, ι, κ, and Rev1 incorporated ETV-TP opposite dC with much lower selection factors (Table 5 and Figure 3) compared to those of the other anti-HBV analogues (Tables 2−4). Except for Rev1, the lack of discrimination between ETV-TP and dGTP was due to the binding affinity of ETV-TP being approximately 20-fold tighter on average. However, the rate of ETV-TP incorporation was on average 120-fold slower than that of dGTP.

for PMEA-DP (44 to 250) than PMPA-DP (40 to >110,000) (Table 2 and Figure 3). The X-family DNA polymerases β and

Figure 3. Comparison of selection factors. The selection factors from Tables 2−5 are plotted for the X- and Y-family DNA polymerases examined herein and the corresponding nucleotide analogue. The selection factors defined as “high” are graphed as being greater than 1,000,000. The bars are color-coded for each nucleotide analogue as follows: PMPA-DP is gray, PMEA-DP is red, L-3TC-TP is green, LTBV-TP is blue, and ETV-TP is black.

λ exhibited the least amount of discrimination for both analogues, whereby the kp value dropped by an average of 14-fold, and the Kd value increased by an average of 5-fold. In contrast, the additional methyl group in PMPA-DP led to a greater degree of discrimination for the Y-family DNA polymerases, although different mechanisms of discrimination were observed for the four enzymes. For Pol η, the rate of PMPA-DP and PMEA-DP incorporation dropped by an average of 420-fold, while the binding was slightly tighter than dATP. For Pols κ, ι, and Rev1, the rate of PMEA-DP incorporation decreased by 60-, 30-, and 4-fold, and the 228



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Table 3. Kinetic Parameters for Nucleotide Incorporation Opposite Template dG at 37 °C human DNA polymerase Pol β Pol λ Pol η Pol κ Pol ι Rev1 Pol γ

dNTP b

dCTP L-3TC-TPb dCTPc L-3TC-TPb dCTPb L-3TC-TPb dCTPb L-3TC-TPb dCTPb L-3TC-TPb dCTPd L-3TC-TPb dCTPe L-3TC-TPe

a

kp (s−1)

Kd (μM)

5.02 ± 0.07 0.00390 ± 0.00010 1.57 ± 0.04 0.00402 ± 0.00008 49 ± 2 0.0296 ± 0.0009 11.8 ± 0.6 0.00045 ± 0.00001 0.075 ± 0.002 0.00356 ± 0.00009 22.4 ± 0.9 0.0236 ± 0.0006 44 ± 2 0.125 ± 0.005

0.71 ± 0.04 0.18 ± 0.02 0.9 ± 0.1 0.106 ± 0.010 25 ± 4 3.3 ± 0.3 32 ± 5 4.0 ± 0.5 50 ± 5 96 ± 8 2.2 ± 0.3 1.04 ± 0.10 1.1 ± 0.1 9.2 ± 0.9

b

kp/Kd (μM−1s−1) 7.1 2.2 1.7 3.8 2.0 9.0 3.7 1.1 1.5 3.7 10 2.3 40 1.4

selection factora

× 10−2

325

× 10−2

46

10−3 10−1 10−4 10−3 10−5

220

× 10−2

450

× 10−2

2,900

× × × × ×

3,300 40

c

Calculated as (kp/Kd)dCTP/(kp/Kd)L‑3TC‑TP. Kinetic parameters are from ref 31. Kinetic parameters are from ref 14. dKinetic parameters are from ref 21. eKinetic parameters are from ref 61.

Table 4. Kinetic Parameters for Nucleotide Incorporation Opposite Template dA at 37 °C human DNA polymerase Pol β Pol λ Pol η Pol κ Pol ι Rev1 a

kp (s−1)

dNTP c

dTTP L-TBV-TP dTTPb L-TBV-TP dTTPc L-TBV-TP dTTPc L-TBV-TP dTTPc L-TBV-TP dTTPc L-TBV-TP

Kd (μM)

12.3 ± 0.5 0.00055 ± 0.00001 3.9 ± 0.2 0.00072 ± 0.00002 35 ± 1 0.0028 ± 0.0003 4.35 ± 0.04 0.00065 ± 0.00006 0.75 ± 0.02 0.00071 ± 0.00006 0.0038 ± 0.0003 No observed incorporation

kp/Kd (μM−1s−1)

3.5 ± 0.5 11 ± 1 2.6 ± 0.4 53 ± 6 41 ± 5 400 ± 100 11.0 ± 0.5 90 ± 20 13 ± 1 100 ± 30 6±1

3.5 5.0 1.5 1.4 8.5 7.0 4.0 7.2 5.8 7.1 6.3

selection factora

× 10−5

70,000

10−5 10−1 10−6 10−1 10−6 10−2 10−6 10−4

11,000

× × × × × × × ×

122,000 55,000 8,100 High

b

c

Calculated as (kp/Kd)dTTP/(kp/Kd)L‑TBV‑TP. Kinetic parameters are from ref 14. Kinetic parameters are from ref 31.

