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The pharmacology of nucleoside analogues in herpesvirus infection Smyrnis, Elie Mario 1999

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THE PHARMACOLOGY OF NUCLEOSIDE ANALOGUES IN HERPESVIRUS INFECTION by Elie Mar io Smyrnis B.Sc, (Microbiology) The University of British Columbia 1982 M.Sc, (Pathology) The University of British Columbia 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology and Therapeutics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1999 ©E. M. SMYRNIS, 1999 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 August, 1999 D E - 6 C 3 / 8 1 ) The Pharmacology of Nucleoside Analogues in Herpesvirus Infection. Abstract: The pharmacological aspect of anti-herpesvirus (HHV) research has entered a new era concomitant with the introduction of the nucleoside analogue-class of anti-viral agent. Recent accession to the specificity of these agents has provided not only substantial improvements in the treatment and prophylaxis of many different HHV infections, but to enhancements in the methods used to diagnose and further study these virus-induced illnesses as well. The disparate applications of such compounds are perhaps best illustrated by the thymidine kinase (^-dependent anti-HHV nucleoside analogues -- agents which, by virtue of their selective uptake and metab-olism, have been used both chemotherapeutically and as molecular indicators of the virus itself. It is clear that a fundamental principle in determining an antiviral's pharmacotherapeutic relevance and of gauging its potential as a diagnostic indicator of disease lies in the ability to accurately detect and quantitate these agents in complex biological matrices. In the present study, radioiodinated l-j8-D-arabino-furanosyl-E-5-(2-iodovinyl)uracil ([*I]-IVaraU) was used to develop an autoradio-graphic technique (PA/IP) that rapidly and accurately demonstrated thefJc- (and thymidylate kinase)-dependent metabolism of this antiviral agent. [*I]-IVaraU-PA/JP was also used to detect antiviral-resistant HHV variants in somatic cell cultures, to examine the growth of HSV in primary cultures of mammalian CNS glial cells and to demonstrate the uptake and metabolism of 9-(4-hydroxy-3-hydroxy-methylbut-l-yl)guanine (PCV) in a human neural (SW) cell line. We further demonstrate limited in vivo utility of [*I]-IVaraU for detecting HHV in tissue auto-radiographs of CNS sections prepared from a rabbit encephalitis model. The use of this agent as a radiodiagnostic indicator of HSE in intact animals using external gamma camera imaging, however, was found to be largely unreliable. Finally, techniques in ESI mass spectrometry (both independent from and in combination with RP-IP HPLC - including the development of requisite volatile buffer systems) were used to determine that the PCV-TP synthesized by HSVW+) type 1-infected-PCV (10 uM and 1 uM)-treated SW cells after a 6 H drug treatment to be 11500 pmole«(10 6)- 1 and 2440 pmole^dO6)-1 SW cells, respectively, and that the intra-cellular stability of PCV-TP in this culture system was roughly 16.5 H. These deter-minations were made by monitoring the nucleotide'^ortho/pyrophosphate transi-tions. We conclude that this technique can be developed further, into a method for monitoring the therapeutic efficacy of this and possibly other antiviral compounds. i i Table of Contents: Page No. Abstract: ii List of Abbreviations: xiii List of Tables: xviii List of Figures: xix Acknowledgments: xxiv Dedication: xxv 1 Introduction: 1.1 Taxonomy of the Herpesviridae: 1 1.2 Physical Characteristics: 1 1.3 Replicative Cycle and Temporal Sequence of Gene Expression: 3 1.4 Clinically Important Herpesvirus-Encoded Enzymes: 5 1.4.1 Herpesvirus Thymidine Kinase: 5 1.4.2 Herpesvirus DNA Polymerase: 9 1.4.3 Thymidine Kinase-Deficient Herpesviruses: ...10 Herpesvirus Neurotropisms: 11 1.5 Biological Properties and Host Range: 12 1.5.1 Animal Models of Human Herpesvirus Infection: 13 Animal Models of Human Herpesvirus CNS Infection: 13 1.5.2 Pathogenesis of the Human Infection: 15 Herpesvirus Latency: 16 Recurrent Herpesvirus Infection: 17 The Immune Response: 17 1.5.3 Herpes Simplex Virus type-1 and type-2: 18 Herpes Simplex Encephalitis: 19 Neonatal Herpes Simplex: 20 1.5.4 Varicella-Zoster Virus: 20 Post Herpetic Neuralgia: .21 1.5.5 Cytomegalovirus: .23 1.5.6 Epstein-Barr Virus: .24 1.5.7 Human Herpesvirus 6: .24 1.5.8 Human Herpesvirus 7: .26 iii Page No. 1.5.9 Kaposi's Sarcoma-Associated Herpes Virus: _ 26 1.6 Laboratory Diagnosis of Herpesvirus Infections: 27 1.6.1 Conventional Diagnostic Techniques: 27 1.6.2 Molecular Diagnostic Techniques: 30 Nucleic Acid Hybridization Techniques: 31 Polymerase Chain Reaction: 32 1.7 Herpesvirus Nucleoside Analogues: 33 1.7.1 The Pyrimidine Nucleoside Analogues: Halogenated Pyrimidines: Iododeoxyuridine: 34 Iododeoxycytidine: 36 Halogenovinyl Pyrimidines: 36 Bromo- and Iodovinyldeoxyuridines: 38 Sugar-Modified 5-Substituted Pyrimidine Analogues: 40 Bromo- and Iodovinylarabinofuranosyluracils: 41 1.7.2 The Purine Nucleoside Analogues. Ara-A (Vidarabine): 44 Acyclovir (and Valaciclovir): 44 Buciclovir: 49 Ganciclovir: 49 Penciclovir (and Famciclovir): 50 1.7.3 Non-Nucleoside Analogue Anti-Herpesvirus Agents: The Phosphonates: Foscarnet & Phosphonoacetic Acid: 53 1.7.4 Acyclic Nucleoside Phosphonates: Phosphonylmethoxyalkyl Purines & Pyrimidines (HPMPA and HPMPC): 54 1.8 Antiviral Resistance: 56 1.8.1 Thymidine Kinase-deficient Herpesvirus Variants as Research Tools: 57 1.8.2 Nucleoside Analogues as Herpesvirus fl-Specific Probes: 58 Radiolabeled Nucleoside Analogues in Plaque Auto-radiography: 58 Radiolabeled Nucleoside Analogues as Radiodiagnostic Indicators of HSE: 59 The 2'-Fluoro-5-Substituted Arabinofuranosyl-pyrimidines: 60 The 5-(2-Halogenovinyl)-Substituted Pyrimidines: .62 1.9 Therapeutic Drug Monitoring: 66 iv Page No. 1.10 The Bioanalysis of Antiviral Agents and their Metabolites: .....67 1.10.1 Sample Preparation: 68 1.10.2 High-Performance Liquid Chromatography (HPLC) of Nucleo-bases, Nucleosides, and Nucleotides: 71 1.10.3 HPLC of Nucleoside Analogues and their Phosphorylated Metabolites: .72 Ion-exchange (IE) HPLC: .73 Reverse-Phase (RP) HPLC: 74 Reverse-Phase Ion-Paired (RP-IP) HPLC: 75 The Use of Volatile Buffers (VB) in HPLC. 78 1.10.4 Mass Spectrometry of Nucleosides, Nucleotides and their Antiviral Homologues: 80 Electron Impact (EI) and Chemical Ionization (CI) Mass Spectrometry: .83 Thermospray Ionization (TSP) Mass Spectrometry: 84 Fast Atom Bombardment/Liquid-Secondary Ionization Mass Spectrometry: .84 Electrospray (Ionspray) Mass Spectrometry: 86 1.10.5 On-Line Mass Spectrometric Analysis of Nucleosides, Nucleo-tides and their Antiviral Homologues: .89 Gas Chromatography Mass Spectrometry: _ .90 High-Performance Liquid Chromatography Mass Spectrometry: .91 1.11 Concluding Remarks: .95 2 M a t e r i a l s a n d M e t h o d s : 2.1 Tissue Culture Methods: 2.1.1 Culture Conditions: .97 2.1.2 Preparation and Maintenance of Cell lines and Primary Cultures: Somatic and Neural Cell Cultures: .97 Preparation of Mixed Primary Glial Cell Cultures: .98 Astrocyte and Oligodendrocyte-Enriched Cultures:____100 2.1.3 Herpesvirus Stocks: Herpes Simplex Virus type-1 (HSV type-1): 101 Herpes Simplex Virus type-2 (HSV type-2): 101 Varicella-Zoster Virus (VZV): 101 2.1.4 Virus Growth and Propagation: 101 2.1.5 Herpes Simplex Encephalitis Model: 102 V Page No. 2.2 Herpesvirus Detection Methods: 2.2.1 Immunological Techniques: Antisera: 103 Immunoperoxidase Staining: 103 Direct Immunofluorescence: 105 Indirect Immunofluorescence: 105 Fluorescence-Activated Cell Sorter Analysis: 106 2.2.2 Radiological Techniques: Radiochemicals: 108 Synthesis and Purification of [l25i]-rVaraU: 108 Autoradiography Standards: 110 Plaque Autoradiography: [125I]-IVaraU Plaque Autoradiography: I l l [4'-3H]-PCV Plaque Autoradiography: 113 Tissue Autoradiography: [l25l]-lVaraU Uptake as a Herpesvirus fTc-Specific Radiotracing Agent: 113 Preparation of HSE Brain Thin-Sections: 115 Thin-Section Autoradiography: 117 2.3 Computer-Assisted Digital Image Densitometry: 117 2.4 High Performance Liquid Chromatography: 2.4.1 H P L C Buffer Reagents: 118 2.4.2 H P L C Standards: Nucleotides, Nucleosides and Nucleobases: 118 Purine H P L C Standards: 119 Pyrimidine H P L C Standards: 119 Nucleoside Analogue H P L C Standards: 119 2.4.3 Microflow-LC Columns: Manufacture of Capillary-LC Columns: 120 Manufacture of Nano-LC Columns: 120 Packing Materials: 120 Slurry Composition/Packing Methods: 122 Capillary Column Efficiencies: 124 2.4.4 Preparative H P L C : Purification of [l25i]-rVaraU: 124 2.4.5 Qualitative H P L C : RP-IP H P L C of Nucleosides, Nucleoside Analogues and Nucleotides Using Non-Volatile (Phosphate) Buffers: 125 Development of a Volatile Buffer System for the RP-IP H P L C of Nucleosides, Nucleoside Analogues and their Phosphorylated Derivatives: 127 vi Page No. 2.5 Mass Spectrometry: 127 2.5.1 Kratos Concept JJHQ EBqQ® Mass Spectrometer Operating Parameters using a Liquid-Secondary Ion Monitoring System (L-SIMS) Interface: 128 Static L-SIMS Mass Spectrometry: 128 L-SIMS Sample Preparation: 128 2.5.2 Fisons VG-Quattro® Triple Quadrupole Mass Spectrometry using an Electrospray (Ionspray) Interface: 129 Preparation of Samples for ESI Analysis: Nucleoside and Nucleoside Analogue Standards used in ESI Analysis: 131 Preparation of Cell Extracts for ESI Analysis: 131 Preparation of FACS-Enriched HSV-Infected/Drug-Treated Cell Extracts for ESI" (MRM) Analysis: 132 Triple Quadrupole ESI Mass Spectrometry. General Operating Parameters: 134 ESI- MS Analyses using a Single Mass Analyzer: Scanning ESI" MS Operating Mode: 135 Single Ion Recording (SIR) Operating Mode: 136 ESI- MS Analyses Using Mass Analyzers in Tandem (MS/MS): 136 Multiple Reaction Monitoring (MRM) Operating Mode: 137 Collision-Induced Dissocia-tion (CID): 137 Daughter-Ion and Parent-Ion ESI- Mass Spectra: 138 2.6 High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS): 138 2.6.1 RP-IP HPLC/ EST MS (SIR) Mass Spectrometry: 138 2.6.2 RP-IP mHPLC/ EST MS (MRM) Mass Spectrometry: 139 RP-IP mHPLC/EST MS/MS of a PCV-TP Standard Solution: 140 RP-IP mHPLC/ ESI" MS/MS of PCV-TP in HSV type-1-Infected/PCV-Treated Cell Extracts: 140 RP-IP mHPLC/ EST MS/MS of PCV-TP in FACS-Enriched/Herpesvirus-Infected (PCV-Treated) SW Cell Extracts: 141 vii Page No. Results: Virus Growth in Somatic Cell Cultures: 142 Demonstration of the Bioactive Metabolites of Radiolabeled Anti-Herpesvirus Nucleoside Analogues: 3.2.1 In Vitro Determinations of Nucleoside Analogues using Plaque Autoradiography: 141 [125i]-lVaraU-PA of Somatic Cell Monolayers: Preparation of Cell Monolayers for [l25l]-lVaraU-Plaque Autoradiography: 144 [l25i]-rVaraU-PA Image Densitometry: 144 [1251] -IVaraU-P A / Immunocy tochemistry: 146 Limits of Detection of [l25I]-IVaraU-PA: 151 [3H]-Penciclovir Autoradiography of HSV-Infected Schwannoma Cell Monolayers: 151 psiJ-rVaraU-PA/IP in the Study of Herpesvirus Neuro-tropisms: 151 Properties of Mixed and Enriched Neural Cell Cultures. 154 Herpesvirus-Infection of Neural Cell Cultures: 159 [!25I]-lVaraU-PA/IP of HSV-Infected Neural Cell Cultures: 164 3.2.2 In Vivo Determinations of Radiolabeled Anti-Herpesvirus Nucleoside Analogue in a Rabbit Model of Herpes Simplex Encephalitis (HSE): 164 Gross Pathologic Changes in the Rabbit HSE Model: 166 In Vivo Demonstration of a Herpesvirus infection in the CNS of a Rabbit Model of HSE using [!25i]-rVaraU-Tissue Autoradiography (TA)/IP: 166 Preparation of Tissue Sections for [!25l]-lVaraU-TA/IP Characterization: 169 Direct Determination (Gamma Counts) of Radio-active HSE Tissue Sections: 169 Image Densitometry of HSE/[l25I]-IVaraU Tissue Autoradiographs: 171 Histopathology and IP Characterization of HSE Tissue Sections:.. 171 In Vivo Demonstration of a Herpesvirus Infection in the CNS of a Rabbit Model of HSE using [l25I]-lVaraU and External Gamma-Camera Scintigraphy: 177 viii Page No. 3.3 Ultratrace Determination of the Bioactive Metabolites of Non-Radiolabeled Anti-Herpesvirus Nucleoside Analogues: 180 3.3.1 Development of a RP-IP HPLC/Mass Spectrometry System for the Separation and Detection of Nucleosides, Nucleoside Analogues, and their Phosphorylated Metabolites: RP-IP HPLC/UV254 using a Non-Volatile Phosphate Buffer (NVB) System: 180 RP-IP HPLC/UV254 using a Volatile Buffer (VB) System:....182 3.3.2 Mass Spectrometric Detection of Nucleosides, Nucleoside Analogues, and their Phosphorylated Derivatives: 182 Direct (Static) Liquid Secondary Ion Monitoring System Quantitative Probe Determinations using a Magnetic-Sector (Kratos Concept®) Mass Spectrometer: 184 sL-SIMS MS of RP-IP H P L C V B Separated Analytes:....184 Electrospray (Ionspray) Mass Spectrometry using a Triple Quadrupole (Fisons VG Quattro®) Mass Spectrometer: 186 Flow-Injection (FI) Electrospray Mass Spectro-metry (ESI MS) of Nucleosides and Nucleoside Analogues using a Single Mass Analyzer: FI-ESI in the Negative-Ion vs. Positive-Ion Mode: 186 FI-ESI- MS (SIR) of Nucleoside Analogue Triphosphates: FI-ESI" MS (SIR) Analysis of PCV-TPN a/xEA Standards: 189 FI-ESI" MS (SIR) Analysis of an A C V - T P N a Standard: 194 Limits of Detection of Nucleotides by FI-ESI- MS (SIR): 199 FI-EST MS (SIR) Determinations of PCV-TP in Cell Extracts. 