ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS Item Type Dissertation Authors DE, CHANDRAV Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 23/09/2021 21:24:43 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1627 ANTIVIRAL ACTIVITY AND MECHANISM OF ACTION OF A NOVEL URACIL ANALOG FOR VARICELLA-ZOSTER VIRUS CHANDRAV DE A dissertation in the Department of Microbiology and Immunology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University. Approved __________________________ (Sponsor’s Signature) Date __________________________ TABLE OF CONTENTS ACKNOWLEDGMENTS……………………………………..……………….………..vi ABSTRACT………………….…………………………………………...……………..vii CHAPTER I: GENERAL INTRODUCTION………...………………………….…..…...1 The Virus…………………...…………………………………………….….…....2 VZV Life Cycle…………...……………………………………………...……….3 VZV Pathogenesis……………………………………………………...….…...…5 Prevention and Treatment strategies……………...…………………….……...….8 Mechanism of Action of antiviral compounds…………………………..…..…...11 Thymidine Kinase………………………………………………………………..13 Drug Resistance…………………………………………...………………….….14 Challenges in VZV research……………………………………………………..15 New technologies…………………………………………..……….………..…..17 L-nucleoside analogues……………………………………………..…….….…..19 Pyrimidine metabolism and Herpesvirus…………………….……......................19 Fig. 1.1. Lethal interaction between birivudine and 5 Fluorouracil…......23 Fig. 1.2. Structures of the compounds used in this study……….……….25 Fig. 1.3. Metabolism and mode of action of anti-VZV compound……...27 Fig. 1.4. Overview of the pyrimidine biosynthesis pathway…..……..….29 References…………………………………………………………………...…...31 CHAPTER II: β-L-1-[5-(E-2-BROMOVINYL)-2-(HYDROXYMETHYL)-1,3- DIOXOLAN - 4-YL)] URACIL (L-BHDU) PREVENTS VARICELLA-ZOSTER i VIRUS REPLICATION IN A SCID-HU MOUSE MODEL AND DOES NOT INTERFERE WITH 5-FLUOROURACIL CATABOLISM………….....………….…..41 Abstract………………………………………………………………………….42 Introduction………………………………………………………………….…..43 Materials and Methods………………………………….…………………….....46 Results…………………………………………………………………………...54 Discussion……………………………………………………………………….60 Acknowledgements……………………………………………………………...64 Table 2.1. Susceptibility of VZV-BAC-Luc to L-dioxolane uracil analogues in HFFs and skin organ culture………………………..65 Table 2.2. Antiviral effects of L-BHDU and valyl-L-BHDU in SCID-Hu mice……………………………………………… ….……….66 Table 2.3. Maximum L-BHDU concentration two hours post final treatment……………………………………...................................67 Fig 2.1. Chemical structures and molecular weights of L-BHDU and analogs……………………………………………………….……..68 Fig. 2.2. Effects of L-BHDU and Valyl-L-BHDU on VZV replication………………………………………………….…………….70 Fig. 2.3. Combined efficacy of L-BHDU with other common anti-VZV drugs…......................................................................................72 Fig. 2.4. Effect of L-BHDU on 5-FU metabolism in-vivo……………….74 Fig. 2.5. Evaluation of L-BHDU and Valyl-L-BHDU in-vivo……….….76 References………………………………………………………………………..78 ii CHAPTER III: PHENOTYPIC AND GENETIC CHARACTERIZATION OF VARICELLA-ZOSTER VIRUS DRUG-RESISTANT ISOLATES AGAINST β-L-1-[5-(E-2-BROMOVINYL)-2-(HYDROXYMETHYL)-1,3-DIOXOLAN-4-YL)] URACIL (L-BHDU) AND OTHER ANTI-HERPETIC COMPOUNDS……………….84 Abstract…………………………………………………………………………..85 Introduction……………………………………………...………………….……86 Materials and Methods……………………………………………………….…..88 Results………………………………………………………………....................97 Discussion……………………………………………………….……...………105 Acknowledgements…………………………………………………………..…108 Table 3.1. Isolation and Genetic characterization of drug-resistant isolates………………………………………..………….109 Table 3.2. Whole genome sequencing of selected drug-resistant isolates………………………………………………..….110 Table 3.3. ORF36 and ORF28 sequencing primers………………….....111 Fig. 3.1. L-BHDU inhibits HSV-1………………………………............112 Fig. 3.2. L-BHDU inhibits SVV…………………………......................114 Fig. 3.3. Cross-resistance pattern of drug resistant VZV mutants…..….116 Fig. 3.4. Mutations in VZV TK……………………….............................118 Fig. 3.5. Thymidine kinase from L-BHDUR strains are enzymatically inactive.............................................................................120 Fig. 3.6. Extracted ion chromatograms for phosphorylated L-BHDU…122 References……………………………………………………………………....124 iii CHAPTER IV: β-L-1-[5-(E-2-BROMOVINYL)-2-(HYDROXYMETHYL)- 1,3-DIOXOLAN-4-YL)] URACIL (L-BHDU) INHIBITS VZV REPLICATION BY DEPLETING THE CELLULAR dTTP POOL……………………………….…......….131 Abstract……………………………………………………………………….….132 Introduction…………………………………………………………………..