Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus 1 Infected Cells 2

3 4

Running Title: Metabolism of CPV and GCV in HCMV-Infected Cells 5 6 7

BRIAN G. GENTRY1#, JOHN C. DRACH2 8 9

1 Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy 10 and Health Sciences, Drake University, Des Moines, Iowa 50311 (brian.gentry@drake.edu) 11 12 2 Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, 13 Ann Arbor, Michigan 48109 (jcdrach@umich.edu) 14 15 16 17 #Address Correspondence to: 18 19 Brian Gentry, Ph.D. 20 Department of Pharmaceutical, Biomedical, and Administrative Sciences 21 Drake University College of Pharmacy and Health Sciences 22 107 Fitch Hall 23 2507 University Ave. 24 Des Moines, Iowa 50310-4505 25 Email: brian.gentry@drake.edu 26 Phone: (515) 271-2980 27 Fax: (515) 271-1867 28 29 30 31 Keywords: Cytomegalovirus, Ganciclovir, Cyclopropavir 32

AAC Accepts, published online ahead of print on 10 February 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02311-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

2

ABSTRACT 33 Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease 34

in immunologically immature and immunocompromised patients. The current standard of 35 therapy for the treatment of HCMV infections is ganciclovir (GCV). However, high incidence 36 rates of adverse effects are prevalent and limit the use of this drug. Cyclopropavir (CPV) is 10-37 fold more effective against HCMV in vitro when compared to GCV (EC50’s = 0.46 and 4.1 μM, 38 respectively) without any observed increase in cytotoxicity. We have previously determined that 39 the viral protein kinase pUL97 and endogenous cellular kinases are responsible for conversion of 40 CPV to a triphosphate, the active compound responsible for inhibiting viral DNA synthesis and 41 viral replication. However, this conversion has not been observed in HCMV-infected cells. To 42 that end, we subjected HCMV-infected cells to equivalently effective concentrations of either 43 CPV or GCV (~5 times the EC50) and observed a time dependent increase in triphosphate levels 44 for both compounds (CPV-TP = 121 ± 11 pmol/106 cells; GCV-TP = 43.7 ± 0.4 pmol/106 cells). 45 A longer half-life was observed for GCV-TP (48.2 ± 5.7 hrs) compared to CPV-TP (23.8 ± 5.1 46 hrs). The area under the curve for CPV-TP produced from incubation with 2.5 μM CPV was 47 8680 ± 930 pmol·hours/106 cells, approximately two fold greater than the area under the curve 48 for GCV-TP of 4520 ± 420 pmol·hours/106 cells produced from incubation with 25 μM GCV. 49 We therefore conclude that the exposure of HCMV-infected cells to CPV-TP is greater than that 50 of GCV-TP under these experimental conditions. 51 52

53

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

3

INTRODUCTION 54 Human cytomegalovirus (HCMV) is a widespread pathogen infecting between 40 and 55

80% of the population worldwide [1]. Although individuals with competent immune systems 56 rarely manifest any symptoms, HCMV can result in severe interstitial pneumonia, encephalitis, 57 and gastroenteritis in immunocompromised individuals [2]. HCMV is also the most common 58 congenital infection in the United States and results in over 4,000 cases of severe mental 59 disabilities, hearing, and/or vision loss in infants each year [3, 4]. Drugs currently approved by 60 the FDA for the treatment of systemic HCMV infections are ganciclovir (GCV; Fig 1) and its 61 oral prodrug valganciclovir, foscarnet (PFA), and cidofovir [5-8]. The mechanism of action for 62 each of these drugs involves inhibition of the viral DNA polymerase resulting in inhibition of 63 HCMV DNA synthesis and viral replication [5]. However, long-term chemotherapy for HCMV 64 is generally required due to recurrence of infection upon cessation of treatment. As such, the 65 selection of strains with decreased drug susceptibility is common [6, 9-11]. Because adverse 66 effects occur with a high rate of incidence (up to 30% of patients) [12], there is a need for new 67 compounds with a greater therapeutic index for the treatment of systemic HCMV. 68