Table 5. Kinetic Parameters for Nucleotide Incorporation Opposite Template dC at 37 °C DNA polymerase Pol β Pol λ Pol η Pol κ Pol ι Rev1 HIV-1 RT a

dNTP

kp (s−1)

Kd (μM)

dGTP ETV-TP dGTPb ETV-TP dGTP ETV-TP dGTP ETV-TP dGTP ETV-TP dGTP ETV-TP dGTPc ETV-TPc

18.8 ± 0.4 0.054 ± 0.002 2.5 ± 0.1 0.034 ± 0.002 38 ± 2 0.24 ± 0.01 4.4 ± 0.1 0.370 ± 0.004 0.095 ± 0.002 0.0097 ± 0.0004 0.0038 ± 0.0002 0.0017 ± 0.0001 18.3 ± 1.30 0.107 ± 0.007

8.7 ± 0.4 0.26 ± 0.04 2.1 ± 0.3 0.09 ± 0.02 80 ± 10 2.4 ± 0.4 10.6 ± 0.9 4.2 ± 0.1 141 ± 8 33 ± 5 3.9 ± 0.8 56 ± 10 1.76 ± 0.51 2.23 ± 0.67

b

kp/Kd (μM−1s−1) 2.2 2.1 × 1.2 3.8 × 4.8 × 1.0 × 4.2 × 8.8 × 6.7 × 2.9 × 9.7 × 3.0 × 10.4 4.8 ×

selection factora

10−1

10

10−1 10−1 10−1 10−1 10−2 10−4 10−4 10−4 10−5

3

10−2

220

5 5 2 32

c

Calculated as (kp/Kd)dGTP/(kp/Kd)ETV‑TP. Kinetic parameters are from ref 14. Kinetic parameters are from ref 58.

Qualitative Analysis of Nonchain Terminators Being Embedded into DNA. ETV and L-TBV are not obligate chain terminators like PMEA, PMPA, and L-3TC (Figure 1) since these two analogues possess the required 3′-hydroxyl group for downstream enzymatic steps such as primer extension or DNA ligation if incorporated into a gapped DNA substrate. Therefore, it is possible that these analogues

could become embedded into the human genome. To examine the extension possibility, we preincubated a solution of the enzyme and the appropriate 5′-[32P]-radiolabeled 21−41-mer DNA substrate before initiating the reaction with either ETVTP or L-TBV-TP (see Experimental Procedures). After allowing sufficient time for the analogue to be incorporated, the four natural dNTPs were added to the reaction mixture. 229



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Representative gel images are shown in Supporting Information, Figures 1 and 2, for an X-family (Pol β) and a Y-family (Pol η) DNA polymerase, respectively. Both Pol β and Pol η efficiently catalyzed the full-length product (41-mer) for the control and ETV-TP as early as 10 s after the addition of the natural dNTPs. In contrast, minimal extension of an L-TBVMP terminated primer was observed after 1 h, and the small amount of 41-mer may be partially due to the extension of the unreacted 21-mer substrate rather than the 22-mer terminated with the analogue. Similar extension activities of both analogues were detected for Pols λ, ι, and κ (data not shown). For Rev1, the main product after ETV-TP incorporation was the 23-mer. Subsequent incorporations likely did not occur because Rev1 can only efficiently catalyze the transfer of dCTP when dG is the template.21 Nonetheless, ETV-TP may be a masked chain terminator for Rev1. Together, these preliminary data suggested L-TBV-TP was a chain terminator, while ETV-TP was not for Pols β, λ, η, ι, κ, and Rev1. Next, we determined whether single-nucleotide gap DNA substrates having primers terminated with ETV-MP or L-TBVMP are substrates for human DNA ligase I. A preincubated solution of Pol β and the appropriate 5′-[32P]-radiolabeled 21− 19−41-mer DNA were treated with either ETV-TP or L-TBVTP before initiating the ligation reaction with human Δ235 DNA ligase I and ATP (see Experimental Procedures). As shown in Supporting Information Figure 3, the ligated DNA product (41-mer) was detected for ETV-TP and the control reactions but not L-TBV-TP. However, the ligation of ETV-MP was less efficient than the dGMP control, for the first visible appearance of the 41-mer was at 10 and 300 s for dGMP and ETV-MP, respectively. On the basis of the extension and ligation assays, these results suggested that ETV-TP can be incorporated and embedded into the genome via primer extension or subsequent ligation, while L-TBV-TP would act as a chain terminator that may lead to single-strand DNA breaks. To eliminate ambiguity in interpreting the likelihood of extension and ligation, we are currently preparing primers with L-TBV-MP or ETV-MP at the 3′-terminus and will directly examine these reactions.

