The Discovery of Antiviral Compounds Targeting

Mårten Strand
The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus
ISBN: 978-91-7601-043-3
ISSN: 0346-6612
Serie nr: 1647
Umeå University
Department of Clinical Microbiology, Virology
Umeå University, SE-901 87 Umeå, Sweden
The Discovery of Antiviral
Compounds Targeting Adenovirus
and Herpes Simplex Virus
Assessment of Synthetic Compounds
and Natural Products
Mårten Strand
Department of Clinical Microbiology, Virology
Umeå University 2014
Umeå University Medical Dissertations, New Series No 1647
The Discovery of Antiviral Compounds
Targeting Adenovirus and Herpes Simplex Virus
Assessment of Synthetic Compounds and Natural Products
Mårten Strand
Akademisk avhandling
som med vederbörligt tillstånd av Rektor vid Umeå universitet för
avläggande av filosofie doktorsexamen framläggs till offentligt försvar i
Betula, byggnad 6M, fredagen den 16:e Maj, kl. 09:00.
Avhandlingen kommer att försvaras på engelska.
Fakultetsopponent: Professor, Lieve Naesens,
Rega Institute, Leuven, Belgien.
Department of Clinical Microbiology, Virology
Umeå University
Umeå 2014
Organization
Umeå University
Clinical Microbiology, Virology
Document type
Doctoral thesis
Date of publication
25 April 2014
Author
Mårten Strand
Title
The Discovery of Antiviral Compounds Targeting Adenovirus and Herpes Simplex Virus
Assessment of Synthetic Compounds and Natural Products
Abstract
There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since
no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most
often associated with respiratory disease, diarrhea and infections of the eye. These infections can be
severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus
infections are associated with morbidity and high mortality rates. These patients are mainly stem
cell or bone marrow transplantation recipients, however solid organ transplantation recipients or
AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated
disease.
Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening
assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11
(RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using
this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One
compound, designated Benzavir-1, was identified with activity against representative types of all
adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral
drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR),
the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2.
To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex
virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity
comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus
infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV
types.
To expand our search for compounds with antiviral activity, we turned to the natural products.
An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated
from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral
activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active
compounds could be isolated. However, several compounds had previously been characterized with
antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this
compound was identified as a butenolide. Additional butenolide analogues were found and we
proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was
characterized and substantial differences in their toxic potential were observed. One of the most
potent butenolide analogues had minimal toxicity and is an attractive starting point for further
optimization of the anti-adenoviral activity.
This thesis describes the discovery of novel antiviral compounds that targets adenovirus and
HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead
to the development of new antiviral drugs for clinical use.
Keywords
Adenovirus, Herpes Simplex Virus, Antiviral, Natural Products, Screening, Drug discovery.
Language
English
ISBN
978-91-7601-043-3
ISSN
0346-6612-1647
Number of pages
104 + 4 papers
The Discovery of Antiviral
Compounds Targeting Adenovirus
and Herpes Simplex Virus
Assessment of Synthetic Compounds and Natural
Products
Mårten Strand
Department of Clinical Microbiology
Umeå 2014
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-368-6
ISSN: 0346-6612
Cover art: Front: An adenovirus virion surrounded by small molecules.
Back: R/V Jan Mayen on the hunt for marine actinobacteria.
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Tryck/Printed by: Print&Media
Umeå, Sverige 2014
Table of Contents
Table of Contents
Abstract
Summary in Swedish – Populärvetenskaplig sammanfattning på svenska
List of papers
Abbreviations
Introduction
1. General introduction
2. Adenovirus
2.1. History
2.2. Taxonomy and classification
2.3. Virion structure
2.4. Genome organization and gene products
2.5. Viral life cycle
2.6. Human adenovirus spread and infections
2.7. Antiviral drugs used in the clinic
3. Herpes Simplex Virus
3.1. History
3.2. Taxonomy and classification
3.3. Virion structure
3.4. Genome organization and gene products
3.5. Viral life cycle
3.6. Latency and reactivation
3.7. HSV spread and infections
3.8. Antiviral drugs used in the clinic
4. Antiviral strategies and drug modes-of-action
4.1. Binding inhibitors
4.2. Inhibitors of DNA replication – Nucleoside/Nucleotide analogues
4.3. Inhibitors of viral maturation - Protease inhibitors
4.4. Antiviral prospects
5. Drug discovery and development
5.1. Targets and assays
5.2. Compound Libraries
5.3. Screening and lead identification
5.4. Lead optimization
5.5. Drug development
5.6. Orphan drugs
5.7. Natural products in drug discovery
Aims
Methods
Results and Discussion
i!
iii!
v!
vii!
viii!
1!
1!
2!
2!
3!
4!
6!
9!
16!
20!
23!
23!
23!
24!
25!
26!
29!
30!
33!
35!
36!
37!
40!
40!
42!
42!
43!
43!
44!
45!
45!
47!
49!
50!
53!
i
Paper I
Paper II
Paper III
Paper IV
Concluding remarks and future prospects
References
Acknowledgements
53!
55!
56!
58!
59!
63!
104!
ii
Abstract
There is a need for new antiviral drugs. Especially for the treatment of
adenovirus infections, since no approved anti-adenoviral drugs are available.
Adenovirus infections in healthy persons are most often associated with
respiratory disease, diarrhea and infections of the eye. These infections can
be
severe, but are
most often
self-limiting. However, in
immunocompromised patients, adenovirus infections are associated with
morbidity and high mortality rates. These patients are mainly stem cell or
bone marrow transplantation recipients, however solid organ
transplantation recipients or AIDS patients may be at risk as well. In
addition, children are at higher risk to develop disseminated disease.
Due to the need for effective anti-adenoviral drugs, we have developed a
cell based screening assay, using a replication-competent GFP expressing
adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay
facilitates the screening of chemical libraries for antiviral activity. Using this
assay, we have screened 9800 small molecules for anti-adenoviral activity
with low toxicity. One compound, designated Benzavir-1, was identified with
activity against representative types of all adenovirus species. In addition,
Benzavir-1 was more potent than cidofovir, which is the antiviral drug used
for treatment of adenovirus disease. By structure-activity relationships
analysis (SAR), the potency of Benzavir-1 was improved. Hence, the
improved compound is designated Benzavir-2. To assess the antiviral
specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex
virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than
Benzavir-1 and had an activity comparable to acyclovir, which is the original
antiviral drug used for therapy of herpes virus infections. In addition,
Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV
types.
To expand our search for compounds with antiviral activity, we turned to
the natural products. An ethyl acetate extract library was established, with
extracts derived from actinobacteria isolated from sediments of the Arctic
Sea. Using our screening assay, several extracts with anti-adenoviral activity
and low toxicity were identified. By activity-guided fractionation of the
extracts, the active compounds could be isolated. However, several
compounds had previously been characterized with antiviral activity.