199 FI-EST Tandem (MS/MS) Analysis. Multiple Reaction Monitoring (MRM): 202 Collision-Induced Dissociation (CID) of Model Nucleotides: 202 The CID of Nucleoside Analogue Tri-phosphates: 205 Parent-Ion Mass Spectra of Nucleoside Analogue Tri-phosphates: .205 FI-EST MS/MS (MRM) Analysis of PCV-TP in SW Cell Extracts: 211 IX Page No. MRM of PCV-TP-Specific Transitions in Relation to HSV moi, tk-Activity, and Parent Drug Concentration: 214 FI-EST MRM Assay Sensiti-vity. Effect of the Extractants and the Pre-Extraction Wash used to Prepare Cell Extracts:.....220 Intracellular Determinations of the Bio-active Metabolites of Nucleoside Anal-ogues using FI-EST MS/MS: .220 Determination of the Intra-cellular Rate of PCV-TP Syn-thesis using FI-EST MS/MS:.....224 Determination of the Intra-cellular Stability (Half-Life) of PCV-TP using FI-ESI" MS/MS: .224 3.3.3 RP-IP H P L C V B with Direct (On-Line) ESI- MS Detection: 227 RP-IP HPLC.VB/UV254/ESI- MS (SIR) Determination of Adenosine and its Nucleotides as Standards in Solution:. .227 RP-IP C - H P L C and N - H P L C V B / E S I - MS (SIR) Determina-tions: 229 RP-IP Mini/Narrow-Bore (m)HPLCvB/ESI- MS/MS of PCV-TP: RP-IP mHPLCvB/ESI- MS/MS Determination of a PCV-TP Standard Solution: 232 RP-IP mHPLCvB/ESI- MS/MS Determination of PCV-TP in Extracts of HSV-Infected/PCV-Treated SW Cells: 232 3.3.4 RP-IP mHPLCvB/ESI- MS/MS Determination of PCV-TP in Extracts of Fluorescence-Activated Cell Sorter (FACS)-Enriched HSV-Infected/PCV-Treated SW Cells: 235 FACS Analysis of HSV-Infected/PCV-Treated SW Cells: 235 Enrichment and Analysis of HSV-Infected/PCV-Treated SW Cells using FACS: 237 FI-ESI- MS/MS Determination of PCV-TP in Extracts of FACS-Enriched HSV-Infected/PCV-Treated SW Cells: .237 RP-IP mHPLCvB/ESI- MS/MS Deter-mination of PCV-TP in Extracts of FACS-Enriched HSV-Infected PCV-Treated Cells: 241 X Page No. 4 Discussion: 4.1 Use of Radioiodinated-IVaraU in the Development of a Herpes-virus fl-Specific Plaque Autoradiography Assay: 243 4.1.1 Plaque Autoradiography of a Diffusable Radioindicator: 245 4.1.2 [125l]-rVaraU-PA: 247 4.1.3 [l25l]-IVaraU-PA/IP: 249 4.1.4 [3H]-Penciclovir Autoradiography: 250 4.2 Use of Radioiodinated-IVaraU-PA/IP as a Method of Demonstrat-ing Herpesvirus Neurotropisms in Primary CNS Neural Cell Cultures: .251 4.2.1 The Composition and In Vitro Properties of Primary CNS Mammalian Neural Cell Cultures: 251 4.2.2 Herpesvirus Infection of Primary CNS Mammalian Neural Cells: 252 4.2.3 Use of [125l]-rVaraU-PA/IP to Demonstrate Herpesvirus in Primary CNS Mammalian Neural Cell Cultures: 257 4.3 Use of Radioiodinated-IVaraU-PA/IP as a Method for Determin-ing the Distribution of Herpesvirus in the CNS of a Rabbit Model of HSE: .259 4.3.1 Neuropathology of the Intranasally-Infected Rabbit Model of HSE: 260 Intrathecal Delivery of [125I]-IVaraU into an Intranasally- , infected Rabbit Model of HSE: 268 Distribution of Herpesvirus ffc-Activity in the CNS of Intranasally-Infected Rabbit Models of HSE as determined by [125i]-rVaraU-TA: 269 Distribution of Herpesvirus Specific Antigens in the CNS of an Intranasally-Infected Rabbit Model of HSE: 272 4.3.2 [!25i]-rVaraU in the CNS of an Intranasally-Infected Rabbit Model of HSE Determined using External Gamma Camera Scintigraphy: 275 4.4 On-Line High-Performance Liquid Chromatography/Mass Spectrometry for the Ultratrace Determination of the Intracellular Levels of Nucleoside Analogues and their Phosphorylated Metabolites: .278 4.4.1 Reverse-Phase Ion-Pair (RP-IP) HPLC of Nucleosides, Nucleo-side Analogues and their Phosphorylated Derivatives: 279 Reconstruction of a Conventional (Non-Volatile/Phos-phate) Buffer System for RP-IP HPLC: 279 xi Page No. Development of a Volatile Buffer System Suitable for On-Line Mass Spectrometric Determinations of Nucleo-tide Standards and their Antiviral Homologues: 282 4.4.2 Mass Spectrometric Analysis of Nucleosides, Nucleoside Analogues and their Phosphorylated Derivatives: sL-SIMS MS Analysis of a RP-IP HPLCyB-Fractionated PCV-TP Standard: 284 Electrospray (Ionspray) Ionization Mass Spectrometry (ESI MS) of Nucleosides, Nucleotides and their Anti-viral Homologues: 285 ESI MS (Single Ion Recordings) of Nucleoside and Nucleotide Standards: ESI MS in the Positive-Ion Mode: 287 ESI MS in the Negative-Ion Mode: 288 Dissociation Pathway of Nucleosides, Nucleotides and their Antiviral Homo-logues: 290 ESI MS of Nucleoside Analogue Standards and their Phosphorylated Derivatives: 294 Discriminating Nucleotides and their Antiviral Homologues in Cell Extracts Using ESI Tandem Mass Spectrometry: 297 ESI MS/MS (Multiple Reaction Monitoring) Profiles of Nucleotides and Nucleotide-Analogue Standards and in Drug-treated Herpesvirus-Infected S W Cell Extracts: .298 Pharmacokinetics of PCV-TP in Drug-treated/Herpesvirus-Infected SW Cell Extracts as Determined by ESI MS/MS: .302 4.4.3 RP-IP HPLC/ESI MS (and MS/MS) of Nucleosides, Nucleotides and their Antiviral Homologues: 310 4.4.4 Demonstrating Penciclovir Triphosphate in Extracts Prepared from Immunocytologically-Defined/FACS-Enriched (HSV-Infected) Cells using RP-IP HPLC/ESI MS MS: 311 4.5 Concluding Remarks: 318 Bibliography: 323 xii List of Abbreviations: 5-FU: 5-Fluorouracil A: Adenine; 6-amiopurine ACV: Acyclovir/Zovirax®; 9-(2-hydroxyethoxymethyl)guanine ACV-TP: Acyclovir-triphosphate Ado: Adenosine; 9-/J-D-ribofuranosyl-adenine ADP: Adenosine 5'-diphosphate AIDS: Acquired immunodeficiency syndrome AMP-PCP: Adenylyl-(/3, ^methylene)-diphosphate AMP: Adenylic acid; Adenosine 5'-monophosphate amu: Atomic mass units AON: Anterior olfactory nucleus APcI: Atmospheric pressure chemical ionization API: Atmospheric pressure ionization Ar: Argon ara-A: Vidarabine; 9-/J-D-Arabino-furanosyladenine ara-C: Cytarabine AST: Astrocytes ATCC: American Type Tissue Collection ATP: Adenosine 5'-triphosphate AU: Absorbance units AUFS: Absorbance units full scale AZT: Zidovudine/Retrovir®; 3'-azido-2',3'-dideoxythimidine [B+2H]+: Quasimolecular/protonated nucleobase in positive ion mode [B]": Quasimolecular/deprotonated nucleobase in negative ion mode BBB: Blood-brain-barrier BCV: Buciclovir; 9-(3,4-dihydroxybutyl)-guanine BDU: 5-Bromo-2'-deoxyuridine BDV: Borna disease virus Bq: Bequerels BSA: Bovine serum albumin Buffer A N V B : Non volatile buffer A (5mM KH 2 P0 4 , ImM Q7) Buffer Ayg: Volatile buffer A (5mM C H 5 N 0 2 ImM DMHA) Buffer B N V B : Non volatile buffer B (15mM KH 2 P0 4 , ImM Q 7 70%MeOH) Buffer Byg: Volatile buffer B (15mM CH5NO2, ImM DMHA 70%MeOH) BVaraU: Sorivudine/Brovavir; 1-/J-D-arabinofuranosyl-£-5-(2-bromovinyl) uracil BVDU: Brivudin; 5-(£)-(2-Bromovinyl)-2'-deoxyuridine BVU: 5-(2-Bromo-E-ethenyl)uracil Cj8 (ODS): Octadecylsilane C8(OS): Octylsilane C: Cytosine; 4-Amino-2-hydroxy-pyrimidine CBV: Carbovir; Carbocyclic 2',3'-dide-hydro-2',3'-dideoxy guanosine CDP: Cytidine 5'-diphosphate CDS: Chemical delivery system CE: Collision energy xiii C E D U : 5-(2-Chloroethyl)-2'-deoxyuridine cf: Continuous flow C G E : Capillary gel electrophoresis cHPLC: Capillary-bore H P L C CI: Chemical ionization CID: Collision induced dissociation C M " : Calcium/Magnesium-free Maximum concentration CMI: Cell mediated immunity CMP: Cytidylic acid; Cytidine 