for a human DNA polymerase in complex with an anti-HBV nucleotide analogue. Potential Relationship between Clinical Toxicity and Drug Incorporation by Host DNA Pols. Nucleoside analogue usage can be limited by drug resistance and unwanted side effects.1 Unfortunately, a striking 15−22% of HBV patients report moderate to severe side effects (e.g., myopathy and nephrotoxicity).32,33 These adverse events may not be derived from mitochondrial toxicity34−36 because assays indicate that anti-HBV nucleoside analogues are weak inhibitors of Pol γ37−40 and/or induce relatively low mitochondrial toxicity.40−44 Since human replicative polymerases (e.g., Pols α, δ, and ε) employ stringent mechanisms of nucleotide selection, these enzymes are unlikely to incorporate PMEA-DP,45 PMPA-DP,46 ETV-TP,47 L-TBV-TP,44 or L-3TC-TP,48 indicating these drugs are relatively weak inhibitors. The selection factors measured for exonuclease-deficient Pol γ38,49 and exonucleasedeficient T7 DNA polymerase,50 two replicative A-family enzymes, incorporating PMPA-DP are 10,800 and 54,400, respectively, which are significantly larger than the values determined for Pol β (40), Pol λ (65), and Pol η (1,800) (Table 2). Similarly, Pol γ discriminates between dCTP and L3TC-TP by 2,900-fold which is larger than most of the selection factors calculated for the noncanonical human pols (46 to 3,300) in Table 3. Overall, the X- and Y-family DNA polymerases exhibit a lower degree of discrimination than a replicative enzyme like Pol γ. To better evaluate the potential of nucleotide analogue incorporation in vivo, it is important to consider the intracellular concentrations of natural nucleotides relative to the nucleotide analogues. Using a liver cell line such as HepG2, the dNTP/nucleotide analog ratios have been determined to be or are predicted to be as follows: 6:1 for dCTP/L-3TC-TP,51 1:0.4 for dATP/PMPA-DP,52 1:1 for dATP/PMEA-DP,52 25:1 for dGTP/ETV-TP,53 and 1,600:1 for dTTP/L-TBV-TP.54 Please note, the concentrations for dATP, dGTP, and dTTP were obtained from Table 1 in reference;55 therefore, the ratios predicted for PMPA-DP, PMEA-DP, ETV-TP, and L-TBV-TP may be different in vivo. Nonetheless, these ratios suggest that intracellular concentrations of nucleotide analogues relative to natural dNTPs can approach 1:1 so that the selection factors (Tables 2−5) reflect insertion frequencies. For example, the selection factor for Pol η incorporating PMEA-DP is 210, thereby estimating that Pol η can incorporate 1 PMEA-DP molecule per 210 incorporations of dATP. Another important consideration is that Pols β, λ, η, ι, κ, and Rev1 are expressed at the mRNA level in most human tissues, including those afflicted with side effects.9,10 Thus, based on the relatively low selection factors measured herein (Tables 2−5 and Figure 3), human X- and Y-family DNA polymerases are likely to incorporate some anti-HBV ncleotide analogues in vivo, which would inhibit base excision repair, nonhomologous end joining, translesion DNA synthesis, V(D)J recombination, and somatic hypermutation pathways. These events would induce a cascade of cellular events associated with nucleoside analogue toxicity: genomic instability → apoptosis/cell death → side effects.5,56 However, the efficacy and toxicity of a drug are a complex function of drug uptake, transport, and metabolism. Kinetic Results Support Entecavir As a Potential Carcinogen. According to the US prescribing information sheet for entecavir, solid tumors were detected in rodents that were exposed to high doses of entecavir.57 Entecavir possesses a 3′-hydroxyl group; therefore, the carcinogenic activity may arise