Nonetheless, one compound had uncharacterized antiviral activity and this
compound was identified as a butenolide. Additional butenolide analogues
were found and we proposed a biosynthetic pathway for the production of
these compounds. The antiviral activity was characterized and substantial
differences in their toxic potential were observed. One of the most potent
iii
butenolide analogues had minimal toxicity and is an attractive starting point
for further optimization of the anti-adenoviral activity.
This thesis describes the discovery of novel antiviral compounds that
targets adenovirus and HSV infections, with the emphasis on adenovirus
infections. The discoveries in this thesis may lead to the development of new
antiviral drugs for clinical use.
iv
Summary in Swedish –
Populärvetenskaplig sammanfattning på
svenska
Det finns ett behov av fler läkemedel för behandling av virussjukdomar. De
antivirala läkemedel som finns idag kan inte effektivt behandla svåra
sjukdomar orsakade av vissa typer av virus. En sådan virustyp är adenovirus.
Hos friska personer orsakar adenovirus oftast luftvägsinfektioner eller
infektioner i ögat. Hos barn är diarrésjukdomar orsakade av adenovirus
vanliga. Dessa infektioner brukar kroppens immunförsvar kunna ta hand om
och de leder sällan till allvarliga komplikationer. Men hos individer med
nedsatt immunförsvar kan en adenovirusinfektion vara direkt dödlig. Dessa
individer är främst patienter som har genomgått en benmärgstransplantation eller en organtransplantation. Man har medvetet försvagat
deras immunförsvar för att undvika att transplantatet ska stötas bort. Detta
innebär att om patienten blir infekterad med adenovirus kan infektionen
spridas obehindrat i kroppen och vara livshotande. Det är främst barn som
drabbas av svåra och livshotande adenovirusinfektioner.
Idag finns det inget godkänt antiviralt läkemedel som kan behandla
adenovirusinfektioner, trots att det finns ett behov. Vi har utvecklat en
screeningmetod som möjliggör att man snabbt kan identifiera substanser
med antiviral aktivitet. Denna metod bygger på ett modifierat adenovirus
som uttrycker ett protein som lyser grönt när viruset förökar sig i cellen. Med
denna metod har vi screenat 9800 syntetiska substanser. Bland dessa
substanser identifierade vi ett flertal med antiviral aktivitet utan att påverka
värdcellen. Vi valde ut en substans, som vi kallar Benzavir-1, och analyserade
dess aktivitet. Benzavir-1 visade kraftfull antiviral aktivitet mot alla
adenovirustyper vi testade och hade låg påverkan på värdcellen. Effekten av
Benzavir-1 var t.o.m. bättre än cidofovir, som är det antivirala läkemedel
som används idag mot adenovirusinfektioner. Genom att modifiera den
kemiska strukturen av Benzavir-1 kunde vi förbättra den antivirala
aktiviteten. Den förbättrade substansen kallar vi Benzavir-2. För att utreda
om den antivirala aktiviteten var specifik för adenovirus, undersökte vi om
Benzavir-1 och -2 kunde hämma en annan typ av virus, nämligen herpes
simplex virus (HSV). Benzavir-1 och -2 var aktiva mot både HSV typ 1 och
typ 2 samt så visade den förbättrade substansen, Benzavir-2, bättre effekt än
Benzavir-1. Benzavir-2 var lika effektiv som acyclovir, som är det läkemedel
som används mot herpes virus idag, och var dessutom aktiv mot acyclovirresistenta kliniska HSV typ 1 och typ 2 isolat.
v
Baserat på den potenta aktiviteten av Benzavir-2, är vår avsikt att utreda
om Benzavir-2 kan vara en möjlig läkemedelskandidat för behandling av
adenovirusinfektioner hos patienter med nedsatt immunförsvar, där det idag
finns ett stort behov.
Parallellt med karaktäriseringen av Benzavir-2, utvidgade vi sökandet av
substanser med antiviral aktivitet mot naturprodukter. Från havsbotten
utanför Norges kust togs sedimentprover. Från dessa sedimentprover
isolerades aktinobakterier, som är kända för sin förmåga att producera
substanser med biologisk aktivitet. Genom att växa aktinobakterier i diverse
stressframkallande lösningar, framtvingas produktion och frisättning av
substanser från bakterierna. Dessa substansrika extrakt, screenades för
antiviral aktivitet med vår screeningmetod. Ett flertal extrakt visade antiviral
aktivitet och genom att fraktionera dessa extrakt kunde vi identifiera de
aktiva substanserna. Några substansers antivirala aktivitet var redan känd
men vi hittade en substans som tidigare inte hade visat antiviral aktivitet.
Denna substans visade sig vara en butenolide. När vi försökte optimera
produktionen av butenoliden upptäckte vi ett flertal analoger till butenoliden
som vi lyckades isolera. Vi kunde även visa i vilken ordning, i relation till
varandra, som analogerna producerades. När vi analyserade den biologiska
aktiviteten av butenoliderna, upptäckte vi stora skillnader. Två butenolider
hade likvärdig antiviral aktivitet men beroende på strukturen av
butenolidens sidokedja, skiljde de sig åt i avseende den toxiska effekten på
värdcellen. Genom att modifiera butenolidernas struktur, kunde vi även
identifiera den del av strukturen som gav upphov till den antivirala
aktiviteten.
Sammanfattningsvis beskriver den här avhandlingen upptäckten av
antivirala substanser som kan leda till att nya antivirala läkemedel kommer
till kliniskt bruk.
vi
List of papers
I
Small-molecule screening using a whole-cell viral replication
reporter gene assay identifies 2-[2-(benzoylamino)benzoylamino]
benzoic acid as a novel antiadenoviral compound.
Andersson EK, Strand M, Edlund K, Lindman K, Enquist PA, Spjut S, Allard
A, Elofsson M, Mei YF, Wadell G.
Antimicrob. Agents Chemother., 2010, 54 (9): 3871-3877.
II
Synthesis,
biological
evaluation,
and
structure-activity
relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid
analogues as inhibitors of adenovirus replication.
Öberg CT, Strand M, Andersson EK, Edlund K, Tran NP, Mei YF, Wadell G,
Elofsson M.
J. Med. Chem., 2012, 55 (7): 3170-3181.
III
2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic
acid, an antiviral compound with activity against acyclovirresistant isolates of herpes simplex virus types 1 and 2.
Strand M, Islam K, Edlund K, Öberg CT, Allard A, Bergström T, Mei YF,
Elofsson M, Wadell G.
Antimicrob. Agents Chemother., 2012, 56 (11): 5735-5743.
IV
Isolation and characterization of anti-adenoviral secondary
metabolites from marine actinobacteria.
Strand M*, Carlsson M*, Uvell H, Islam K, Edlund K, Cullman I, Altermark
B, Mei YF, Elofsson M, Willassen NP, Wadell G, Almqvist F.