We have previously demonstrated that cyclopropavir (CPV; Fig 1), a 69 methylenecyclopropane guanosine nucleoside analog, is approximately 10-fold more active in 70 vitro (EC50 = 0.46 μM) than GCV (EC50 = 4.1 μM) without any observed increase in cytotoxicity 71 [13]. In addition, CPV is also active against several HCMV strains that are resistant to GCV or 72 PFA [14]. Further experimentation in vivo with CPV demonstrated a 2-5 log10 reduction in titers 73 of murine cytomegalovirus resulting in reduced mortality in severe combined immunodeficient 74 (SCID) mice and reduced viral replication in human fetal tissue implanted in SCID mice infected 75 with HCMV [15]. Toxicology studies performed in vivo demonstrated little to no adverse effects 76

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

4

at therapeutic concentrations making CPV a good clinical candidate for the treatment of systemic 77 HCMV infections [16]. 78

We and others have previously established that the mechanism of action of CPV is 79 similar to that of GCV; namely, phosphorylation to a monophosphate by the viral pUL97 protein 80 kinase [17-19], additional phosphorylation to a triphosphate by an endogenous cellular kinase 81 [20], and viral DNA synthesis inhibition resulting in inhibition of viral replication [14, 21]. 82 Although enzymatic conversion of CPV to a triphosphate by the viral protein kinase pUL97 and 83 an endogenous cellular kinase has been established [17, 20], this conversion has not been 84 observed in virus-infected cells. Therefore, the goal of this study is a comparison of the 85 metabolism of CPV and the current standard for HCMV chemotherapy, GCV, in HCMV-86 infected cells. 87 88 89

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

5

MATERIALS AND METHODS 90 Viral strain and chemicals. HCMV Towne strain was kindly provided by M. F. Stinski, 91

University of Iowa. GCV was kindly provided by Hoffman La Roche (Palo Alto, CA). 92 Cyclopropavir [(Z)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}guanine, CPV] along 93 with its mono-, di-, and triphosphates were kindly provided by Dr. Jiri Zemlicka (Karmanos 94 Cancer Center, Wayne State University, Detroit, MI) [13, 22]. 8-3H-GCV (19 Ci/mmol) and 8-95 3H-CPV (0.3 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA) and provided 96 through the courtesy of Microbiotix, Inc. (Worchester, MA). 97

Cell culture procedures. Human foreskin fibroblasts (HFF) were grown in minimal 98 essential medium with Earle’s salts and 10% fetal bovine serum. They were grown at 37 oC in a 99 humidified atmosphere of 3-5% CO2 – 97-95% air and were regularly passaged at 1:2 dilutions 100 using conventional procedures with 0.05% trypsin and 0.02% EDTA in HEPES-buffered saline 101 [23]. 102

Nucleoside analog triphosphate biosynthesis. HFF cells were seeded at 250,000 cells 103 per well in a 6-well cluster dish and infected the following day at a multiplicity of infection 104 (M.O.I) of ~5 plaque forming units (pfu) per cell. Two hours post-infection, 2.5 μM 3H-CPV or 105 25 μM GCV (both 3H- (~2%) and unlabeled-GCV) were added to the cells. At designated times, 106 cells were removed from plates using trypsin (0.05% with 0.02% EDTA), counted, lysed using 107 water, and proteins were precipitated using perchloric acid (final concentration 0.4 N). Samples 108 were neutralized using 10 N potassium hydroxide and stored at -20 oC until analysis by high 109 pressure liquid chromatography (HPLC). 110

Nucleoside analog triphosphate half-lives. HFF cells were seeded at 250,000 cells per 111 well in a 6-well cluster dish and infected the following day at a M.O.I. of ~5 pfu per cell. Two 112

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

6

hours post-infection, 2.5 μM 3H-CPV or 25 μM GCV (both 3H- (~2%) and unlabeled-GCV) 113 were added to the cells. Following five days of drug incubation, media containing drug was 114 removed and replaced with fresh media without drug. Samples were collected at the time of and 115 at designated times following media replacement and processed as described above. 116