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DISCUSSION Overall Trends of Anti-HBV Drugs by Noncanonical Human DNA Polymerases. Ranking the selection factors of the five anti-HBV analogues for each enzyme (Tables 2−5 and Figure 3) generated the following profiles: the order is identical for Pols λ, η, and Rev1 (ETV-TP < PMEA-DP < L-3TC-TP < PMPA-DP < L-TBV-TP) and Pols κ and ι (ETV-TP < PMEADP < L-3TC-TP < L-TBV-TP < PMPA-DP), while Pol β is unique (ETV-TP < PMPA-DP < PMEA-DP < L-3TC-TP < LTBV-TP). Consistently, the lowest level of discrimination occurred with ETV-TP, whereas PMPA-DP and/or L-TBV-TP had the highest selection factors for the six human pols examined in this work. The different chemical modifications resulted in unique mechanisms of incorporation. Some modifications (e.g., L-stereochemistry and acyclic ribose) were unfavorable for incorporation, while others (e.g., oxathiolane and methylene moieties in the sugar ring) enhanced the incorporation efficiency by forming a more stable ternary complex through tighter binding. The structural basis for the wide range of incorporation efficiencies exhibited by the human DNA polymerases with these anti-HBV drugs remains uncertain because structures have not been determined 230


Chemical Research in Toxicology when this drug is embedded into genomic DNA. It has been reported that entecavir functions as a masked chain terminator for HBV pol and cellular Pols α, β, γ, δ, and ε.47 However, our efficient incorporation (Table 5), extension (Supporting Information, Figures 1B and 2B), and ligation (Supporting Information Figure 3B) results suggested that there is a high potential for entecavir to become embedded into DNA. Importantly, entecavir is not a masked chain terminator for Pols β, λ, η, ι, and κ; therefore, these polymerases can likely rescue a stalled replication fork due to entecavir incorporation. Together, these processes are likely to contribute to a putative mechanism of carcinogenicity, especially if the embedded drug induces higher error rates during subsequent rounds of replication. Langley et al.47 did not provide information on their assays with Pol β; therefore, we are unsure of the discrepancy in our incorporation and extension results. Also, ETV-TP has been shown to be a substrate for HIV-1 reverse transcriptase (RT), although the enzyme discriminates between ETV-TP and dGTP by 220-fold,58 a selection factor that is greater than those measured for the human pols (2 to 32 in Table 5). HIV-1 RT is able to bypass a site-specific entecavir in a DNA template, although pausing is detected near the entecavir site.59 These kinetic results suggested that entecavir would not be a good drug candidate for treating HIV-1 infected patients. Telbivudine Is Unlikely to Become Embedded in DNA. Unlike entecavir, telbivudine exhibited an extremely low potential to become embedded in the human genome since the selection factors were large (8,100 to >122,000), and an LTBV-MP-terminated primer was poorly extended (Supporting Information, Figures 1C and 2C) and was not a substrate for human DNA ligase I (Supporting Information, Figure 3C). The unnatural L-stereochemistry of telbivudine is likely problematic for extension and ligation because of the unfavorable alignment of the 3′-hydroxyl group for catalysis. Thus, L-stereochemistry is less problematic for the incorporation step than subsequent enzymatic steps. The incorporation and extension of L-dCTP or L-dTTP (i.e., L-TBV-TP) at low concentrations was not observed for Pols α, β, and ε,60 which is likely due to the weak binding affinity and slow rate of incorporation for unmodified L-nucleotides as observed for the noncanonical pols (Table 4). Interestingly, the oxathiolane ring of L-3TC and L-FTC, an anti-HIV nucleoside analogue which has a fluoro group at the C5 position of the L-3TC chemical structure,31 improves the incorporation efficiency for L-dCTP analogues (Table 3).