Mar. Drugs, 2014, 12(2): 799-821. (*equally contributing authors).
vii
Abbreviations
Adpol
Adenovirus DNA polymerase
AIDS
Aquired immunodeficiency syndrome
ADP
Adenovirus death protein
BMT
Bone marrow transplant
CAR
Coxsackie and adenovirus receptor
CDK
Cyclin-dependent kinase
CC50
Half maximal cytotoxic concentration
CMV
Cytomegalovirus
DBP
DNA binding protein
DSG-2
Desmoglein-2
EBV
Epstein-Barr virus
EC50
Half maximal effective concentration
EMA
European medicines agency
EKC
Epidemic keratoconjunctivitis
FDA
Food and drug administration
GFP
Green fluorescent protein
HCS
High content screening
HSCT
Hematopoetic stem cell transplantation
HSV
Herpes simplex virus
HTS
High throughput screening
PK
Pharmacokinetics
pTP
Pre-terminal protein
qPCR
Quantitative polymerase chain reaction (PCR)
RCAD11GFP Replication competent GFP-expressing adenovirus 11
SAR
Structure activity relationship
SCT
Stem cell transplant
TP
Terminal protein
TK
Thymidine kinase
viii
Introduction
1. General introduction
The word virus is Latin for poison or toxin. A virus is an intracellular
molecular parasite that is unable to replicate outside the cell. Viruses can
infect all forms of life and many viruses have co-evolved with their host
organisms. The origin of viruses is not clear, however three main hypotheses
are defined. The first hypothesis states that virus arose from mobile cellular
genetic elements that gained the ability to exit one cell and enter another.
The second hypothesis states that virus originates from cellular organisms
that have lost genetic information over time and have adopted a parasitic
replication approach. The third and last hypothesis postulates that virus was
the first self-replicating unit that was evolved to form cells [1].
The first infectious agent that was designated as virus, was the tobacco
mosaic virus. In 1898, Martinus Beijerinck was convinced that the filtrated
solution from infected tobacco plants contained a new form of agent, distinct
from bacteria. He believed that the infectious agent was a liquid, thus with
the development of the plaque assay in 1917, and when the first electron
micrographs of tobacco mosaic virus in 1939, it became more obvious that
viruses were particles [2].
Virus may have beneficial functions in the evolution of organisms. It is
estimated that about 5 % of the human genome originates from retroviruses
[3]. Hence, viruses may have a role in the evolution of man. However,
viruses are most commonly associated with a variety of diseases and have
been described as “a piece of bad news wrapped in a protein coat” by Peter
Medawar, who is regarded as the father of transplantation [4]. This
description could have arisen from the fact that viral infections are more
difficult to treat compared to e.g. bacterial infections. This is mainly due to
that it is challenging to target the intracellular replication of viruses without
negative effects on the cell and the host organism. Thus, the limited number
of antiviral drugs today generates a need for development of new antiviral
drugs.
1
2. Adenovirus
2.1. History
In 1953, Wallace Rowe and Robert Huebner cultured human tonsils and
adenoids in tissue culture with the aim of identifying the agent causing
“common cold”. Rowe noted that cells derived from adenoids changed their
morphology and degenerated over time [5]. The causative agent for this was
considered to be a virus. Almost coincidently in 1954, Hilleman and Werner,
isolated an agent that presumably also was a virus, but not an influenza
virus, that caused acute respiratory disease in military recruits [6].
Subsequently, it was shown that these viruses were related [7]. In 1956, it
was proposed that this group of viruses should be commonly named as
“adenoviruses” based on the cell type where it was first isolated [8].
This was the onset of an era in virology research that has led to great
achievements and impacts in understanding both fundamental virological
events such as: viral structure and viral replication as well as understanding
cellular events and the interplay between virus and cell and ultimately the
effect on the whole organism. For example, in 1977, Phillip Sharp and
Richard Roberts discovered that genes were not merely parts of a long
continuous segment of DNA, as previously believed, but could consist of
several small segments of DNA [9, 10]. This was discovered using adenovirus
as a model system, since its genes display important similarities to higher
organisms, including man. The phenomenon that genes could originate from
discontinuous segments of DNA is called splicing, and is a fundamental
principle in modern molecular biology. Sharp and Roberts were jointly
awarded the 1993 Nobel Prize in Physiology/Medicine for their discovery.
Another important event in the field of adenovirus is the usage of
adenovirus in gene therapy, which has both scientific implication and great
clinical impact. Gene therapy is when a functional gene (DNA) is introduced
to the organism to replace a dysfunctional gene. Most often is the gene
packaged into a virus (vector) that delivers the gene to target cells.
Adenoviruses are today the most commonly used gene therapy vectors,
followed by retrovirus vectors, and different types of cancers are by far the
most common indication [11]. There are several reasons why adenoviruses
are so commonly used as gene delivery vectors; i) adenovirus can give a high
gene expression. ii) adenovirus can infect both dividing and non-dividing
cells without incorporation of viral DNA into the host genome. iii) the overall
viral biology is well understood. However, despite successful clinical
therapies with adenovirus vectors, a major drawback was in 1999, when 18year-old Jesse Gelsinger, died after a large dose of an adenovirus vector that
was administrated into arteria hepatica [12]. Gelsinger died of multi-organ
2
failure after that substantial amounts of the vector for unknown reasons had
spread to the circulation and triggered a massive inflammatory response.
Although Gelsinger was exposed to a replication incompetent vector, it is
important to be able to suppress and control the infection when performing
clinical gene therapy trials. An effective antiviral drug is obviously also
needed in the field of gene therapy.
2.2. Taxonomy and classification
The International Committee on Taxonomy of Viruses (ICTV) has classified
adenoviruses to the family Adenoviridae that is divided into five genera;
Atadenovirus (contains A and T-rich genomes, capable of infecting a broad
range of hosts), Aviadenovirus (infecting birds), Ichtadenovirus (currently
only one species isolated from sturgeon), Siadenovirus (infecting frogs and
some birds) and Mastadenovirus (infecting mammals). Human adenovirus
is within the genus of Mastadenovirus and consists of over 54 types, divided
in seven species, A to G (Table 1). The classification of the different types is
originally based on serology, their hemagglutination patterns and their
oncogenicity in rodents among other biological attributes [13, 14]. However,
it is currently debated whether the types should be classified according to
whole-genome sequencing instead [15-17]. Depending on the method of
classification, the number of adenovirus types varies. According to the
whole-genome sequencing technique, over 60 isolated adenovirus types have
been characterized so far [18]. As seen in Table 1, there is a correlation
between the species and their tropism, e.g. the tropism of species F and G is
only enteric and species D does not infect respiratory tissue, reasons for
these speificities are discussed in section 2.5.1.
3
Table 1. Categorization of adenovirus types within species and their
respective tropisms.