5'-mono-phosphate CNPase: 2',3'-cyclic nucleotide 3'-phosphodiesterase CNS: Central nervous system CoA: Anterior cortical nucleus of the amygdala CPE: Cytopathic effect CRBC: Glutaraldehyde-fixed chicken red blood cells CSF: Cerebrospinal fluid CTP: Cytidine 5'-triphosphate CV: Capillary voltage Cyd: Cytidine; cytosine j3-D-riboside C Z E : Capillary zone electrophoresis D j : Intermediate detector d4T: Stavudine/Zerit®; l-(2,3-Dide-hydro-3-deoxy-/3-D-eryf7zro-pento-furanosyl)thymine D: Deuterated D A B : Diaminobenzidine dd: Double-distilled/deionized ddA: 2',3'-dideoxy-9-/3-ribofuranosyl-9H-purine-6-amine ddC 2',3'-dideoxycytidine ddl: 2',3'-dideoxyinosine DHPG: GCV/Ganciclovir; (9-(l,3-di-hydroxy-2-propoxymethyl)guanine DI- (FI- /LI-): Direct-injection (flow injec-tion / loop injection) DIV: Days in vitro; D L E A : Lateral division entorhinal area D M H A : N,N-Dimethylhexylamine D N A : Deoxyribonucleic acid DNAPol+/Po1-. Herpesvirus D N A poly-merase (competent/deficient) DPC: Dorsal peduncular cortex DPI: Days post-infection dThd: Thymidine; l-(2-deoxy-jf3-D-ribo-furanosyl)-5-methyluracil dTTP: Thymidine 5'-triphosphate EBV: Epstein-Barr virus EDU: Ethyldeoxyuridine EHDI: Electrohydrodynamic ionization EI: Electron impact E M : End Mass ES MS: Electrospray mass spectrometry ES: Exanthema Subitum ESI: Electrospray (ionspray) ionization EtOHr: Reagent ethanol FAB: Fast atom bombardment FACS: Fluorescence activated cell sorter FALS: Forward angle light scatter FB: Fibroblasts FBS: Fetal bovine serum FCV: Famciclovir; 2-amino-9-(4-acetoxy-3-[acetoxymethyl]but-l-yl)purine FD: Field desorption xiv F H P G : 9-(3-Fluoro-l-hydroxy-2-propoxy-methy 1) guanine FIAU: Fialuridine; 2'-fluoro-2'deoxy-l-j3-D-arabinofuranosyl-5-iodouracil FITC: Fluorescein isothiocyanate F M A U : 2'-fluoro-5-methyl-l-j3-D-ara-binosyluracil F N : Fibronectin FW: Formula weight G: Guanine; 2-amino-6-hydroxypurine G C : Gas chromatography G C V : DHPG/Ganciclovir; 9-(l,3-di-hydroxy-2-propoxymethyl)guanine GDP: Guanosine 5'-diphosphate G F A P : Glial Fibrillary Acidic Protein GIBCO: Grand Island Biologicals Co. G M P : Guanosine 5'-monophosphate (guanylic acid) GTP: Guanosine 5'-triphosphate Guo: Guanosine; 9-/J-D-ribofuranosyl-guanine HBSS: Hank's balanced salt solution HBV: Hepatitis B virus H C M V : Human cytomegalovirus H E L F : Human embryonic lung fibroblast HEPES: N-hydroxyethyl piperazine-N'-2-ethanesulfonic acid HFF: Human foreskin fibroblasts HHV-(l-8): Human herpesvirus (1-8) HI: Heat-inactivated HIV: Human immunodeficiency virus H M resolution: High mass resolution H P L C : High-performance liquid chromatography H P M P A : (S)-9-(3-hydroxy-2-phosphonyl-methyoxypropyl) adenine H P M P C : Cidofovir/Vistide; (S)-9-(3-hydroxy-2-phosphonylmethyoxy-propyl)cytosine HSE: Herpes simplex encephalitis HSV: Herpes simplex virus (type-1 -2) H V : High voltage lens I C A M j : Cell adhesion molecule ID: Internal diameter IdC: 5-Iodo 2'-deoxycytidine IDU: Idoxuridine Ie: Ion energy IE: Ion-exchange IF: Immunofluorescence IL: Interleukin INF: Interferon IP: Immunoperoxidase IPA: isopropanol IVaraU: l-/J-D-Arabinofuranosyl-E-5-(2-iodovinyl)uracil IVDU: 5-(E)-(2-Iodovinyl)-2'-deoxy-uridine rVTaraU: (E)-5-(2-iodo-vinyl)-l-(2-deoxy-2-fluoro-j3-D-arabinofuranosyl)uracil IVFRU: (E)-5-(2-iodovinyl)-l-(2-deoxy-2-fluoro-/3-D-ribofuranosyl)uracil k': Column capacity factor Ki: Inhibition kinetics constant K S H V : Kaposi's sarcoma-associated herpes virus L A T : Latency-associated transcript L M resolution : Low mass resolution LTR: Long terminal repeat xv [M+H]+: Protonated quasimolecular form of the analyte [M-H] -: Deprotonated quasimolecular form of the analyte m/z: Mass to charge ratio M E M : Eagle's minimal essential media mHPLC: Minibore HPLC LiHPLC: Microbore HPLC moi: Multiplicity of infection MRM: Multiple reaction monitoring MS/MS: Tandem mass spectrometry MSj+2: Mass analyzers ( 1 and 2 ) MS: Mass spectrometry _MS: Negative ion mode MS + M S : Positive ion mode MS Ni/2'- Nominal theoretical plate number NDP: Nucleotide diphosphate N F 6 8 : Neurofilaments ng: Nanogram NGF: Nerve growth factor NGS: Normal goat serum nHPLC: Nano-bore HPLC NK: Natural killer NLOT: Lateral olfactory tract NTP: Nucleotide triphosphate OB: Olfactory bulb OD: Optical density OD: Outer diameter ODS (C 1 8): Octadecylsilane OL: Oligodendrocytes ORF: Open reading frame OS(C 8): Octylsilane OT: Olfactory tubercle PA: Plaque autoradiography PAA: Phosphonoacetic acid PAC: Periamygdaloid cortex PAGE: Poly aery lamide gel electrophoresis PBS: Phosphate buffered saline PC^: Anterior piriform cortex PCp: Posterior piriform cortex PCR: Polymerase chain reaction PCV: Penciclovir; 9-(4-hydroxy-3-hydroxymethylbut-l-yl)guanine P C V - T P N a : Sodium salt of PCV-TP P C V - T P T E A : Triethylamine salt of PCV-TP pEC: Pseudoelectrochromatography PET: Positron emission tomography PFA: Foscarnet; Phosphonoformic acid PFC: Plaque forming cell PFU: Plaque forming unit pg: Picogram PHN: Postherpetic neuralgia PMT: Photomultiplier tube PNS: Peripheral nervous system ppb: Part per billion (ultratrace) ppm: Part per million (trace) ppt: Part per trillion (ultratrace) PRK: Primary rabbit kidney cells psi: Pounds per square inch PVA: Polyvinyl alcohol Qi: Single/first quadrupole Q 2 : Collision cell Q 3 : Third quadrupole Q 7 : Heptyltriethylammonium phosphate Q: Quadrupole RA: Radiometric assay xvi RD: Radiometric detection RE: Vacuum-assisted rotary evaporator RITC: Rhodamine isothiocyanate RNA: Ribonucleic acid RP-IP: Reverse-phase ion-pair RP: Reverse-phase RT: Retention time sL-SIMS: Static liquid-secondary ioniza-tion Base+[C2H30] (+42 amu) S2: Base+[CHO]) (+28 amu) SIR: Single Ion Recording SM: Start Mass Sp Act: Specific activity SPE: Solid-phase extraction SPECT: Single-photon emission tomo-graphy SW cell: Schwannoma cell tj/2- Elimination half-life T: Thymine; 2,4-dihydroxy-5-methyl-py rimidine / 5-methy lur acil TA: Tissue autoradiography TCID 5 0: Infectious dose 50 TDP: Thymidine 5-diphosphate TFA: Trifluoroacetate TFT: Trifluorodeoxythymidine tk+/tk-: Herpesvirus thymidine kinase (competent/ deficient) TK^: Cellular (cytosolic) thymidine kinase TK2: Cellular (mitochondrial) thymidine kinase TLC MS: Thin-layer chromatography mass spectrometry TMP: Thymidylic acid; Thymidine 5'-monophosphate TMS: Trimetylsilylation TNF: Tumor necrosis factor TSP: Thermospray ionization TTP: Deoxythymidine triphosphate TT V : Ventral aspect of the tenia tecta U: Uracil; 2,4-dihydroxypyrimidine UDP: Uridine 5'-diphosphate UL97: HCMV-specified cytoplasmic phosphotransferase UMP: Uridylic acid; Uridine 5'-mono-phosphate Urd: Uridine; 9-/3-D-ribofuranosyl-uracil USFDA: United States Food and Drug Administration UTP: Uridine 5'-triphosphate UV: Ultraviolet UV: Ultraviolet HPLCUV254 Valaciclovir: 2-[(2-amino-l,6-dihydro-6-oxo-9H-purin-9-yl)methoxy]ethyl