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Article

* Supporting Information Extension of ETV-MP and L-TBV-MP catalyzed by Pol β, extension of ETV-MP and L-TBV-MP catalyzed by Pol η, and ligation of ETV-MP and L-TBV-MP catalyzed by human DNA ligase I. This material is available free of charge via the Internet at http://pubs.acs.org. S

Corresponding Author *880 Biological Sciences, 484 West 12th Avenue, Columbus, OH 43210, U.S.A. Tel: +1 614 688 3706. Fax: +1 614 292 6773. E-mail: [email protected]. Funding This work was supported by National Institutes of Health Grant (GM079403) to Z.S. J.A.B was supported by an American Heart Association Predoctoral Fellowship (Grant 0815382D) and a Presidential Fellowship from The Ohio State University. L.R.P. was supported by an REU Supplemental Grant from a National Science Foundation Career Award (Grant MCB-0447899 to Z.S.).

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ACKNOWLEDGMENTS We thank Dr. Tom Ellenberger for providing human Δ235 DNA ligase I, Gilead Sciences, Inc. for providing us PMPA-DP, PMEA-DP, L-3TC-TP, and ETV-TP, and Drs. Michael Miller and Joy Feng for critical reading of the manuscript.

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ABBREVIATIONS BSA, bovine serum albumin; dNTP, 2′-deoxyribonucleotide 5′triphosphate; DP, diphosphate; ETV, entecavir; FDA, Food and Drug Administration; HBV, hepatitis B virus; HIV-1, human immunodeficiency virus type 1; L-3TC, β-L-2′,3′dideoxy-3′-thiacytidine or lamivudine; L-FTC, β-L-2′,3′-dideoxy-5-fluoro-3′-thiacytidine or emtricitabine; L-TBV, telbivudine or β-L-2′-deoxythymidine 5′-triphosphate; MP, monophosphate; PMEA, adefovir or 9-[2-(phosphonomethoxy)ethyl]adenine; PMPA, tenofovir or 9-[2(phosphonomethoxy)propyl]adenine; Pol, DNA polymerase; Pol β, DNA polymerase beta; Pol γ, DNA polymerase gamma; Pol η, DNA polymerase eta; Pol ι, DNA polymerase iota; Pol κ, DNA polymerase kappa; Pol λ, DNA polymerase lambda; RT, reverse transcriptase; TP, triphosphate

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REFERENCES

(1) Lok, A. S., and McMahon, B. J. (2007) Chronic hepatitis B. Hepatology 45, 507−539. (2) Fontana, R. J. (2009) Side effects of long-term oral antiviral therapy for hepatitis B. Hepatology 49, S185−195. (3) Lee, H., Hanes, J., and Johnson, K. A. (2003) Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 42, 14711−14719. (4) Copeland, W. C. (2010) The mitochondrial DNA polymerase in health and disease. Subcell. Biochem. 50, 211−222. (5) Moyle, G. (2000) Toxicity of antiretroviral nucleoside and nucleotide analogues: is mitochondrial toxicity the only mechanism? Drug Saf. 23, 467−481. (6) Sommadossi, J. P., Carlisle, R., and Zhou, Z. (1989) Cellular pharmacology of 3′-azido-3′-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. Mol. Pharmacol. 36, 9−14.

CONCLUDING REMARKS

Unfortunately, similar transient state kinetic approaches have not been used to determine the selection factors for the HBV polymerase incorporating nucleotide analogues, so it is difficult to discern how selective the anti-HBV drugs are for the viral enzyme versus the human DNA polymerases. Nonetheless, these findings highlight the importance of initiating an in vivo investigation to confirm whether the noncanonical human DNA polymerases contribute to drug toxicity. In addition, this work established structure−function relationships that will be important in designing nucleoside analogues to overcome the limitations of clinical toxicity and drug resistance associated with current FDA-approved drugs. 231