Reverse-phase HPLC. CPV and its phosphorylated derivatives (CPV-MP, CPV-DP, 117 CPV-TP) were separated by reverse-phase HPLC [Beckman Coulter, Inc., (Indianapolis, IN) 118 System Gold Programmable Solvent Module 125 and System Gold Programmable Detector 119 Module 166 controlled by 32 Karat Software (version 7.0)]. Before injection, each sample was 120 centrifuged at 14,000 rpm for 10 min to remove any remaining particulate matter. Samples were 121 loaded onto a 10 μm Alphabond C18 300 x 3.9 mm reverse phase column (Alltech, Deerfield, 122 IL) at a flow rate of 1.0 mL/min. Base-line separation of CPV and its phosphorylated derivatives 123 was achieved by eluting with 150 mM ammonium phosphate (pH 3.0) and 100% methanol 124 (linear gradient of 20% methanol over 30 min followed by 10 min of isocratic conditions (80% 125 ammonium phosphate and 20% methanol)). One-minute fractions were collected, analyzed, and 126 tritium quantified by liquid scintillation spectrometry using a Beckman LS 6500 (Beckman 127 Coulter, Inc., Indianapolis, IN). Concentrations of CPV-TP were calculated based upon the 128 amount of label in the HPLC effluent fractions corresponding to the known position of CPV-TP 129 plus the specific activity of 3H-CPV. Final concentrations were adjusted for differences in cell 130 confluency and infection. 131

Strong anion exchange HPLC. GCV and its phosphorylated derivatives (GCV-MP, 132 GCV-DP, GCV-TP) were separated by strong anion exchange HPLC [Beckman Coulter, Inc., 133 (Indianapolis, IN) System Gold Programmable Solvent Module 125 and System Gold 134 Programmable Detector Module 166 controlled by 32 Karat Software (version 7.0)]. Before 135

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

7

injection, each sample was centrifuged at 14,000 rpm for 10 min to remove any remaining 136 particulate matter. Samples were loaded onto a 5 μm Hypersil 250 x 4.6 mm strong anion 137 exchange column (Thermo Scientific, Waltham, MA) at a flow rate of 1.0 mL/min. Base-line 138 separation of GCV and its phosphorylated derivatives was achieved by eluting with 10 mM 139 ammonium phosphate (pH 3.0) and 500 mM ammonium phosphate (pH 3.0) (10 mM ammonium 140 phosphate isocratic conditions for 12 minutes followed by 25% gradient of 500 mM ammonium 141 phosphate over 24 minutes). One-minute fractions were collected, analyzed, and tritium 142 quantified by liquid scintillation spectrometry using a Beckman LS 6500 (Beckman Coulter, 143 Inc., Indianapolis, IN). Concentrations of GCV-TP were calculated as described above for CPV-144 TP. 145

Data analysis. Upon collection of data and calculation of triphosphate concentrations, 146 results were graphed and analyzed using Prism (version 5.0; GraphPad Software, San Diego, 147 CA) to determine standard deviation, linear regressions, statistical significance (Student’s t-test), 148 and areas under the curve. 149

150 151

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

8

RESULTS 152 Metabolism of CPV and GCV to their respective triphosphates in HCMV-infected 153

HFF cells. We have hypothesized that the mechanism of action of CPV involves 154 phosphorylation to a triphosphate, the active compound that inhibits the viral DNA polymerase 155 [14, 17, 20]. However, the production of CPV-TP in HCMV-infected cells has not been 156 demonstrated. Therefore to test for the biosynthesis of CPV-TP in vitro, HFF cells infected with 157 the Towne strain of HCMV (M.O.I. ~5) were incubated with either 2.5 μM CPV or 25 μM GCV 158 (positive control) and cell extracts were analyzed for the presence of nucleoside analog 159 triphosphates (Fig 2, Table 1). HCMV-infected cells incubated with 2.5 μM CPV demonstrated 160 a time dependent increase in CPV-TP levels resulting in a maximum of 121 ± 11 pmol/106 cells 161 at 120 hours. HCMV-infected cells incubated with 25 μM GCV demonstrated a similar time 162 dependent increase in triphosphate levels. However, the maximum quantity of GCV-TP (43.7 ± 163 0.4 pmol) is 2.5-fold lower and occurs earlier (96 hours) than that of CPV-TP even though the 164 concentration of GCV with which HCMV-infected cells were incubated was 10-fold greater than 165 CPV. No mono- or di-phosphates of CPV or GCV were detected indicating that the rate-limiting 166 step in the biosynthesis of both triphosphates is the initial phosphorylation step. Uninfected HFF 167 cells incubated with 2.5 μM CPV demonstrated no measureable level of CPV-TP (limit of 168 detection ~0.6 pmol/106 cells) indicating that the presence of virus, and more specifically the 169 viral protein kinase pUL97 [17] is necessary for the conversion of CPV to CPV-TP. 170