L-valinate hydrochloride VB: volatile buffer VERO: African green monkey kidney cells VLEA: Ventral division entorhinal area VTM: Virus transport media VZV: Varicella-zoster virus w/v: Weight per volume w/w: Weight per weight WGA-HRP: Wheat germ agglutinin-horseradish peroxidase xvii List of Tables: Page No. Table 2.1: RP C - H P L C and N - H P L C column description and determination of nominal efficiencies: 123 Table 3.1a: Discrimination of thymidine kinase-deficient H S V type-1 by [l25i]-iVaraU plaque autoradiography (PA) in combination with H S V type-l-specific (IP) antigen expression: 149 Table 3.1b: Uptake of [125i]-iVaraU by herpesvirus-infected P e r c o l ® - e n r i c h e d Lapine neural cells: 149 Table 3.2: Summary of the immunohistopathology and [!25i]-iVaraU tissue autoradiography in a rabbit HSE model: 167 Table 3.3: Comparison of volatile and non-volatile buffers for the reverse-phase ion-pair H P L C of select nucleosides, nucleoside analogues and their nucleotides: 181 Table 3.4: Static liquid secondary ion monitoring system (sL-SIMS) analysis of a penciclovir triphosphate aqueous standard: 185 Table 3.5a: Limits of detection of adenosine-triphosphate using flow-injection electrospray ionization mass spectrometry: 201 Table 3.5b: Limits of detection of penciclovir-triphosphate using flow-injection electrospray ionization mass spectrometry: 201 Table 3.6: Electrospray ionization (MRM) of PCV-treated Schwannoma cell extracts from cells infected with thymidine kinase (f/c)-wild type and fi:-deficient herpesvirus: 219 Table 3.7: Relative ESI M S / M S intensities of PCV-TP-specific transitions obtained from Herspevirus-infected/PCV-treated SW cells with the use of various extractants: 221 xviii List of Figures: Page No. Fig. 1.1: Taxonomy of the Herpesviridae: 2 Fig. 1.2: Schematic representation of a herpesvirus: 4 Fig. 1.3: The salvage and de novo pathway in the biosynthesis of thymidine nucleotides: 7 Fig. 1.4: Nucleoside analogues demonstrating anti-herpesvirus activity: 35 Fig. 1.5 Mechanism of action of iododeoxycytidine (ITJCyd), and iododeoxy-uridine (IDU): 37 Fig. 1.6 Mechanism of action of iodovinyl- and bromovinyl-deoxyuridine 39 Fig. 1.7 Mechanism of action of bromo- and iodo-vinylarabinofuranosyl-uracils: _ 42 Fig. 1.8 Mechanism of action of vidarabine (ara-A): 45 Fig. 1.9 Mechanism of action of acyclovir (and valacyclovir): 47 Fig. 1.10 Uptake and phosphorylation (bioactivation) of penciclovir in Herpes Simplex Virus-infected Schwannoma cells:, 52 Fig. 2.1 Preparation of Percoll-enriched mixed neural (glial) primary cell cultures: 99 Fig. 2.2 Synthesis of no-carrier-added [*I]-IVaraU: 109 Fig. 2.3 [*I]-IVaraU plaque autoradiography of thymidine kinase-positive herpesvirus-infected cells: 112 Fig. 2.4 Ultratrace determination of nucleoside analogues and their metabolites in neural-cell extracts using electrospray ionization tandem mass spectrometry: 114 Fig. 2.5 [*I]-IVaraU uptake in a rabbit model of Herpes Simplex Encephalitis:! 16 Fig. 2.6 Preparation of reverse-phase microbore capillary HPLC columns:. 121 Fig. 2.7 Preparative reverse-phase high-performance liquid chromato-graphy for the separation of radioiodinated IVaraU from unlabeled BVaraU: 126 Fig. 2.8 Ion optics of a triple quadrupole tandem mass spectrometer: 130 Fig. 2.9 PCV-TP in FACS-enriched HSV 1-infected neural cells determined using RP-IP HPLC/ESI-MS/MS: 133 Fig. 3.1 Photomicrograph of herpesvirus-infected SW cells: 143 xix Page No. Fig. 3.3 [l25i]-iVaraU plaque autoradiography of an HSV-type-1-infected cell monolayers: 145 Fig. 3.4 [125i]-iVaraU plaque autoradiography and immunocytochemistry of Varicella-Zoster Virus-infected cell monolayers: 147 Fig. 3.5 Immunocytochemistry and [125i]-rVaraU plaque autoradiography of Varicella-Zoster Virus (ffc")-infected cell monolayers: 148 Fig. 3.6 Comparison of image densities generated from [125i]-iVaraU plaque autoradiography and immunocytochemistry: 150 Fig. 3.7 [l25i]-iVaraU-PA determination of thymidine kinase (tk) positive herpesvirus in experimentally reconstructed populations of tk-positive and -negative HSV. _ 152 Fig. 3.8 [3H]-PCV autoradiography of VZV-infected cell monolayers: 153 Fig. 3.9 Photomicrograph of fluorescent antibody (CNPase+/FITC)-labeled freshly prepared Percoll-enriched oligodendrocytes: 155 Fig. 3.10 Photomicrograph of Percoll-enriched mixed glial cell cultures: 156 Fig. 3.11 Photomicrograph of fluorescent antibody (GFAP/FITC)-labeled astrocytes in mixed glial cultures: 157 Fig. 3.12 Photomicrograph of immunoperoxidase-labeled CNPase+ oligo-dendrocytes in mixed glial cultures: 158 Fig. 3.13 Photomicrograph of immunoperoxidase-labeled (GFAP+) astrocytes in a large aggregate of oligodendrocytes: 160 Fig. 3.14 Phase-contrast photomicrographs of herpesvirus infected (and uninfected) mixed glial cell culture: 161 Fig. 3.15 Photomicrograph of immunoperoxidase (HSV+)-labeled HSV type-1 and type-2-infected glial cultures: 162 Fig. 3.16 Photomicrograph of fluorescent antibody (GFAP/FITC)-labeled H S V type-1 and type-2-infected astrocytes in mixed glial cultures: 163 Fig. 3.17 [125i]-iVaraU plaque autoradiography of herpesvirus-infected Lapine oligodendrocytes: _ 165 Fig. 3.18 Gross pathology of the left-side temporal lobe of a rabbit Herpes Simplex Encephalitis model: _ 168 Fig. 3.19 [125i]-lVaraU uptake in a rabbit Herpes Simplex Encephalitis model:__170 Fig. 3.20 Digitized high-contrast X-ray film images of the differential uptake of [l25i]-iVaraU (left vs. right hemispheres) in fresh-frozen/air-dried (7 L i m ) serial sections of rabbit HSE olfactory bulbs: 172 XX Page No. Fig. 3.21 Comparative optical densities of tissue autoradiographs prepared from HSV type-1-infected [!25l]-lVaraU-treated rabbit CNS cryostat sections: 173 Fig. 3.22 Photomicrograph of a histological thin section of brain tissue of a rabbit Herpes Simplex Encepnalitis model demonstrating micro-pathology (perivascular cuffing): 174 Fig. 3.23 Photomicrographs of immunohistologically stained (HSV/IP+)-positive thin sections of olfactory bulbs and pre-frontal brain regions of a rabbit model of Herpes Simplex Encepnalitis: 175 Fig. 3.24 Photomicrograph of an immunohistologically stained (HSV/IP +)-positive thin-section of a temporal lobe of a rabbit model of Herpes Simplex Encephalitis: 176 Fig. 3.25 Photomicrograph of a fresh-frozen thin (7 urn) section of olfactory bulb infected with Herpes Simplex Virus type-1 (KOS-SB) tk-deletion mutant in a rabbit model of HSE: 178 Fig. 3.26 [!3ll]-lVaraU/external gamma-camera image of a rabbit model of HSE: 179 Fig. 3.27 Reverse-phase ion-pair high-performance liquid chromatography of Eenciclovir and its phosphorylated derivatives using a volatile uffer system: 183 Fig. 3.28 RP-IP HPLC of penciclovir standard and its derivatives. Concentrated fractions used for sL-SIMS analysis: 187 Fig. 3.29 