Article

(7) Copeland, W. C., Chen, M. S., and Wang, T. S. (1992) Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA. J. Biol. Chem. 267, 21459−21464. (8) Olivero, O. A., Shearer, G. M., Chougnet, C. A., Kovacs, A. A., Landay, A. L., Baker, R., Stek, A. M., Khoury, M. M., Proia, L. A., Kessler, H. A., Sha, B. E., Tarone, R. E., and Poirier, M. C. (1999) Incorporation of zidovudine into leukocyte DNA from HIV-1-positive adults and pregnant women, and cord blood from infants exposed in utero. AIDS 13, 919−925. (9) Yamtich, J., and Sweasy, J. B. (2010) DNA polymerase Family X: Function, structure, and cellular roles. Biochim. Biophys. Acta 1804, 1136−1150. (10) Guo, C., Kosarek-Stancel, J. N., Tang, T. S., and Friedberg, E. C. (2009) Y-family DNA polymerases in mammalian cells. Cell. Mol. Life Sci. 66, 2363−2381. (11) Patterson, T. A., Little, W., Cheng, X., Widen, S. G., Kumar, A., Beard, W. A., and Wilson, S. H. (2000) Molecular cloning and highlevel expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes. Protein Expression Purif. 18, 100−110. (12) Brown, J. A., Duym, W. W., Fowler, J. D., and Suo, Z. (2007) Single-turnover kinetic analysis of the mutagenic potential of 8-oxo7,8-dihydro-2′-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta. J. Mol. Biol. 367, 1258− 1269. (13) Brown, J. A., Pack, L. R., Sanman, L. E., and Suo, Z. (2011) Efficiency and fidelity of human DNA polymerases lambda and beta during gap-filling DNA synthesis. DNA Repair 10, 24−33. (14) Fiala, K. A., Duym, W. W., Zhang, J., and Suo, Z. (2006) Upregulation of the fidelity of human DNA polymerase lambda by its non-enzymatic proline-rich domain. J. Biol. Chem. 281, 19038−19044. (15) Brown, J. A., Fiala, K. A., Fowler, J. D., Sherrer, S. M., Newmister, S. A., Duym, W. W., and Suo, Z. (2010) A novel mechanism of sugar selection utilized by a human X-family DNA polymerase. J. Mol. Biol. 395, 282−290. (16) Duym, W. W., Fiala, K. A., Bhatt, N., and Suo, Z. (2006) Kinetic effect of a downstream strand and its 5′-terminal moieties on single nucleotide gap-filling synthesis catalyzed by human DNA polymerase lambda. J. Biol. Chem. 281, 35649−35655. (17) Sherrer, S. M., Fiala, K. A., Fowler, J. D., Newmister, S. A., Pryor, J. M., and Suo, Z. (2011) Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases. Nucleic Acids Res. 39, 609−622. (18) Kirouac, K. N., and Ling, H. (2009) Structural basis of errorprone replication and stalling at a thymine base by human DNA polymerase iota. EMBO J. 28, 1644−1654. (19) Masuda, Y., Takahashi, M., Tsunekuni, N., Minami, T., Sumii, M., Miyagawa, K., and Kamiya, K. (2001) Deoxycytidyl transferase activity of the human REV1 protein is closely associated with the conserved polymerase domain. J. Biol. Chem. 276, 15051−15058. (20) Masuda, Y., and Kamiya, K. (2006) Role of single-stranded DNA in targeting REV1 to primer termini. J. Biol. Chem. 281, 24314− 24321. (21) Brown, J. A., Fowler, J. D., and Suo, Z. (2010) Kinetic basis of nucleotide selection employed by a protein template-dependent DNA polymerase. Biochemistry 49, 5504−5510. (22) Pascal, J. M., O’Brien, P. J., Tomkinson, A. E., and Ellenberger, T. (2004) Human DNA ligase I completely encircles and partially unwinds nicked DNA. Nature 432, 473−478. (23) Fiala, K. A., and Suo, Z. (2004) Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV. Biochemistry 43, 2106−2115. (24) Fiala, K. A., Abdel-Gawad, W., and Suo, Z. (2004) Pre-steadystate kinetic studies of the fidelity and mechanism of polymerization catalyzed by truncated human DNA polymerase lambda. Biochemistry 43, 6751−6762. (25) Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) Global conformational dynamics of a Y-family DNA polymerase during catalysis. PLoS Biol. 7, e1000225.