Species
A
B:1
B:2
Type
12, 18, 31
3, 7, 16, 21, 50
14, 11, 34, 35
C
1, 2, 5, 6
D
8-10, 13, 15, 17, 19, 20, 22-30,
32, 33, 36-39, 42-49, 51, 53, 54
4
40, 41
52
E
F
G
Tropism
Enteric and respiratory
Respiratory and ocular
Renal, respiratory and
ocular
Respiratory, ocular,
hepatic and lymphoid
Ocular and enteric
Respiratory and ocular
Enteric
Enteric
!
2.3. Virion structure
Adenovirus is between 70 – 100 nanometers in size and its outer structure
is composed of a non-enveloped capsid with icosahedral architecture. The
most abundant protein in the capsid is the hexon protein with a total of 240
hexon trimers distributed to 12 trimers at each of the side of the twentysided structure. At each of the 12 vertices of the capsid is a penton that
consists of a penton base and a protruding fiber (Fig. 1). The fiber is a
trimeric protein with a distal globular knob domain that functions as the
cellular attachment site. The length of the fiber varies between adenovirus
types, depending on the number of amino acid sequence repetitions in the
shaft, from six repeats in Ad3 to 22 repeats in Ad2 and Ad5 [19, 20]. In
addition, Ad40 and Ad41 have two different lengths of fibers and some avian
adenovirus can have two different fibers attached on the same penton base
[21-23].
The minor proteins, IIIa, VI, VIII and IX are capsid associated proteins
and are important for virion assembly, the transient virion stability and also
when the virion disassembles on infection [24]. For example, protein IIIa is
involved in the viral DNA packaging and protein VI may aid the virion to
escape from the intracellular endosome and facilitates the nuclear import of
hexon proteins [25, 26]. Protein IX is located in the cavities between the
hexon proteins and stabilizes the capsid formation [27]. Furthermore, IX has
also been reported to be involved in transcriptional activation of late
adenoviral genes [28].
4
Figure 1. The structure of the adenovirus virion. Reprinted from [29] with
permission from the publisher.
Inside the core of the capsid is a double-stranded DNA genome with a
covalently attached terminal protein at each 5´ end [30]. The linear genome
is condensed by three basic DNA binding proteins, V, VII and µ [31]. Protein
VII is a histone-like protein that in a sequence-independent fashion binds to
the DNA and wraps it up in nucleosome-like structures. Protein V connects
the DNA to the capsid via protein VII and the penton base [32, 33]. Protein µ
is a small protein that is tightly associated with the DNA and facilitates the
DNA condensation [34]. Moreover, the core protein IVa2 binds sequencespecific to motifs in the major late promoter-region of the adenovirus
genome and is suggested to be a transcriptional activator and/or involved in
the packaging of DNA in the virion core [35, 36]. The last, to date, known
protein inside the core is the adenovirus protease. The adenovirus protease
5
(adpol) is a monomeric cysteine protease that is essential for the virion to be
infective [37]. The protease is involved in both the entry of the adenovirus
into cells and the assembly of new virion particles [38, 39]. The protease
requires two cofactors to be active. One cofactor is the 11-amino acid Cterminal part of the precursor protein VI and the other cofactor is the viral
DNA [40, 41]. The precursor protein VI has been suggested as a “molecular
sled” that carries the protease along the viral DNA to efficiently locate its
substrates [42] . The adenovirus protease is considered to be an attractive
target for antiviral drug development [43] (discussed in section 4).
2.4. Genome organization and gene products
The adenovirus genome is a linear, double-stranded DNA molecule with two
inverted terminal repeats (ITR) and two covalently bound terminal proteins
at each end of the DNA. The size of the genome in the Mastadenovirus genus
is between 34-36 kb, although the genome size can vary between 26-45 kb in
the other genera in the Adenoviridae family [44]. The genome organization
is well conserved among the presently known adenoviruses, except for the
avian adenovirus CELO with some distinctive differences [45]. Henceforth,
the genome of Mastadenovirus will be discussed.
The size of the genome is critical in the biology of adenoviruses, since it
has to fit into the virion capsid. It is quite remarkable that the ~36 kb
genome can produce over 40 proteins. What makes this possible is the
effective organization of the genome. There are 8 RNA polymerase II
transcription units that are located at either strand of the genome. These
units are divided in five early (E1A, E1B, E2, E3, E4), two intermediate (IX
and IVa2) and one late (L1-L5). In addition, there are two virus-associated
RNAs (VA RNAI and II) (Fig. 2).
6
Figure 2. Schematic genome organization of adenovirus type 5. Figure
reprinted from [46] with permission from the publisher.
E1A is the first transcription unit to be expressed during infection. E1A
products be detected in the cell as early as one hour post-infection [47]. The
gene products are two major proteins (243R and 289R) that are
transcriptional modulators and stimulate the cell to enter S-phase [46]. E1A
is considered as an oncogene since it has potential to immortalize cells [48].
E1B is transcribed shortly after E1A, after approximately 1.5-2 hours postinfection is the mRNA in the cytoplasm [47]. The E1B encodes the 19K and
55K proteins that prevent the cell from apoptosis. The 55k protein binds to,
and inhibits the function of the tumor suppressor protein p53 [49]. The 19K
protein is a viral analog to the cellular Bcl-2 protein and can inhibit both p53
dependent and p53-independent apoptosis [50].
E2 is expressed from two regions (E2A and E2B) and yields three proteins
by alternative splicing. The E2 gene products are directly involved in, and
initiate the viral DNA replication. The proteins are the terminal binding
protein (TP), the DNA polymerase (Adpol) and the DNA binding protein
(DBP). TP is part of a quite unique feature in DNA replication. Adenovirus
replicates by protein-priming mechanism instead of a RNA/DNA single
stranded primer mechanism [51] (DNA replication is discussed in more
detail in section 2.5.4). This feature is dependent on TP, which after cleavage
of the adenovirus protease, is linked to each end of the viral DNA by a
phospodiester bond from a serine to the terminal genomic dCMP by the
7
adenovirus DNA polymerase [52, 53]. In addition to functioning as a primer
for the viral DNA, this protein linkage may also protect from exonucleases
and also prevent that cellular DNA end-binding proteins bind to the viral
DNA and inhibit the replication [54]. The second protein expressed from E2
is the 140-kD viral DNA polymerase. The polymerase has an intrinsic 3´- 5´
proofreading exonuclease activity and belongs to the Pol α family. The last
expressed protein is the DBP, which is a 72-kDa protein with affinity to
single-stranded DNA and RNA as well as double stranded DNA [55].
E3 encodes between 6-9 mRNAs, depending on adenovirus type [56]. All
of these proteins function as immunomodulatory proteins to avoid the
immune system of the host. Hence, the E3 region is not essential for virus
growth in cell culture [57]. Due to the variation in the E3 region among
adenovirus types it is believed that the E3 region is important in the speciesspecific characteristics. The E3 proteins are mostly transmembrane proteins,
except 14.7 kD, 12.5 kD. The 11.6 kD is the adenovirus death protein (ADP)
that induces cell lysis, maybe through interaction with the mitotic spindle
assembly checkpoint protein, MAD2B [58]. ADP is also believed to be the
switch between lytic and persistent adenovirus infection [59].
