mechanisms of action of drugs with dual or multiple antiviral activities

268 Pages·2009·5.29 MB·English

MECHANISMS OF ACTION OF DRUGS WITH DUAL OR MULTIPLE
ANTIVIRAL ACTIVITIES

Egor Petrovitch Tchesnokov

Department of Microbiology & Immunology
McGill University, Montreal

June, 2009

A thesis submitted to McGill University in partial fulfillment of the requirements
of the
degree of Doctor of Philosophy

© Egor Petrovitch Tchesnokov

i
ABSTRACT

Viral enzymes that catalyze replication of the viral genome remain the main
subject of currently available antiviral therapies. This work focuses on three anti-
viral agents that target viral DNA polymerases: foscarnet, acyclovir and entecavir.
Though the broad spectrum of antiviral activities of foscarnet has been recognized
for decades, only recently has it been shown that the anti-herpesvirus drug
acyclovir and the anti-hepatitis B virus drug entecavir are also active against the
human immunodeficiency virus (HIV) 1. The clinical benefits of the multiple
antiviral activities of foscarnet have been demonstrated in treating
HIV/herpesvirus co-infections. Foscarnet is currently approved for treating human
cytomegalovirus infections. But the precise molecular mechanism of foscarnet
action and resistance remains poorly understood. The study of foscarnet’s mode
of action against the HCMV DNA polymerase (UL54) is complicated in part by
the difficulty in expressing and purifying the viral enzyme. To address the first
issue, I conducted biochemical studies of foscarnet/UL54 interactions using an
unpurified viral enzyme. I then proposed a model based on these studies for the
presumptive foscarnet binding site within UL54. To address the second issue I
generated the RB69 bacteriophage/HCMV polymerase chimera, which is easily
purifiable and displays the UL54 phenotype with respect to foscarnet and
acyclovir. The recent discovery of the anti-HIV activity of acyclovir has been
followed by a report showing the selection of the V75I mutation within HIV-1
reverse transcriptase (RT) under the selective pressure of acyclovir. My own
biochemical studies have revealed that the major effect of V75I substitution
involves reducing the catalytic rate of acyclovir incorporation. The recent report
of entecavir anti-HIV-1 activity has demonstrated the selection of M184V-
containing HIV-1. Subsequent biochemical studies have revealed that M184V-
containing RT discriminates against entecavir at the level of incorporation. My
own studies show that the major effect of entecavir incorporation has been the
delayed chain termination (DCT) of the DNA synthesis. DCT protects the
incorporated drug from excision. The research project described in this thesis
ii
provides novel tools and reveals new concepts for future drug design and
development.

iii
RÉSUMÉ

Les enzymes virales qui sont responsables pour la réplication du génome viral
constituent l’objet principal des thérapies antivirales actuelles. Ce travail se
concentre sur trois agents anti-viraux qui inhibitent les polymérases d’ ADN
virales. Malgré le fait que plusieurs activités antivirales du foscarnet sont connues
depuis des décennies, il a éte démontré seulement récemment que l’acyclovir, un
agent contre le virus de l`herpes, et entecavir, un agent contre le virus de l`hepatite
B, ont une activité anti-VIH-1. Les avantages cliniques des multiples activités
antivirales du foscarnet dans le traitement des co-infections VIH/herpes ont été
démontrés. Le foscarnet est présentement approuvé pour traiter les infections du
cytomégalovirus humain. Le mécanisme précis de l’action antivirale du foscarnet,
ainsi que le mécanisme de résistance au foscarnet demeurent mal compris.
L’étude du mécanisme d’action de foscarnet contre l’ADN polymérase du VCMH
(UL54) est compliquée en partie par deux problématiques : l’expression de
l’enzyme virale et la purification de cette protéine. Pour adresser le premier
problème, j’ai poursuivis des études biochimiques sur les interactions foscarnet-
UL54 en utilisant une enzyme virale non-purifiée. En se basant sur ces études, j’ai
ensuite proposé un model du site de liaison du foscarnet. Pour addresser le
deuxième problème, j’ai généré une protéine chimère bactériophage/HCMV
polymérase. Cette protéine est facilement purifiable et possède le profile de la
UL54 polymérase envers foscarnet et acyclovir. La découverte récente de
l’activité anti-VIH de l’acyclovir a été suivie d`une étude démontrant la sélection
de la mutation V75I dans la transcriptase inverse (TI) du VIH-1 sous la pression
sélective de l’acyclovir. Mes propres études biochimiques ont révélées que l’effet
majeur de la substitution V75I implique la réduction du rythme catalytique de
l’incorporation de l’acyclovir. L`étude récente portant sur l’activité anti-VIH-1
d’entecavir a démontré la sélection de TI contenant la mutation M184V. Des
études biochimiques subséquentes ont révélées que la TI contenant la M184V
démontre une préférence sélective envers l’entecavir au niveau de l’incorporation.
Mes propres études démontrent que l’effet majeur se situe au niveau de
iv
l’abrogation de la synthèse d’ADN ultérieure (ASAU). L’ASAU protège
l’entecavir incorporé de l’excision. Le projet de recherche décrit dans cette thèse
apporte de nouveaux outils et révèle de nouvelles approches pour la conception et
le développement de médicaments.