(26) Fowler, J. D., Brown, J. A., Kvaratskhelia, M., and Suo, Z. (2009) Probing conformational changes of human DNA polymerase lambda using mass spectrometry-based protein footprinting. J. Mol. Biol. 390, 368−379. (27) Brown, J. A., Pack, L. R., Sherrer, S. M., Kshetry, A. K., Newmister, S. A., Fowler, J. D., Taylor, J. S., and Suo, Z. (2010) Identification of critical residues for the tight binding of both correct and incorrect nucleotides to human DNA polymerase lambda. J. Mol. Biol. 403, 505−515. (28) Johnson, K. A. (1992) Transient-State Kinetic Analysis of Enzyme Reaction Pathways, in The Enzymes (Sigman, D. S., Ed.) pp 1−61, Academic Press, Inc., New York. (29) Brown, J. A., Newmister, S. A., Fiala, K. A., and Suo, Z. (2008) Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase. Nucleic Acids Res. 36, 3867−3878. (30) Fowler, J. D., Brown, J. A., Johnson, K. A., and Suo, Z. (2008) Kinetic investigation of the inhibitory effect of gemcitabine on DNA polymerization catalyzed by human mitochondrial DNA polymerase. J. Biol. Chem. 283, 15339−15348. (31) Brown, J. A., Pack, L. R., Fowler, J. D., and Suo, Z. (2011) Presteady state kinetic analysis of the incorporation of anti-HIV nucleotide analogs catalyzed by human X- and Y-family DNA polymerases. Antimicrob. Agents Chemother. 55, 276−283. (32) Matthews, S. J. (2007) Telbivudine for the management of chronic hepatitis B virus infection. Clin. Ther. 29, 2635−2653. (33) Scott, L. J., and Keating, G. M. (2009) Entecavir: a review of its use in chronic hepatitis B. Drugs 69, 1003−1033. (34) Brinkman, K., and Kakuda, T. N. (2000) Mitochondrial toxicity of nucleoside analogue reverse transcriptase inhibitors: a looming obstacle for long-term antiretroviral therapy? Curr. Opin. Infect. Dis. 13, 5−11. (35) Tanji, N., Tanji, K., Kambham, N., Markowitz, G. S., Bell, A., and D’Agati, V, D. (2001) Adefovir nephrotoxicity: possible role of mitochondrial DNA depletion. Hum. Pathol. 32, 734−740. (36) Zhang, X. S., Jin, R., Zhang, S. B., and Tao, M. L. (2008) Clinical features of adverse reactions associated with telbivudine. World J. Gastroenterol. 14, 3549−3553. (37) Bryant, M. L., Bridges, E. G., Placidi, L., Faraj, A., Loi, A. G., Pierra, C., Dukhan, D., Gosselin, G., Imbach, J. L., Hernandez, B., Juodawlkis, A., Tennant, B., Korba, B., Cote, P., Marion, P., CrettonScott, E., Schinazi, R. F., and Sommadossi, J. P. (2001) Antiviral Lnucleosides specific for hepatitis B virus infection. Antimicrob. Agents Chemother. 45, 229−235. (38) Johnson, A. A., Ray, A. S., Hanes, J., Suo, Z., Colacino, J. M., Anderson, K. S., and Johnson, K. A. (2001) Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase. J. Biol. Chem. 276, 40847−40857. (39) Feng, J. Y., Murakami, E., Zorca, S. M., Johnson, A. A., Johnson, K. A., Schinazi, R. F., Furman, P. A., and Anderson, K. S. (2004) Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2′,3′-dideoxy-5-fluoro-3′-thiacytidinetriphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase. Antimicrob. Agents Chemother. 48, 1300−1306. (40) Mazzucco, C. E., Hamatake, R. K., Colonno, R. J., and Tenney, D. J. (2008) Entecavir for treatment of hepatitis B virus displays no in vitro mitochondrial toxicity or DNA polymerase gamma inhibition. Antimicrob. Agents Chemother. 52, 598−605. (41) Birkus, G., Hitchcock, M. J., and Cihlar, T. (2002) Assessment of mitochondrial toxicity in human cells treated with tenofovir: comparison with other nucleoside reverse transcriptase inhibitors. Antimicrob. Agents Chemother. 46, 716−723. (42) Biesecker, G., Karimi, S., Desjardins, J., Meyer, D., Abbott, B., Bendele, R., and Richardson, F. (2003) Evaluation of mitochondrial DNA content and enzyme levels in tenofovir DF-treated rats, rhesus monkeys and woodchucks. Antiviral Res. 58, 217−225. (43) Venhoff, N., Setzer, B., Melkaoui, K., and Walker, U. A. (2007) Mitochondrial toxicity of tenofovir, emtricitabine and abacavir alone 232