E4 is located at the 5´ end of the genome with a single promoter. This
promoter generates at least 18 distinct mRNAs after alternative splicing and
at least six proteins have been demonstrated to exist in infected cells named
after their open reading frames: ORF1, ORF2, ORF3, ORF4, ORF6 and
ORF6/7 [60]. These gene products are involved in DNA replication, viral
transcription, apoptosis and regulation of cell cycle signaling [61, 62]. The
interplay between the proteins seems to be complex since only the deletion
of the entire E4 region impairs virus growth but individual mutations within
the proteins have minimal or modest effect [63, 64].
The virus associated RNAs (VA RNAI and II) are small and highly
structured low molecular weight regulatory RNAs found in infected cells.
These are RNA polymerase III transcripts that are produced with different
kinetics [65, 66]. However, some adenovirus types encode only one VA RNA
and not all encoded VA RNAs contribute to the virus infectivity [67]. The
functions of the VA RNAs are mostly studied in adenovirus type 2 and 5,
where it has been shown that VA RNAI inhibits the interferon-induced PKR
protein and thereby keeps the eIF-2α transcription factor and the general
protein translation active [68]. Additionally, the VA RNAs can be processed
to small RNAs (mivaRNA) by the Dicer complex, and be incorporated into
the RNA-induced silencing complex (RISC). This results in inhibition of the
host cell antiviral defense by suppression of the RNA interference (RNAi)
machinery that can degrade viral mRNA [69].
The intermediate transcript gene products (IX and IVa2) are partly
discussed in section 2.3. These are expressed prior to the late gene
expression, and it has been shown that the IVa2 product contributes to the
8
late gene expression whereas the IX product serves not only as a capsid
protein but also as a transcriptional activator [70, 71].
The major late transcriptional unit (MLTU) is expressed with the onset of
viral DNA replication from the major late promoter (MLP). However, the
MLP is also active in the early phase of the infection but to a lower
transcriptional rate [72]. Coincidentally with the onset of viral DNA
replication the transcription rate from MLP is increased dramatically [73].
The transcriptional units from MLTU are grouped into five families (L1-L5)
and represent mostly structural proteins needed for progeny virus, e.g.
penton base (L2), hexon (L3) and fiber (L5). Additionally, the adenovirus
protease gene is in the L3 family.
2.5. Viral life cycle
2.5.1. Binding
The initial step of the virus life cycle is the recognition of the target cell.
For adenovirus, this recognition is mediated by the fiber sand the fiberknobs.
This initial contact is also the decisive factor for the adenovirus type-specific
tissue tropism, since different types have different fibers that target specific
cell surface proteins (receptors) (Fig. 3). Hence, receptor characterization is
important, especially in gene therapy where specific cell types (most often
cancerous) are targeted. Furthermore, with the techniques in molecular
cloning that are available today, it is possible to retarget an adenovirus type
to a certain receptor, and cell type, by modulation of the fiber and fiberknob.
The first receptor that was characterized was for adenovirus type 2 and 5
in 1997. It was shown that the adenovirus and coxsackie virus shared the
same receptor, this protein was then named coxsackie and adenovirus
receptor (CAR) [74]. CAR is a transmembrane tight-junction protein
involved in the cell-to-cell attachment and is abundantly expressed in a wide
variety of tissues, including the intestine, prostate, heart, testis and pancreas.
The fiber of adenovirus species A and C-F (not species B) have affinity for
CAR but only species C have been found to use CAR as a functional receptor
[75, 76]. However, since CAR is located in the tight-junction of the cells and
is not accessible on the apical surface of the cells, the biological CAR-fiber
interaction is debated [77]. It has been suggested that lesions in the epithelial
layer would be needed prior infection or that adenovirus first infect nonpolarized cells that spreads the infection to neighboring polarized cells [78].
Furthermore, the interaction with CAR has been considered to function as an
exit mechanism instead of an entry mechanism in vivo. This is due to that
during infection, an excess of fibers and pentons are produced that could
9
bind to CAR and thereby disrupt the cellular tight-junctions allowing virus
escape [79].
Figure 3. Receptors used by human adenoviruses. Figure reprinted from
[80] with permission from the publisher.
Several additional receptors have subsequently been identified. CD46,
Desmoglein-2 (DSG-2) and the sialic acid-containing proteins are the most
characterized. CD46 is expressed on all nucleated cells and is a regulator of
the complement activation (RCA) family of proteins. Adenovirus types from
species B, and species D types Ad37 and Ad49, have been demonstrated to
use CD46 as a receptor [81-86]. Moreover, CD46 is used as receptor for
several other pathogens as well, and is considered to be a pathogen magnet
[87]. Interestingly, it has been shown that when the fiberknob of Ad11 binds
to CD46, this alters the confirmation of CD46 from bent to a planar structure
[88]. Quite recently, the cadherin protein Desmoglein-2 that is important in
cell adhesion, was found to be the receptor of the species B types 3, 7, 11 and
14 [89]. As noted, Ad11 and other species B types can utilize more than one
receptor depending on the affinity/avidity characteristics of the interaction
[90, 91]. This implies also for type Ad37 from species D, which is reported to
use CD46 and/or sialic acid proteins as cellular receptor(s) [84, 92].
However, the molecular interaction between the Ad37 fiberknob and
ganglioside GD1a has been characterized, strongly suggesting that sialylated
glycoproteins are functional receptors [93]. Ad37, Ad8, and Ad19, all from
10
species D, can infect the eye and cause epidemic keratoconjunctivitis. Their
primary interactions with sialylated proteins are currently considered as a
target for development of novel antiviral therapeutics for treatment of eyeinfections caused by adenovirus [94].
2.5.2. Internalization
In general, after the initial binding of the virion to e.g. CAR, the virion needs
to interact with an additional receptor to be internalized by the cell (Fig 4).
These receptors are designated co-receptors and belong to the integrin
family. Integrins are, similarly to CAR, involved in the cell-to-cell adhesion,
although integrins are also engaged in adhesion to the extracellular matrix.