Half-life of CPV-TP and GCV-TP in HCMV-infected HFF cells. Enzymatic 171 conversion of CPV to CPV-TP, while necessary for the drug to elicit an anti-viral effect, is not 172 the sole determinant of efficacy. Drug half-life, or the length of time the virus is exposed to 173 active compound, is another major determinant of efficacy. Therefore we measured the half-life 174

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

9

of CPV-TP and GCV-TP in HCMV-infected cells following five days of exposure to either 2.5 175 μM CPV or 25 μM GCV, respectively (Fig 3, Table 1). The results for both compounds 176 demonstrated first-order kinetics and the half-lives were calculated from their respective linear 177 regression lines. Although triphosphate levels for both compounds persisted through 96 hours, 178 GCV-TP had an approximately 2-fold longer half-life (48.2 ± 5.7 hrs) compared to CPV-TP 179 (23.8 ± 5.1 hrs). Thus, while the accumulation of GCV-TP is not as large as that of CPV-TP, 180 GCV-TP appears to persist longer than that of CPV-TP. In fact, even though the dose of GCV 181 was 10-times greater than that of CPV, the level of GCV-TP (10.2 ± 1.3 pmol/106 cells) at 96 182 hours post wash-out is statistically greater than that of CPV-TP (6.5 ± 1.0 pmol/106 cells) (P < 183 0.05), although this difference is less than two-fold. 184

Total exposure of CPV-TP and GCV-TP to HCMV-infected HFF cells. Since the 185 administration of CPV resulted in greater biosynthesis of triphosphate when compared to GCV 186 but with a shorter half-life, combining the data into one single plot and measuring areas under 187 the curve was used to determine which combination of properties resulted in greatest exposure of 188 HCMV-infected cells to active compound (Fig 4, Table 1). The results demonstrate that under 189 the conditions used in these experiments the area under the curve for CPV-TP (8680 ± 930 190 pmol·hours/106 cells) is approximately two fold greater than the area under the curve for GCV-191 TP (4520 ± 420 pmol·hours/106 cells) even though cells were exposed to 10-times more GCV 192 than CPV. We therefore conclude that the exposure of HCMV-infected cells to CPV-TP is 193 greater than that of GCV-TP under equivalently effective concentration (EC) conditions - 194 approximately five times the EC50 for both drugs [13]. 195

196 197

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

10

DISCUSSION 198 The formation of CPV-TP has been assumed to be an essential element for CPV to inhibit 199 viral replication and elicit an anti-viral effect [14, 17, 18, 20]. Since our previous experiments 200 demonstrated that CPV is a better substrate for the viral protein kinase pUL97 than GCV, and 201 that the initial phosphorylation to a monophosphate catalyzed by this enzyme is the rate-limiting 202 step in the formation of triphosphate [17], we hypothesize that the production of CPV-TP would 203 be greater than that of GCV-TP under equivalent concentrations. Consistent with this 204 hypothesis, the conversion of CPV to CPV-TP occurred to a greater extent than that of GCV to 205 GCV-TP despite being administered at a lower concentration (2.5 μM CPV verses 25 μM GCV; 206 equivalently effective concentrations (~five times the EC50)). 207 Our results demonstrated a significant difference in the half-lives of CPV-TP (23.8 ± 5.1 208 hours) and GCV-TP (48.2 ± 5.7 hours) (Fig 3, Table 1). In these experiments, GCV-TP appears 209 to reach equilibrium within the HCMV-infected cell (a point at which the rate of 210 dephosphorylation is equal to the rate of phosphorylation) at 48 hours post drug wash-out. The 211 half-life of GCV-TP before this state of equilibrium occurs is approximately 27 hours and not 212 statistically different from that observed for CPV-TP. It also appears that this state of nucleoside 213 analog triphosphate equilibrium is common between the two compounds; the levels of CPV-TP 214 towards the end of the half-life experiment (84 hours post wash-out) also appear to have reached 215 an equilibrium state. We hypothesize that if the duration of the experiment was longer than 96 216 hours post wash-out, we would also observe a persistent, stable level of CPV-TP. 217 In contrast to our results, a previous study by Biron et al. reported that the half-life of 218 GCV-TP in HCMV-infected cells was approximately 12 hours [24], a significant variance from 219 what we have observed (48.2 hours). There is, however, a significant difference between the two 220