…133 Materials and Methods……………………………………………….…….…...134 Results………………………………………………………………………….....139 Discussion…………………………………………………………………..…..143 Acknowledgements………………………………………………………..……146 Table 4.1. Changes in relative concentration of deoxynucleotides (dNPs) during VZV infection and L-BHDU treatment…………………………147 Fig. 4.1. VZV replication is restored by addition of Thymidine and uridine…………………………………………………………………..148 Fig. 4.2. VZV replication is restored by addition of pyrimidine bases only in dividing HFFs…………………………………………...150 Fig. 4.3. Active L-BHDU does not inhibit cellular proliferation……….152 Fig. 4.4. L-BHDU treatment decreases cellular dTTP pool……............154 Fig. 4.5. VZV infection does not induce cellular TS expression in quiescent cells…………………………………………………………..156 Fig. 4.6. Uridine cannot restore VZV 13S replication in dividing HFFs ...………………………………………..........................158 Fig. 4.7. Proposed model for mechanism of action of L-BHDU…….…160 References………………………….…………………………………………...162 iv CHAPTER V: DISCUSSION AND FUTURE DIRECTION………………….............168 Fig. 5.1. Structures of L-BHDU phosphorylated forms and related compounds……………………………………………………...175 Fig. 5.2. Model for mechanism of action of L-BHDU……………........177 References……………………………………………………………….……….……..179 v ACKNOWLEDGMENTS First and foremost I want to thank my advisor Dr. Jennifer Moffat whose support and guidance made my thesis work possible. It has been an honor to be her Ph.D. student. I am very grateful for her patience, motivation, and mentorship. I sincerely thank my qualifying and thesis committee members for their support, and recommendations throughout the years. A special acknowledgement goes to Dr. Mark Prichard for his scientific advice and knowledge and many insightful discussions and suggestions. I want to thank the present and past members of the Moffat lab, especially Dongmei Liu, for giving me an introduction into the Moffat lab and for sharing their knowledge. The thesis would not have come to a successful completion, without the help I received from the past and present members of the Department of Microbiology and Immunology and College of Graduate Studies. My time at graduate school was made enjoyable in large part due to my wonderful friends for their friendship. I want to thank my past mentors and teachers, for their advise and encouragements. Last, but very importantly, I would like to thank my family for their love and support throughout these years - especially my grandmother, Ava Choudhury. Thank you. vi ABSTRACT Antiviral activity and mechanism of action of a novel uracil analog for varicella-zoster virus Chandrav De Jennifer F. Moffat The alphaherpesvirus varicella-zoster virus (VZV) is widespread in humans. VZV causes primary and recurrent diseases that are preventable with live vaccines and are treatable with antiviral drugs. New antiviral drugs for varicella-zoster virus (VZV) with increased potency are needed, especially to prevent post-herpetic neuralgia. The purpose of this project was to evaluate β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4- yl)] uracil (L-BHDU) and 5′-O-valyl-L-BHDU for efficacy, safety, resistance, and mechanism of action in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. We found that L-BHDU and valyl-L-BHDU were safe and effective against VZV in culture and in a mouse model. Herpes simplex virus Type 1 was also sensitive to L- BHDU in cultured cells. The mechanism of action of L-BHDU and its effect on drug- drug interactions were not known. Given its similar structure to brivudine (BVdU), we addressed whether L-BHDU, like BVdU, inhibits 5-fluorouracil (5-FU) metabolism. L- BHDU did not interfere with 5FU metabolism, indicating that L-BHDU is a safer drug than BVdU. However, L-BHDU antagonized the activity of acyclovir (ACV), BVdU and foscarnet (PFA) in cultured cells, which was due to competition for phosphorylation by VZV thymidine kinase (TK). The mechanism of action of L-BHDU was studied by evaluating its activity against related α-herpesviruses and by analyzing resistant VZV vii strains. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to ACV and BVdU but not to PFA and cidofovir (CDV). Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV TK. The purified L-BHDUR TKs were enzymatically inactive and failed to phosphorylate the drug. In wild type VZV- infected cells, L-BHDU was converted to L-BHDU mono- and diphosphate forms; cells infected with L-BHDUR virus did not phosphorylate the drug. We also investigated whether addition of nucleosides reversed L- BHDU inhibition of