Article

and in combination with additional nucleoside reverse transcriptase inhibitors. Antiviral Ther. 12, 1075−1085. (44) Charuworn, P., and Keeffe, E. B. (2009) A review of the use of telbivudine in the treatment of chronic hepatitis B. Clinical Medicine: Therapeutics 1, 157−166. (45) Kramata, P., and Downey, K. M. (1999) 9-(2phosphonylmethoxyethyl) derivatives of purine nucleotide analogs: A comparison of their metabolism and interaction with cellular DNA synthesis. Mol. Pharmacol. 56, 1262−1270. (46) Birkus, G., Hajek, M., Kramata, P., Votruba, I., Holy, A., and Otova, B. (2002) Tenofovir diphosphate is a poor substrate and a weak inhibitor of rat DNA polymerases alpha, delta, and epsilon*. Antimicrob. Agents Chemother. 46, 1610−1613. (47) Langley, D. R., Walsh, A. W., Baldick, C. J., Eggers, B. J., Rose, R. E., Levine, S. M., Kapur, A. J., Colonno, R. J., and Tenney, D. J. (2007) Inhibition of hepatitis B virus polymerase by entecavir. J. Virol. 81, 3992−4001. (48) Hart, G. J., Orr, D. C., Penn, C. R., Figueiredo, H. T., Gray, N. M., Boehme, R. E., and Cameron, J. M. (1992) Effects of (-)-2′-deoxy3′-thiacytidine (3TC) 5′-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma. Antimicrob. Agents Chemother. 36, 1688−1694. (49) Johnson, A. A., and Johnson, K. A. (2001) Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase. J. Biol. Chem. 276, 38090−38096. (50) Suo, Z., and Johnson, K. A. (1998) Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor, (R)-9-(2Phosphonylmethoxypropyl)adenine. J. Biol. Chem. 273, 27250−27258. (51) Kewn, S., Hoggard, P. G., Sales, S. D., Johnson, M. A., and Back, D. J. (2000) The intracellular activation of lamivudine (3TC) and determination of 2′-deoxycytidine-5′-triphosphate (dCTP) pools in the presence and absence of various drugs in HepG2 cells. Br. J. Clin. Pharmacol. 50, 597−604. (52) Delaney, W. E. t., Ray, A. S., Yang, H., Qi, X., Xiong, S., Zhu, Y., and Miller, M. D. (2006) Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob. Agents Chemother. 50, 2471−2477. (53) Levine, S., Hernandez, D., Yamanaka, G., Zhang, S., Rose, R., Weinheimer, S., and Colonno, R. J. (2002) Efficacies of entecavir against lamivudine-resistant hepatitis B virus replication and recombinant polymerases in vitro. Antimicrob. Agents Chemother. 46, 2525−2532. (54) Hernandez-Santiago, B., Placidi, L., Cretton-Scott, E., Faraj, A., Bridges, E. G., Bryant, M. L., Rodriguez-Orengo, J., Imbach, J. L., Gosselin, G., Pierra, C., Dukhan, D., and Sommadossi, J. P. (2002) Pharmacology of beta-L-thymidine and beta-L-2′-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus. Antimicrob. Agents Chemother. 46, 1728−1733. (55) Traut, T. W. (1994) Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1−22. (56) Igoudjil, A., Begriche, K., Pessayre, D., and Fromenty, B. (2006) Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors. Anti-Infect. Agents Med. Chem. 5, 273−292. (57) Bristol-Myers Squibb Co. (2010) Baraclude (entecavir) US prescribing information [online], http://www.baraclude.com/, accessed May 28, 2010. (58) Domaoal, R. A., McMahon, M., Thio, C. L., Bailey, C. M., Tirado-Rives, J., Obikhod, A., Detorio, M., Rapp, K. L., Siliciano, R. F., Schinazi, R. F., and Anderson, K. S. (2008) Pre-steady-state kinetic studies establish entecavir 5′-triphosphate as a substrate for HIV-1 reverse transcriptase. J. Biol. Chem. 283, 5452−5459. (59) Tchesnokov, E. P., Obikhod, A., Schinazi, R. F., and Gotte, M. (2008) Delayed chain termination protects the anti-hepatitis B virus drug entecavir from excision by HIV-1 reverse transcriptase. J. Biol. Chem. 283, 34218−34228. (60) Semizarov, D. G., Arzumanov, A. A., Dyatkina, N. B., Meyer, A., Vichier-Guerre, S., Gosselin, G., Rayner, B., Imbach, J. L., and

Krayevsky, A. A. (1997) Stereoisomers of deoxynucleoside 5′triphosphates as substrates for template-dependent and -independent DNA polymerases. J. Biol. Chem. 272, 9556−9560. (61) Feng, J. Y., Johnson, A. A., Johnson, K. A., and Anderson, K. S. (2001) Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs. J. Biol. Chem. 276, 23832−23837.

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Presteady State Kinetic Investigation of the Incorporation of Anti

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