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

11

studies that can partially account for the variance in results. Biron et al. conducted their 221 experiments using a M.O.I. of 0.5 pfu per cell which would result in approximately 50% HCMV 222 infection rate [24]. We conducted these experiments using a M.O.I. >5 pfu per cell, which 223 would result in nearly 100% of the cells being infected with HCMV. This difference in M.O.I. 224 would not only result in different intracellular concentrations of GCV-TP, but could also affect 225 half-life. In fact, Gentry et al. previously demonstrated that a culture in which only 50% of the 226 cells were able to produce GCV-TP resulted in about a 2-fold reduction in half-life when 227 compared to a culture in which 100% of the population of cells could produce GCV-TP [25]. 228 This reduction in half-life was hypothesized to be the result of two cells dephosphorylating 229 GCV-TP per every cell that could produce GCV-TP in comparison to a culture in which all cells 230 produce and dephosphorylate GCV-TP. The presumed mechanism by which this occurs is 231 transfer of phosphorylated GCV metabolites from virus-infected cells to uninfected cells through 232 gap junctions, intracellular communication channels capable of direct transfer of small molecules 233 from the cytoplasm of one cell to another [26]. In addition, if you remove the latter time points 234 from our experiment in which the level of GCV-TP was stable (48 hours post-drug removal), the 235 calculated half-life would be 27 hours or approximately 2-fold greater than what was previously 236 observed. Therefore, this combination of factors could account for the variance in GCV-TP half-237 life reported here and by Biron et al. 238

By performing these experiments at equivalently effective concentrations, we are able to 239 speculate about the relative efficacy for each compound acting at the enzymatic and whole cell 240 level. Regarding the action of the triphosphates against HCMV DNA polymerase and assuming 241 a cell volume of ~5 picoliters [27], our determination of 121 pmol CPV-TP/106 cells and 43.7 242 pmol GCV-TP/106 cells would translate into an intracellular concentration of 5.3 µM and 1.2 243

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

12

μM, respectively. For a whole cell comparison, the area under the curve for CPV-TP (8680 ± 244 930 pmol·hrs/106 cells) is approximately 2 times greater than the area under the curve for GCV-245 TP (4520 ± 420 pmol·hrs/106 cells). The inference is that it requires two to four times the 246 amount of CPV-TP when compared to GCV-TP to elicit the same anti-viral effect. However, it 247 has been previously reported that CPV (EC50 = 0.46 μM) is more efficacious than GCV (EC50 = 248 4.1 μM) since it requires less drug to elicit the same anti-viral response [13]. We therefore 249 hypothesize that the superior efficacy of CPV when compared to GCV does not come from 250 better efficacy of active compound (CPV-TP verses GCV-TP), but stems from the fact that CPV 251 is a better substrate for the viral protein kinase pUL97 and thus is phosphorylated to a 252 monophosphate (rate-limiting step) to a much greater extent than GCV. This would result in a 253 greater production of CPV-TP compared to GCV-TP under equivalent concentration conditions. 254 Consistent with this hypothesis, we have previously determined that the HCMV protein kinase 255 pUL97 phosphorylates CPV to CPV-MP at a rate 45 times greater than that of GCV to GCV-MP 256 at equivalent concentrations [17]. 257

Our current results demonstrate that at equivalently effective concentrations of CPV and 258 GCV, HCMV-infected cells are exposed to CPV-TP to a greater extent than GCV-TP, the active 259 compounds that elicit an anti-viral response. In addition, the greater efficacy of CPV observed 260 in vitro and in vivo without any increase in toxicity [13, 16] and the ability to achieve therapeutic 261 concentrations in vivo without prodrug modification [7, 16] are a few reasons why CPV appears 262 to be superior to GCV for the treatment of HCMV disease. 263 264