v
PREFACE

This thesis was written in accordance with McGill University’s “Guideline for
Thesis Preparation”. The format of this thesis conforms to the “Manuscript-based
thesis” option which states:

“Candidates have the option of including, as part of the thesis, the text of one or
more papers submitted, or to be submitted, for publication, or the clearly-
duplicated text (not the reprints) of one or more published papers. These texts
must conform to the "Guidelines for Thesis Preparation" with respect to font size,
line spacing and margin sizes and must be bound together as an integral part of
the thesis.”

Manuscripts included in this thesis:

Tchesnokov EP, Gilbert C, Boivin G, Götte M.
Role of helix P of the human cytomegalovirus DNA polymerase in resistance and
hypersusceptibility to the antiviral drug foscarnet.
J Virol. 2006 Feb;80(3):1440-50.

Tchesnokov E.P., Obikhod A., Schinazi I. R. F. and Götte M.
Engineering of a chimeric RB69 DNA polymerase sensitive to drugs targeting the
cytomegalovirus enzyme.
Journal of Biological Chemistry. Submitted

Tchesnokov E.P., Obikhod A., Massud I., Lisco A., Vanpouille C., Brichacek
B., Balzarini J., McGuigan C., Derudas M., Margolis L., Schinazi R. F. and
Götte M.
Mechanisms associated with HIV-1 resistance to acyclovir by the V75I mutation
in reverse transcriptase.
Journal of Biological Chemistry. Submitted

Tchesnokov EP, Obikhod A, Schinazi RF, and Götte M.
Delayed chain-termination protects the anti-HBV drug entecavir from excision by
HIV-1 reverse transcriptase.
J Biol Chem. 2008 Dec 5;283(49):34218-28

vi
The contribution of co-authors to published or submitted articles appears in the
section “CONTRIBUTIONS OF AUTHORS”. The journal of submission and
information from published articles can be found on the title page of the
concerned chapters.

Other manuscripts not included in this thesis, but to which a significant
contribution was made by the candidate, are listed as follows:

Marchand B, Tchesnokov EP, Götte M.
The pyrophosphate analogue foscarnet traps the pre-translocational state of HIV-1
reverse transcriptase in a Brownian ratchet model of polymerase translocation.
J Biol Chem. 2007 Feb 2;282(5):3337-46.

Lisco A, Vanpouille C, Tchesnokov EP, Grivel JC, Biancotto A, Brichacek B,
Elliott J, Fromentin E, Shattock R, Anton P, Gorelick R, Balzarini J, McGuigan
C, Derudas M, Götte M, Schinazi RF, Margolis L.
Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-
infected human tissues.
Cell Host Microbe. 2008 Sep 11;4(3):260-70.