They consists of two subunits, α and β, and today the αvβ3, αvβ5, αvβ1, α5β1
and α3β1 integrins have been shown to facilitate cell entry [95, 96]. The
virion interaction with the integrins is mediated by the penton base, which
possesses a tripeptide amino acid sequence, Arginine-Glycine-Aspartic acid
(RGD), which can bind to integrins. This RGD amino acid sequence is
conserved in all adenovirus types, except the typical enteric types 40 and 41
of species F, suggesting that the interaction with integrins is functioning
independently from the initial receptor specificity [97]. The RGD sequence is
part of a flexible loop in the penton base. However, the penton base is part of
the capsid in a distal direction from the fiberknob, where the binding to CAR
takes place. Hence, the length of the fibershaft would be expected to affect
the physical interaction between the penton base and integrin. However, it
has been suggested that the type 5 (and all species C) fibers contain a “hinge
region” that facilitates the interaction [98]. The penton base–integrin
interaction triggers phosphatidylinositol-3-OH kinase (PI3K), which is an
intracellular lipid kinase [99]. The phospholipid products act as second
messengers that lead to actin reorganization and promote endocytosis of the
virion by clathrin-coated vesicles [100-102]. In less than 5 min after the
initial receptor binding, adenovirus can be observed in endosomes (Fig. 4).
Although, clathrin-mediated endocytosis of adenovirus is the most
characterized entry pathway, macropinocytosis entry has been described as
well. Macropinocytosis leads to vacuole formation at the cellular periphery
and has been reported for both species C (type 2 and 5) and species B (type 3
and 35) adenovirus. For species C adenovirus, a simultaneous
macropinocytosis and clathrin-mediated uptake was reported, that both
were integrin-dependent [103]. For the species B adenovirus, it was reported
that both CD46 and integrins was needed and macropinocytosis was
suggested to be the major entry mechanism for type 3 and 35 [104, 105].
11
Figure 4. Illustration of adenovirus uptake and transport to nucleus. Figure
adopted from [106] with permission.
When the endocytosis is initiated, a stepwise dismantling of the virion
starts in parallel. The fiber proteins starts to detach from the penton base at
the plasma membrane followed by a conformational change in the penton
base protein that exposes hydrophobic parts and weakens the structure of
the capsid [101, 107]. This is followed by release of internal proteins, such as
IIIa, V, VIII and VI [108]. Protein VI plays an essential role in the early
endosome escape [25]. The virion uncoating process and endosomal escape
is very rapid, within 15 min after the initial extracellular binding, free virions
can be detected in the cytosol [38]. Although, the rate of the endosomal
escape seem to differ between adenovirus types [109]. The exact mechanism
for the endosomal escape is not known, although it is known that an acidic
pH is essential [110]. This acidification is believed to activate the adenovirus
protease that cleaves protein VI, thus activating the membrane lytic capacity
of VI [38].
12
2.5.3. Transportation to nucleus
After the endosomal escape to the cytoplasm, the virion is now partly
disassembled and consists of the structural proteins hexon, a fraction of
penton base and protein IX that secure the capsid formation. Inside the
capsid is the DNA condensed with protein V and VII. The cytoplasm is a very
crowded and viscous milieu, hampering a spontaneous diffusion to the
nucleus. The virion overcomes this obstacle by active transportation to the
nucleus along the microtubules. This transportation is facilitated by a motor
protein, called dynein, which literally “walks” on the microtubule towards
the microtubule-organizing center (MTOC) near the nucleus [111]. However,
kinesin, which is a motor protein but is directed away from the nucleus, has
been shown to compete for the intracellular transportation of species C
adenovirus [112]. It has been demonstrated that the interaction between the
capsid and dynein is mediated through the hexon protein [113]. The
transport towards the nucleus is rapid, after approximately 60 min postinfection virions can be located around the nucleus [114]. When the capsid
arrives at the MTOC, near the nucleus, the capsid cannot enter the nucleus
since the nuclear pores are roughly half the size of the capsid, but need to
dock to the nuclear pore [115]. This docking is facilitated by interaction
between the capsid and the nuclear pore proteins CAN/Nup214 that are
proteins of the cytoplasmic filaments of the nuclear pore (Fig. 4) [114]. This
interaction allows nuclear histone H1 proteins to dock to hexon proteins
followed by interaction to importin β proteins that tear the capsid apart. The
viral DNA can then freely diffuse through the nuclear pore into the nucleus.
Alternatively, chaperone protein Hsc70 or protein VII interaction with
transportin mediate the translocation of the viral DNA into the nucleus [116,
117].
2.5.4. DNA replication
The replication of the viral DNA starts 6 hours post infection and is a highly
efficient process that generates high amounts of progeny molecules. Within
40 hours after infection, one cell can produce approximately one million
copies [118]. Prior to the onset of DNA replication the E1a proteins must
have been transcribed and triggered the cell to enter S-phase. Also, the E2gene products (TP, DBP and adpol) must have been expressed. The
adenovirus DNA replication utilizes a protein-priming mechanism, in which
the pre-terminal protein (pTP) acts as the protein primer. The pTP and the
adenovirus DNA polymerase (adpol) are present in the infected cell as a
heterodimer [119]. The onset of the DNA replication is when the adenovirus
polymerase covalently attaches a dCMP nucleotide to the hydroxyl group of a
serine residue of the pTP [54]. This facilitates binding of the pTP-adpol-
13
complex to specific DNA sequences in the origin of replication that is located
at the initial 1-50 bp of either end of the viral genome (Fig 5A). Two cellular
proteins enhance the initiation of replication vastly; nuclear factor-1 (NF1)
and octomer-binding transcription factor-1 (Oct-1), which bind to the
auxiliary region, close the pTP-adpol-complex [120, 121]. Subsequently, the
unwinding of the DNA is needed. The mechanism for this is not fully
understood, though the adenovirus DNA binding protein (DBP) has an
essential role due to its high affinity to single-stranded DNA [122]. After
DNA unwinding, the pTP-adpol complex forms a trinucletide CAT sequence
that upon formation “jumps back” three basepairs to the 3´end of the
genome [123] (Fig 5B). Sequentially, the adpol is released from the pTP and
starts the chain elongation [124]. Later in infection the adenovirus protease
cleaves pTP to TP, that is the covalently attached protein at each end of the
viral DNA [125]. The newly synthesized double stranded DNA can serve as a
template for subsequent replication rounds. In addition, the displaced single
strand is protected by the DBP protein and can form pan-handles that could
serve as replication templates [126, 127].
Figure 5. The initiation of adenovirus DNA replication. A, The origin of
replication. B, The protein priming and “jumping back” mechanisms. Figure
reprinted with permission from [118], copyright to Cold Spring Harbor
Laboratory Press.
14
2.5.5. Assembly and release
The assembly of progeny virions takes place in the nucleus. However, the
protein translation occurs in the cytoplasm, meaning that there is both an
import and export trafficking over the nuclear pore complex. It has been
shown that adenovirus proteins utilizes the cellular pathways for nuclear
transportation by presenting nuclear localization signals (NLSs) and nuclear
export signals (NESs), that are short stretches of basic amino acid residues.
Although, no NLS has been found on the hexon protein, instead protein VI is
suggested to shuttle the hexon proteins between the cytoplasm and nucleus
[26]. Inside the nucleus the hexon proteins trimerize to form capsomeres
that together with the other capsid proteins form the empty capsid structure.