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

13

ACKNOWLEDGMENTS 265 We thank Andrew Samann and James Simmer for assistance with the use of Dr. 266

Simmer’s HPLC system and liquid scintillation spectrometer system. We especially thank the 267 inventor of CPV, Dr. Jiri Zemlicka, for his interest, encouragement and support. We also thank 268 James Sacco for his help with the half-life data analysis. This work was supported by the gift of 269 tritiated CPV and GCV from Microbiotix, Inc. plus funds from the University of Michigan and 270 Drake University. 271

272 273 274

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

14

REFERENCES 275 1. Ho, M., The History of Cytomegalovirus and its Diseases. Medical Microbiology and 276

Immunology, 2008. 197: p. 65-73. 277 278 2. Landolfo, S., Gariglio, M., Gribaudo, G., Lembo, D., The Human Cytomegalovirus. 279

Pharmacology & Therapeutics, 2003. 98: p. 269-297. 280 281 3. Fortunato, E.A., Spector, D. H., Regulation of Human Cytomegalovirus Gene Expression. 282

Advances in Virus Research, 1999. 54: p. 61-128. 283 284 4. Manicklal, S., Emery, V.C., Lazzarotto, T., Boppana, S.B., and Gupta, R.K., The "silent" 285

global burden of congenital cytomegalovirus. Clin Microbiol Rev, 2013. 26(1): p. 86-286 102. 287

288 5. Andrei, G., De Clercq, E., Snoeck, R., Drug Targets in Cytomegalovirus Infection. 289

Infectious Disorders - Drug Targets, 2009. 9(2): p. 201-222. 290 291 6. Biron, K.K., Antiviral Drugs for Cytomegalovirus Diseases. Antiviral Research, 2006. 292

71: p. 154-163. 293 294 7. Cocohoba, J.M., McNickoll, I. R., Valganciclovir: An Advance in Cytomegalovirus 295

Therapeutics. The Annals of Pharmacotherapy, 2002. 36: p. 1075-1079. 296 297 8. Balfour, H.H., Antiviral Drugs. New England Journal of Medicine, 1999. 340(16): p. 298

1255-1268. 299 300 9. Baldanti, F., Lurain, N., Gerna, G., Clinical and Biologic Aspects of Human 301

Cytomegalovirus Resistance to Antiviral Drugs. Human Immunology, 2004. 65: p. 403-302 409. 303

304 10. Chou, S., Lurain, N.S., Thompson, K.D., Miner, R.C., and Drew, W.L., Viral DNA 305

polymerase mutations associated with drug resistance in human cytomegalovirus. J 306 Infect Dis, 2003. 188(1): p. 32-9. 307

308 11. Erice, A., Resistance of human cytomegalovirus to antiviral drugs. Clin Microbiol Rev, 309

1999. 12(2): p. 286-97. 310 311 12. Morris, D.J., Adverse effects and drug interactions of clinical importance with antiviral 312

drugs. Drug Saf, 1994. 10(4): p. 281-91. 313 314 13. Zhou, S., Breitenbach, J.M., Borysko, K.Z., Drach, J.C., Kern, E.R., Gullen, E., Cheng, 315

Y.C., and Zemlicka, J., Synthesis and antiviral activity of (Z)- and (E)-2,2-316 [bis(hydroxymethyl)cyclopropylidene]methylpurines and -pyrimidines: second-317 generation methylenecyclopropane analogues of nucleosides. J Med Chem, 2004. 47(3): 318 p. 566-75. 319

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

15

320 14. Kern, E.R., Kushner, N.L., Hartline, C.B., Williams-Aziz, S.L., Harden, E.A., Zhou, S., 321

Zemlicka, J., and Prichard, M.N., In vitro activity and mechanism of action of 322 methylenecyclopropane analogs of nucleosides against herpesvirus replication. 323 Antimicrob Agents Chemother, 2005. 49(3): p. 1039-45. 324

325 15. Kern, E.R., Bidanset, D.J., Harline, C.B., Yan, Z., Zemlicka, J., Quenelle, D.C., Oral 326

Activity of a Methylenecyclopropane Analog, Cyclopropavir, in Animal Models for 327 Cytomegalovirus Infections. Antimicrobial Agents and Chemotherapy, 2004. 48: p. 4745-328 4753. 329