Direct static liquid secondary-ion monitoring system (sL -SIMS) mass spectrometric detection of penciclovir-diphosphate following fractionation by R P - I P H P L C V B : 188 Fig. 3.30 Scanning electrospray ionization of penciclovir and two model nucleosides in the positive ion-mode: 190 Fig. 3.31 Scanning electrospray ionization of adenosine and guanosine in the negative-ion mode: 191 Fig. 3.32 Scanning electrospray ionization of penciclovir in the negative-ion mode: 192 Fig. 3.33 Scanning electrospray ionization and collision-induced dissociation of penciclovir triphosphate in the negative-ion mode: 193 Fig. 3.34 Scanning ESI" demonstrating the presence of the ion m/z 984.3 in a standard solution of penciclovir triphosphate 195 Fig. 3.35 Scanning ESI" of penciclovir triphosphate also demonstrating parent-ion and daugnter-ion spectra: 196 xxi Page No. Fig. 3.36 Parents of m/z 492 (PCV-TP) in a concentrated standard solution: 197 Fig. 3.37 Collision-induced dissociation of acyclovir triphosphate in the negative-ion mode: 198 Fig. 3.38 Determination of the limits of detection of adenosine triphosphate by SIR EST in the negative ion mode: 200 Fig. 3.39 Flow-injection electrospray ionization mass spectrometric detection of PCV and its phosphorylated derivatives in Schwannoma cell extracts: .203 Fig. 3.40 Scanning (MSi) ESI" of HSV type-l-infected/PCV-treated SW cell extract: .204 Fig. 3.41 Collision-induced dissociation of nucleotides demonstrating a m/z 78 and m/z 158 transition: .206 Fig. 3.42 Collision-induced dissociation of acyclovir triphosphate in the positive-ion mode: .207 Fig. 3.43 Collision-induced dissociation of penciclovir triphosphate in the positive-ion mode: 208 Fig. 3.44 Parents of the transitions m/z 158 and m/z 78 from the CID of acyclovir triphosphate in the negative-ion mode: _ 209 Fig. 3.45 Parents of the transitions m/z 158 and m/z 78 from the CID of penciclovir triphosphate in the negative-ion mode: .210 Fig. 3.46 Parents of m/z 158 and m/z 78 in cell extracts prepared from HSV type-l-infected/PCV-treated SW cells: 212 Fig. 3.47 Flow-injection electrospray ionization (ionspray) tandem mass spectrometry (FI-EST MS/MS) of Schwannoma cell extracts: .213 Fig. 3.48 Extraction of PCV-TP from HSV type-l-infected Schwannoma cells using various solvents: .215 Fig. 3.49 Flow-injection ESI" (MS/MS) of ACV-treated and PCV-treated uninfected SW cell extracts: 216 Fig. 3.50 Multiple reaction monitoring (MRM) of the transition m/z 984»*m/z 491 (PCV-TP dimer) in HSV type-l-infected/PCV-treated SW cell extracts: 217 Fig. 3.51 Flow-injection electrospray ionization (ionspray) tandem mass spectrometric detection of (FI-EST MS/MS) of HSV type-l-infected/PCV-treated Schwannoma cell extracts: _ .218 Fig. 3.52 MRM of the m/z 492»*m/z 158 transition (PCV-TP specific) in VZV-infected/PCV-treated SW cell extracts: Volatile buffer vs. non-volatile buffer pre-extraction wash: 222 xxii Page No. Fig. 3.53 Comparison of the PCV-TP specific transition (m/z 492»*m/z 158) and ACV-TP specific transition (m/z464ll*m/z 158) in VZV-infected drug treated SW cell extracts: .223 Fig. 3.54 Rate of synthesis of PCV-TP in HSV type-l-infected/PCV-treated SW cells determined using EST (MS/MS):. 225 Fig. 3.55 Intracellular pharmacokinetics (formation and stability) of PCV-TP in HSV type-1-infected PCV-treated SW cells determined using ESI-(MS/MS): 226 Fig. 3.56 RP-IP HPLC of Adenosine and its phosphorylated derivatives detected using UV254 absorption and SIR (EST) MS: 228 Fig. 3.57 RP-IP HPLC of Adenosine and its phosphorylated derivatives by SIR (ESL) mass spectrometry at the limit of detection by UV254: 230 Fig. 3.58 Representative UV254 absorption profile of an in-house manufact-ured reverse-phase capillary-HPLC column: _ .231 Fig. 3.59 Reverse-phase ion-pair HPLC of the phosphorylated derivatives of PCV standards detected with MRM (EST) MS: .233 Fig. 3.60 RP-IP HPLC/MRM (EST) MS determination of PCV-TP in PCV-treated/HSV type 1-infected SW cell extracts: 234 Fig. 3.61 Determination of positively immunolabeled HSV type-l-infected and uninfected/PCV-treated Schwannoma cells Fluorescence Act-ivated Cell Sorter: 236. Fig. 3.62 Fluorescence-Activated Cell Sorter (FACS) analysis of HSV type-l-infected Schwannoma cells: .238 Fig. 3.63 Photomicrographs of experimentally reconstructed populations of herpesvirus-infected and uninfected, and Fluorescence-Activated Cell Sorter (FACS)-enriched Schwannoma cells: 239 Fig. 3.64 Ion-suppression by inorganic salts in cell sample extracts of FACS-enriched HSV-1 infected/PCV-treated SW cells analyzed by flow-injection ESI" MS/MS: .240 Fig. 3.65 Detection of PCV-TP in extracts of FACS-enriched (HSV type-l-infected)/PCV-treated SW cells by RP-IP HPLC/EST MS-MS: .242 xxiii Acknowledgments: The present study would not have been possible had it not been for the considerable guidance and support given to me by a number of individuals. I am deeply indebted to my research supervisor Dr. Stephen Sacks (Pharmacology), both, for giving me the opportunity to become involved in his research project and for the very generous financial support that I was provided with over the majority of these studies. I also thank him for his patience, understanding and friendship when times were difficult. I am also especially thankful to my co-supervisor Dr. Frank Abbott (Pharmaceutical Sciences), for his guidance, friendship and unwavering confidence not only in the project but in my ability to follow this work to its successful completion. I most gratefully acknowledge my supervisory committee (Dr. Lome Kastrukoff [Medicine/Neurology], Dr. Max Cynader [Surgery/Ophthalmology], and Dr. Richard Wall [Pharmacology]) for always being available, for their expert instruction and for allowing me unrestricted access to their resources and facilities. I would also like to acknowledge my sincerest appreciation to my Graduate Advisor Dr. Ismahil Laher and by my Department Head Dr David Godin for the considerable efforts that they made on my behalf. Special thanks to Dr. Guenther Eigendorf (Chemistry) and Mr. Roland Burton (Pharmacy) for their expert instruction on the theory and use of the mass spectrometer, to Tariq Aziz in helping me in any and ever way possible and to Dr. Norman Wong for his continuous support and helpful discussion throughout the duration of my studies. Finally, I would also like to express my heartfelt thanks to Tassie Antonopoulous, George Andersen and Robert McRae for their continued support and most helpful assistance. xxiv To my beloved family To my darling children Samantha and Natasha, my loving wife Kosovka and my dearest Mother Despina. In loving memory of my brother Alexander Does the road wind up-hill all the way? Yes, to the very end. Will the day's journey take the whole long day? From morn to night, my friend Christina Rossetti xxv 1 Introduction: 1.1 