It is believed that the viral DNA is “injected” into the empty capsids, though
it is also suggested that the capsid is formed around the DNA core [128, 129].
In the infected cell, a large amount of empty capsids and capsids with
incomplete DNA are produced, in parallel to mature virions [130-132]. These
incomplete virions have the potential to induce inflammation without any
gene expression [133]. The complete virions need a maturations process to
be infective progeny virions. This virion maturation is processed by the adpol
which cleaves pIIIa, pVI, pVII, pVIII, pre-µ and pTP, possibly also the L1
52/55k polypeptide, to their functional forms [134]. The protease is also
encapsidated and is essential for the progeny virus entry into new cells when
released. When the nucleus is packed with virions, the cytoskeleton is
disrupted and the cell rounds up to the typical cytophatic effect (CPE) that
ends in cell lysis. It has been shown that adpol can cleave the cellular
cytokeratin K18 in HeLa cells, which results in perturbation of the formation
of cytoskeleton filaments, thus adpol is involved in the CPE and promotes
cell lysis [135]. However, the precise mechanism of adenovirus-caused cell
lysis is poorly understood. Though, the adenovirus death protein (ADP), also
known as E3-11.6K protein, has a major role in this process. ADP is
abundantly expressed late in infection and is, at least partly, involved in cell
lysis since virus lacking ADP replicates as well as wild type virus, but the cell
lysis phase is delayed [136]. This corresponds to that ADP levels have been
suggested to be the switch between lytic and persistent infections [59].
Furthermore, both autophagy and apoptosis mechanisms have been
reported to be involved in the adenovirus induced cell killing [137, 138]. In
the end when the cell lyse, up to 104 progeny virions are produced per cell,
along a vast excess of viral proteins and DNA that have not been
encapsidated [139]
15
2.6. Human adenovirus spread and infections
Adenoviruses are easily spread from one person to another through aerosols
when sneezing or coughing, or through the fecal-oral route. The virus is also
very stable outside the host, on surfaces and in water. Most types are still
infectious at 36 °C for a week and several weeks at room temperature [140].
In addition, the virus can be viable on dry surfaces for several months [141].
The adenoviruses are also resistant to lipid disinfectants (soap) but are
sensitive to 70 % ethyl alcohol, heat, formaldehyde and chlorine [142, 143].
Due to the stability of the virus and that it is easily spread, large outbreaks of
adenovirus infections have been described, especially in hospital settings,
child-care centers and among military recruits [144-146]. However,
community-wide outbreaks have also been reported [147].
2.6.1. Adenovirus infections in immunocompetent
persons
In otherwise healthy, immunocomptetent persons, adenovirus infections can
be severe but is most often self-limiting and can even be asymptomatic.
Adenovirus infections are common in society and can occur throughout the
year. Respiratory infections are often associated with species C adenovirus,
and account for 5-10 % of all respiratory infections in children [143]. Fever,
pharyngitis, and conjunctivitis are symptoms of pharyngoconjuctival fever
(PCF) caused by adenovirus type 3, 7 and 14. PCF affects mostly children and
can cause localized outbreaks in e.g. primary schools and swimming pools
[148, 149]. Acute respiratory disease (ARD), caused predominately by
species C and type 7, 14 from species B plus type 4 from species E, is
associated with fever, rhinorrhea, cough and sore throat that last for 3-5
days, usually with tonsillitis and otitis media. In 1971 an oral vaccine was
implemented to reduce ARD in military recruits where it was a major
concern with occasional fatalities. The vaccine reduced adenovirus-related
respiratory illnesses in recruits, by more than 95 %, however the vaccine
production ceased in 1996 [150, 151]. Adenovirus can cause pneumonia with
sporadic fatalities [152, 153]. Moreover, adenovirus type 40 and 41 infect the
gastrointestinal tract and are responsible for 5-15 % acute diarrheal illness in
children [143]. Adenovirus type 11 and 21 from species B can infect the
urinary tract and cause haemorrhagic cystitis that occurs mostly in boys. It is
characterized by gross hematuria and adenovirus accounts for 20 % of all
reported hemorrhagic cystitis cases in US [154]. Myocarditis, hepatitis and
meningoencephalitis are rarely reported manifestations of adenovirus
infections [155-158]. More common is the highly contagious eye infection
epidemic keratoconjunctivitis (EKC) that is mainly caused by type 8, 19 and
16
37 from species D. EKC cause significant morbidity for the patient, with
photophobia, pain, red-eyes, tearing, and blurred sight. EKC can lead to
prolonged impaired vision or blindness. Due to the infectiousness, outbreaks
are common in densely populated areas. In Japan, between half a million to
one million people acquire EKC every year [159].
Table 2. Predominant adenovirus types associated with adenovirus disease.
Table adopted from [143] with permission from the publisher.
Diseases caused by adenoviruses
Predominant types
Infants:
Pharyngitis, pneumonia
Otitis media
Diarrhea
1, 2, 5
1, 2, 5
40, 41
Children:
Pharyngoconjunctivial fever
3, 7
Pneumonia
3, 7, 21
Diarrhea, mesenteric adenitis
40, 41, 2
Haemorrhagic cystitis
11, 21
Myocarditis
1, 2, 5
Young adults:
Acute respiratory disease
4, 7, 14
Adults:
Epidemic keratoconjuctivitis
8, 19, 37
Immunocompromised patients:
Pneumonia
1, 2, 5
Gastroenteritis, hepatitis
5
Haemorrhagic cystitis, nephritis
11, 34, 35
Meningoencephalitis
1, 2, 5
17
As seen in Table 2, the age of the person, when infected, and the
adenovirus type are closely correlated to the clinical manifestation of the
infection. Although, most adults have acquired immunity to multiple
adenovirus types due to infections in childhood [160]. Furthermore,
adenoviruses can establish persistent infections in lymphatic tissues (tonsils
and adenoids), the gastrointestinal tract (lymphocytes) and in the urinary
tract [161-163]. These infections lead to shedding of the virus in the stool or
urine, for a long time after the acute infection [164, 165]. In addition, since
adenovirus infections can be asymptomatic, shedding of virus without prior
clinical manifestation has been observed [166]. Adenovirus type 36 has been
shown to be associated with obesity, both in animals and humans, however
the medical implications of these findings are debated [167-169].
2.6.2. Adenovirus infections in immunocompromised
patients
Opportunistic viral infections cause considerable morbidity and mortality in
immunocompromised patients. Adenoviruses are increasingly recognized as
pathogens causing severe infections and death, in these patients. The clinical
manifestations of adenovirus infections in persons with insufficient immune
system are more severe and prolonged with risk of disseminated disease and
multi-organ failure. Adenovirus infections are associated with high
morbidity and mortality rates in these patients [170]. The insufficient
immune system could either be inborn deficiencies (congenital or primary
immunodeficiency) or due to immunosuppressant medication when
undergoing haematopoetic stem cell (HSCT), bone marrow (BMT) or solid
organ transplantations (acquired or secondary immunodeficiency). In
addition, the adenovirus infection can either be a primary infection or a
reactivation of a persistant infection. However, due to the limited number of
persons with severe congenital immunodeficiencies, reports of adenovirus
disease within this group are limited. Several case reports describe fatal
adenovirus infections in patients with severe combined immunodeficiency
syndrome (SCID), Bruton´s X-linked agammaglobulinemia, DiGeorge
syndrome [171-174].