330 16. Bowlin, T.L., Brooks, J.L., Zemlicka J., Preclinical Pharmacokinetic, Toxicokinetic and 331

Toxicology Results for Cyclopropavir, a Promising New Agent for the Treatment of Beta- 332 and Gamma-herpesviruses. Antiviral Research, 2009. 82: p. A46-A47. 333

334 17. Gentry, B.G., Kamil, J.P., Coen, D.M., Zemlicka, J., Drach, J.C., Stereoselective 335

Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir. 336 Antimicrobial Agents and Chemotherapy, 2010. 54: p. 3093-3098. 337

338 18. Gentry, B.G., Vollmer, L.E., Hall, E.D., Borysko, K.Z., Zemlicka, J., Kamil, J.P., and 339

Drach, J.C., Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base 340 Pair Deletion in the Open Reading Frame of UL97. Antimicrob Agents Chemother, 341 2013. 57(9): p. 4343-8. 342

343 19. Chou, S. and Bowlin, T.L., Cytomegalovirus UL97 mutations affecting cyclopropavir and 344

ganciclovir susceptibility. Antimicrob Agents Chemother, 2011. 55(1): p. 382-4. 345 346 20. Gentry, B.G., Gentry, S.N., Jackson, T.L., Zemlicka, J., Drach, J.C., Phosphorylation of 347

Antiviral and Endogenous Nucleotides to Di- and Triphosphates by Guanosine 348 Monophosphate Kinase. Biochemical Pharmacology, 2011. 81: p. 43-49. 349

350 21. Chou, S., Marousek, G., and Bowlin, T.L., Cyclopropavir susceptibility of 351

cytomegalovirus DNA polymerase mutants selected after antiviral drug exposure. 352 Antimicrob Agents Chemother, 2012. 56(1): p. 197-201. 353

354 22. Li, C., Gentry, B.G., Drach, J.C., Zemlicka, J., Synthesis and Enantioselectivity of 355

Cyclopropavir Phosphates for Cellular GMP Kinase. Nucleosides, Nucleotides, & 356 Nucleic Acids, 2009. 28: p. 795-808. 357

358 23. Shipman, C., Jr., Evaluation of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 359

(HEPES) as a tissue culture buffer. Proc Soc Exp Biol Med, 1969. 130(1): p. 305-10. 360 361 24. Biron, K.K., Stanat, S.C., Sorrell, J.B., Fyfe, J.A., Keller, P.M., Lambe, C.U., and 362

Nelson, D.J., Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-363 (hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human 364 cytomegalovirus. Proc Natl Acad Sci U S A, 1985. 82(8): p. 2473-7. 365

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

16

366 25. Gentry, B.G., Im, M., Boucher, P.D., Ruch, R.J., and Shewach, D.S., GCV phosphates 367

are transferred between HeLa cells despite lack of bystander cytotoxicity. Gene Ther, 368 2005. 12(13): p. 1033-41. 369

370 26. Evans, W.H. and Martin, P.E., Gap junctions: structure and function (Review). Mol 371

Membr Biol, 2002. 19(2): p. 121-36. 372 373 27. Mastrocola, T., Lambert, I.H., Kramhøft, B., Rugolo, M., and Hoffmann, E.K., Volume 374

regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the 375 activation of the Cl− efflux. The Journal of Membrane Biology, 1993. 136(1): p. 55-62. 376

377 378 379

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

17

Table 1. Comparison of CPV-TP and GCV-TP 380 381 Compound Peak Triphosphate Level Half-Life (t1/2) Area Under the Curve 382 CPV-TP 121 ± 11 pmol/106 cells* 23.8 ± 5.1 hrs* 8680 ± 930 pmol·hrs/106 cells‡ 383 GCV-TP 43.7 ± 0.4 pmol/106 cells* 48.2 ± 5.7 hrs* 4520 ± 420 pmol·hrs/106 cells‡ 384 * Mean ± standard deviation from at least two experiments 385 ‡ Value calculated as a result of combining the data from the triphosphate biosynthesis and half-386 life studies. 387

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

on May 14, 2018 by guest

http://aac.asm.org/

Dow

nloaded from