Taxonomy of the Herpesviridae: Few pathogens demonstrate a wider distribution in nature or hold greater clinical significance than the viruses which constitute the family Herpesviridae*. Taxonomic data compiled by Roizmanef. al. in the 6th International Committee on Taxonomy of Viruses Report describes three subfamilies (alpha-, beta- and gamma-herpesvirinae) along with 49, as yet, unassigned viruses that make up the Herpesviridae. Each subfamily, in turn, is comprised of the genus Simplexvirus, Varicellovirus and the unassigned (and tentatively assigned) viruses of the genus and of the species; the genus Cytomegalovirus, Muromegalovirus, Roseolovirus and the unassigned viruses of the genus; and finally, the genus Lymphocryp to virus, Rhadinovirus and the unassigned viruses of the genus, respectively (Fig. 1.1). Of the more than one hundred different species of virus which make up the Herpesviridae, nine are currently recognized as a cause of disease in humans (Chen-Osmond and Dallwit, 1996). The human herpesviruses include herpes simplex virus type-1 (HSV type-1) and type-2 (HSV type-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (HCMV), human herpesvirus-6, human herpesvirus-7, and Kaposi's sarcoma-associated herpes virus (KSHV); human herpesvirus (HHV)-l to HHV-8, respectively. Occasionally, although particularly rare, the simian herpes B virus (a "non-human" herpesvirus) has also been the cause of human illness (Whitley and Schlitt 1991). Although most human herpesvirus infections present as clinically benign, any member of this family of virus has the potential for producing serious, even life-threatening, disease. 1.2 Physical Characteristics: Inclusion into the family Herpesviridae, by definition, is based on a combination of biological and architectural criteria (Roizman 1990). The viruses of this family tend to share many physical and biological properties, and are morphologically indistinguishable from one another under the electron microscope (Pilling et al. 1996). Although some degree of antigenic cross-reactivity has been shown to exist between at least some of the herpesviruses (Balachandran et al. 1987), the extent of this cross-reactivity (with the possible exception of HSV-1 and HSV-2 [Fujinaga et al. 1987], and HHV 6 and HHV-7 [Neipel et al. 1996]), tends to be + The term herpes is derived from the Greek word, herpein, meaning to spread or creep. 1 rather inconsiderable (Pango and Lemon 1980; Balachandran et al. 1987). The use of immunological or even molecular techniques (Sec. 1.6) are, therefore, often necessary to unambiguously distinguish between the herpesvirus species found in a clinical specimen or between HHV strains in populations which have been experimentally constructed (Whitley and Schlitt 1991). Having a diameter in the range of 150 to 250 nm, the herpesviruses are among some of the largest known viruses (Straus 1990). The mature virion (Fig. 1.2) is comprised of a central core (or nucleoid), containing protein and a linear double-stranded DNA genome. Herpesvirus nucleic acid ranges in molecular weight from between 90x106 Daltons (Epstein Barr virus) to 150x106 Daltons (cytomegalovirus) (Pango and Lemon 1980). The viral genome is approximately 120 kilobases to 230 kilobases in length, and encodes for over 70 proteins (Whitley and Schlitt 1991). The inner core of the virion is surrounded by an icosadeltahedral nucleocapsid consisting of 162 symmetrical/repeating capsomeric protein subunits. Trans-capsomeric channels of the nucleocapsid are believed to play an important role in the transport of genomic DNA and scaffolding proteins during capsid morphogenesis (Zhou 1996). The capsomere is surrounded by, and tightly associated to, an amorphous, sometimes asymmetric proteinaceous material called the tegument. The entire structure is finally enveloped within a trilaminar outer-lipid structure of modified host-cell membrane. The viral envelope is encrusted with virus-encoded glycoprotein protrusions or spikes, which, when deposited into the host-cell membrane, are incorporated into the envelope of the mature virion as it egresses the host. These glycoprotein spikes are believed to be important in the process of attachment and penetration of the herpesviruses. They are also believed to function as Fc receptors, and have been shown to bind complement as well (Straus 1990). 1.3 Replicative Cycle and Temporal Sequence of Gene Expression: A herpesvirus infection is initiated with the recognition and attachment of the virus to the host-cell's plasmalemma-associated heparan sulfate proteoglycan receptor molecule. Attachment is rapidly followed by fusion of the viral envelope with the plasmalemma and the subsequent release of the viral nucleocapsid into the host cytoplasm. Capsids are then transported across the cytosol to the nuclear pore complex by a mechanism mediated by the cytoskeleton (microtubules; Sodeik et al. 1996). The capsids then uncoat and the DNA genome is released into the cellular nucleoplasm. Once inside the nucleus, the viral genome circularizes and progeny 3 Figure 1.2: Schematic representation of a Herpesvirus • T h e h e r p e s v i r u s e s a r e e n v e l o p e d i n a s t r u c t u r e d e r i v e d l a r g e l y f r o m h o s t - c e l l m e m b r a n e s . • T h e v i r a l e n v e l o p e is e n c r u s t e d w i t h v i r u s - e n c o d e d g l y c o p r o t e i n s p i k e s . • B e t w e e n t h e o u t e r e n v e l o p e a n d t h e n u c l e o c a p s i d is t h e t e g u m e n t . • T h e i c o s a d e l t a h e d r a l n u c l e o c a p s i d c o n s i s t s o f 1 6 2 s y m e t r i c a l r e p e a t i n g c a p s o m e r i c p r o t e i n s u b u n i t s . • T h e c e n t r a l c o r e c o n t a i n s v i r a l p r o t e i n s a n d t h e l i n e a r d o u b l e - s t r a n d e d D N A g e n o m e . • T h e v i r a l g e n o m e is 1 2 0 t o 2 3 0 k i l o -b a s e s i n l e n g t h a n d e n c o d e s f o r as m a n y as 70 ( + ) v i r a l p r o t e i n s . 