Ehteshami M, Scarth BJ, Tchesnokov EP, Dash C, Le Grice SF, Hallenberger S,
Jochmans D, Götte M.
Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against
nucleotide-competing reverse transcriptase inhibitors.
J Biol Chem. 2008 Oct 31;283(44):29904-11.

Ehteshami M, Beilhartz GL, Scarth BJ, Tchesnokov EP, McCormick S,
Wynhoven B, Harrigan PR, Götte M.
Connection domain mutations N348I and A360V in HIV-1 reverse transcriptase
enhance resistance to 3'-azido-3'-deoxythymidine through both RNase H-
dependent and -independent mechanisms.
J Biol Chem. 2008 Aug 8;283(32):22222-32.

vii
ACKNOWLEDGEMENTS

I would first like to thank my supervisor, Matthias Götte, for his unconditional
support throughout my studies, especially when good data was scarce.

I am grateful to my parents, Petr Petrovitch Chesnokov and Maria Lvovna
Chesnokova.

I would like to thank my wife, Yesim Sökmen, without whom this thesis would
have never happened.

I would like to thank Paula Lavery for teaching me the discipline of a lab life.

I would like to thank Dr. Jérome Deval for teaching me the pre-steady state
kinetics as well as his involvement in my other projects.

I would also especially like to thank Dr. Bruno Marchand and Dr. Claudia
D’Abramo for the assistance that they have given me in my earlier days in
Matthias’ lab.

I would like to thank Suzanne McCormick for her kind assistance throughout the
years.

I would like to thank the current and former members of Matthias’ lab for their
friendship and their support: Stephen Barry, Aneeta Mistry, Rose Chen, , Meoin
Hagege, Maryam Ehteshami, Dimitry Coutsinos, Brian Scarth, Greg Beilhartz,
Dr. Konstantin Ivanov, Megan Powdrill, Jordan Gagnon, Colin Vasquez, Mia
Biondi and Jean Bernatchez.

viii
I would also like to thank Colin Vasquez, Greg Beilhartz, Brian Scarth for
proofreading this manuscript and Jean Bernatchez for translating the abstract in
French.

I would like to thank Dr. Jan Ryder for final proofreading and editing the
manuscript.

ix
CONTRIBUTIONS OF AUTHORS

The manuscript entitled “Role of helix P of the human cytomegalovirus DNA
polymerase in resistance and hypersusceptibility to the antiviral drug foscarnet”
was written in collaboration with Dr. C. Gilbert and Dr. G. Boivin, who provided
the nucleic acid sequences of the HCMV DNA polymerase.

The manuscripts entitled “Engineering of a chimeric RB69 DNA polymerase
sensitive to drugs targeting the cytomegalovirus enzyme” and “Delayed chain-
termination protects the anti-HBV drug entecavir from excision by HIV-1 reverse
transcriptase” were written in collaboration with Dr. A. Obikhod and Dr. R.F.
Schinazi who synthesized acyclovir.

The manuscript entitled “Mechanisms associated with HIV-1 resistance to
acyclovir by the V75I mutation in reverse transcriptase” was written in
collaboration with Dr. A.Obikhod, Dr. I. Massud, Dr. A. Lisco, Dr. C. Vanpouille,
Dr. B. Brichacek, Dr. J. Balzarini, Dr. C. McGuigan, Dr. M. Derudas, Dr. L.
Margolis and Dr. R.F. Schinazi. Dr. A. Obikhod and Dr. R.F. Schinazi
synthesized acyclovir. Dr. C. McGuigan and Dr. M. Derudas synthesized CF2648.
Dr. I. Massud, Dr. A. Lisco, Dr. C. Vanpouille, Dr. B. Brichacek, Dr. J. Balzarini
and Dr. L. Margolis were responsible for the selection of resistance with CF2648
and genotyping of the drug-exposed HIV -1 strains (Table 1). Tchesnokov E.P.
conducted the rest of the experimental work (Tables 2 and 3, Figures 2, 3, 4, 5, 6,
7 and S1).

x