4 DNA molecules are synthesized through a "rolling-circle" mechanism (Ben-Portat and Tokazewski 1977). Temporal expression of the immediate-early, early and late genes (perhaps, more appropriately designated as a, (3 [f5\ and /%] and y [yi and 72] genes) can then take place (Honnes and Roizman 1972; Wolf et al. 1982). The immediate-early (a) gene products are considered essential for the replication of the herpesviruses. Immediate-early gene expression, instituted by a protein constituent of the tegument, is responsible for down-regulating host cell macromolecular synthesis, as well as for inducing the subsequent transcriptional activation of the ft- and 7-genes. Immediate-early products are also believed to play an important role in the mechanism of latency. Early (/?i and P2) gene expression has been shown to coincide with, and to possibly contribute to, the shut-off of host cell protein synthesis. /J-gene expression precedes, and is largely associated with, the replication of the viral genome. The late (^ J-gene products (which are, for the most part, structural) tend to be expressed only after DNA synthesis has terminated. Gamma gene products are incorporated, or may assist in, the final assembly of progeny virus (Piette et al. 1995). Herpesvirus DNA is synthesized as a multimer within the host's nucleus, and then cleaved and eventually packaged into newly formed capsids. Mature virions are produced as they bud off from the inner surface of the nucleolemma and are transported through the cytoskeleton to the plasma membrane where they are finally released (Roizman and Sears 1991). The generation of infectious progeny (in such a "productive" infection) invariably results in the destruction of the infected host cell (Roizman 1991). 1.4 Clinically Important Herpesvirus-Encoded Enzymes: 02 gene expression is of special pharmacological consequence as these genes specify enzymes (including thymidine kinase, thymidylate kinase and DNA polymerase) which are central to the metabolism and synthesis of herpesvirus nucleic acid and, as such, are frequently targeted by what have proven to be the more clinically effective nucleoside analogue-class of anti-herpesvirus agents. 1.4.1 Herpesvirus Thymidine Kinase: Cellular and virus-encoded thymidine kinases (tk) are enzymes that are central to the biosynthesis of nucleotides and, therefore, to the formation of nucleic 5 acid. Nucleotides can be synthesized from newly formed purine and pyrimidine bases (denovo synthesis), as well as from the re-use of free bases recovered from the degradation of pre-existing nucleic acid materials, also known as the "salvage" path-way (Fig. 1.3). Thymidine kinases are one of the principal enzymes associated with the nucleotide salvage pathway (Falke et al. 1979; Arner et al. 1992; Perigaud et al. 1992). Human cells and the herpesviruses are both able to reclaim pyrimidine deoxyribonucleosides through a process of 5'-phosphorylation, a process which is also the major route in the bioactivation of several chemotherapeutic nucleoside analogues. In human cells, the key enzymes responsible for this activity are the cytosolic thymidine kinase (TKi), the mitochondrial thymidine kinase (TK2) as well as a cytosolic deoxycytidine kinase (Arner et al. 1992). To a large extent, human TKi (and possibly TK2) resemble the enzymes of all eukaryotes, the poxviruses (i.e. vaccinia virus), and those of the bacteria (Black and Hruby 1990; Gentry 1992). The thymidine kinase specified by the herpesviruses (once believed to be wholly unique; Cheng et al. 1979) have been shown to resemble, to some extent, the human (and possibly all eukaryotic) deoxycytidine kinases (Gentry 1992). Herpesvirus-specified thymidine kinase has a native molecular weight of from 80,000 Da to 90,000 Da (Black and Hruby 1990). Polyacrylamide gel electrophoresis (PAGE), under specific denaturing conditions, has shown that the functional herpesvirus tk is actually a homodimeric protein comprised of two 40,000 Da to 45,000 Da subunits. Parenthetically, there is also evidence to suggest that (unlike herpesvirus-encoded enzymes), poxvirus thymidine kinase — and by analogy the thymidine kinase expressed by eukaryotic cells, may be a tetrameric complex of four identical 20, 000 Da subunits (Black and Hruby 1990). Although all thymidine kinases tend to promote a similar function, the enzymes specified by the herpesviruses possess several very important biological properties that tend to distinguish them from the cellular enzymes. For example, herpes simplex virus thymidine kinase, although originally believed to only phosphorylate thymidine, is now known to phosphorylate other pyrimidine deoxyribonucleosides, including, deoxyuridine and deoxycytidine (Jamieson et al. 1974; Crumpacker 1993). Another important property demonstrated by these enzymes is the ability of the herpesvirus thymidine kinase to utilize, as the phosphate donor molecule (in addition to ATP), triphosphates such as CTP (Chen and Prusoff 1978). Finally, the thymidine kinase of certain herpesviruses (the human alphaherpesvirinae) are also know to express thymidylate kinase activity as 6 Figure 1.3: The salvage and de novo pathways in the biosynthesis of thymidine nucleotides. U N 5 N H 2 HO-II H X L I II II I P - O - P - O - i v ^ >v| H O - P - 0 - P - 0 - i > » ' >J OH O H ^~JT OH OH ^^Jr O H (dUDP) o II H O - P - O -o o o d^isr II II II o - P - O - P - O - P - O - J ^ >0 O H O H O H ^ ^H' H N O ^ N A ^ , C H 3 O H (Deoxy thymidine) 1 O H O H O H O H o O^INT N H 2 O II H O - P - O -O H o O H O H O ^ J ^ II II II O I - P - O - P - O - P - O - T - / ' vJ OH OH O H ^^^T CDP Deoxycytidine kinase ADP ATP r dCMP deaminase dUTPase 5,10-Methylene-THF (dUMP) Thymidine kinase Thymidylate DHF )C7 ADP ATP H N O ^ N ' Thymidine kinase Cl A T P -/ ADP OH (dTMP) Thymidylate kinase o •CH 3 II II X L I • P - O - P - O — OH O H ^^^r O H (dTDP ) a A T P -/ NDP kinase O O O H N O ^ N ' A ^ C H 3 ll ll ll T X L I • P - O - P - O - P - O — i > ' V J O H OH OH T""T N H 2 O ^ ^ Deoxy-cytidine o H N O ^ N ' XS1 Deoxy-uridine an appendage of their herpesvirus-* fc enzyme function, although such enzyme activity is not a property of cellular thymidine kinases, nor is it one shared by other species of the herpesviruses (Gentry 1992). Herpesvirus thymidylate kinase activity (which is expressed predominantly by HSV type-1 and V Z V — but only weakly by HSV type-2), functions to diphosphorylate endogenous as well as synthetic nucleotide monophosphates (See De Clercq 1993). It is these differences which have made it possible to develop bioassays that discriminate between cellular and herpesvirus-induced enzymes, as well as assays which can be used to distinguish between the various species (and even strains) of the herpesviruses themselves (Campione-Piccardo et al. 1979; Cheng et al. 1982; Tenser et al. 1983; Martin et al. 1985; Tenser and Edris 1987; Chatis and Crumpacker 1991). From a pharmacological perspective, perhaps the most compelling property of the herpesvirus-induced thymidine kinase lies in the enzyme's range of acceptable substrates, a range which is very much broader than what is naturally exhibited by the corresponding cellular enzymes (Black and Hruby 1990; Folkers et al. 1991; Eriksson et al. 1991; Gentry 1992). Herpesvirus-induced thymidine kinases, therefore, generally have a much greater propensity for metabolizing compounds that are structurally cognate of the naturally occurring nucleosides, thus, prompting the development and use of these analogue substrates as agents that can selectively target herpesvirus-infected cells. With few exceptions, most of the clinically relevant anti-herpesvirus agents are nucleoside analogues, agents t