Adenovirus infections in patients with acquired immunodeficiencies have
been more characterized, though HSCT and BMT is also performed in
patients with congenital immunodeficiences, together with hematological
malignancies and other types of cancers. The incidence of adenovirus
infections in HSCT and BMT patients varies depending the age of the
patient,
T-cell
counts,
HLA-match
and
more.
In
general,
immunocompromised patients are a very heterologous group with expected
variations in disease incidence, severity and outcome. A recent review of
several reports concludes that adenovirus infections were reported in 5 - 47
18
% of the BMT and SCT patients [175]. Of these, 10 - 89 % had symptomatic
adenovirus disease with diarrhea as the most common manifestation
followed by respiratory disease and hemorrhagic cystitis. And ultimately, 6 70 % of the infected patients died due to the adenovirus infection [175]. In
children, general mortality rates greater than 50 % are frequently reported
[176]. Baldwin et al. reported 26 % adenovirus positive children and 9 % of
the adults were positive, following BMT [177]. This is similar to Howard et al.
that reported adenovirus in 23 % for children and 9 % of adults [178]. This
illustrates that adenovirus infections are more common in pediatric than
adult BMT and SCT patients, hence associated with higher mortality rates.
However, the underlying reason for this is currently unclear, though it is
believed that primary adenovirus infections are more frequent in children
compared to adults. In addition, primary infections are associated with
higher viral loads. Adenovirus infections are also a more pronounced
problem in children than adult solid organ transplant recipients.
In the solid organ transplant recipients, the adenovirus disease is usually
related to the transplanted organ [179]. For liver transplant recipients,
incidence of adenovirus infection is 6-11 % with symptoms as jaundice and
hepatitis, and associated with 53 % mortality rates if hepatitis is developed
[170]. Notably, it is mainly species C adenoviruses that are associated with
hepatitis in the immunocompromised host, whereas in immunocompetent
persons species C is associated with respiratory disease. Hence, reactivation
or transmission from the donor liver has been demonstrated in serum
negative recipients that have received donor liver from serum positive donor
[180]. In renal transplant recipients, adenovirus incidence has been reported
to 12 % leading to haemorrhagic cystitis and nephritis. Mortality rates of 18
% in these patients are reported, caused by species B adenovirus types [170].
In lung transplant recipients, adenoviruses have been reported in 50 % of the
recipients and were associated with respiratory failure, graft loss and death
[181].
Adenovirus infections in AIDS patients can be fatal, although since the
introduction of effective antiretroviral therapy the mortality rates have
decreased. Adenoviruses are most commonly isolated from the stool in AIDS
patients and may be the cause of diarrhea that frequently is observed in
AIDS patients [182, 183]. In addition, adenovirus-associated prostatitis,
hepatitis, nephritis, parotitis and encephalitis, have also been reported in
these patients [184-188].
To conclude, adenovirus infections in immunocompromised patients are
associated with severe morbidity and mortality rates. However, it is difficult
to assess how many patients that are affected by severe adenovirus infections
worldwide. European Medicines Agency (EMA) reported 2001 in an orphan
drug designation for ribavirin, that 0.6 – 3 people per 10,000 in EU are
affected [189]. Converted to current number of citizens in EU, this
19
corresponds to 30,000 to 150,000 people in EU. In addition, EMA reported
in 2014 that adenovirus infection in allogenic HSCT recipients affected less
than 0.2 in 10,000 people in the EU [190]. Furthermore, considering the
growing number of patients undergoing blood stem cell- and solid organ
transplantations worldwide, severe and potentially fatal adenovirus
infections will become an increasing threat without effective antiviral
therapies.
2.7. Antiviral drugs used in the clinic
Today there are no approved specific antiviral drugs available for treatment
of adenovirus infections. However, cidofovir and ribavirin are used offlicense for adenovirus therapy, while no other alternatives are available (Fig.
6). Cidofovir is a nucleoside analog that suppresses the replication of viral
DNA by inhibiting the viral DNA polymerase (mode-of-action for antiviral
drugs is addressed in more detail in section 4). Cidofovir displays broad
antiviral activity against several DNA viruses but is only regulatory approved
for treatment of cytomegalovirus retinitis in AIDS patients [191]. Several
reports describe the usage of cidofovir in immunocompromised patients with
adenovirus infections, however both successes and failures are reported. In
addition, cidofovir has low bioavailability when administrated orally, thus
intravenous administration is needed. Intravenous administration of
cidofovir is associated with nephrotoxicity due to intracellular accumulation
of cidofovir to toxic levels, in renal tubule cells [192]. Consequently, frequent
monitoring of renal function is required and potentially cidofovir needs to be
co-administrated with probenicid to reduce nephrotoxicity [193]. Initial
administration of cidofovir is recommended when adenovirus viremia is
observed but cidofovir is not recommended to use prophylactically [194]. In
general, monitoring adenovirus DNA load in blood is essential to be able to
foresee dissemination of the infection. However, Lion et al. reported that
adenovirus levels in stool could be observed 11 days prior viremia. Hence,
early initiation of antiviral therapy is possible for preventing progression of
the infection [195].
Ribavirin is also a nucleoside analog, with broad antiviral activity against
several viruses, both RNA- and DNA viruses. The exact mode-of-action of
ribavirin is not clearly understood and several mechanisms have been
suggested (see section 4). Ribavirin is approved in the US for inhalation
therapy of lower respiratory tract respiratory syncytial virus infections in
children and for systemic treatment, in combination with interferon, of
Hepatitis C infections, both in EU and US. As with cidofovir, several reports
describe the treatment of adenovirus, though with both successes and
failures [196-198]. The antiviral activity of ribavirin has also been suggested
to differ between adenovirus types in vitro and differences were also
20
observed in clinical isolates [199, 200]. According to “European guidelines
for treatment of adenovirus infections 2011”, by Matthes-Martin et al.,
ribavirin is not recommended for treatment of adenovirus infections [194].
Figure 6. Structures of cidofovir and ribavirin.
Other nucleoside analog drugs have been clinically used for treatment of
adenovirus infections, including ganciclovir and vidarabine [201, 202] (Fig.
7). However, the reports are sporadic case reports and more clinical
information is needed for conclusive data. Recent developments of lipid
conjugates of existing antiviral drugs have resulted in Brincidofovir
(CMX001) (Fig. 7). Brincidofovir, is an orally bioavailable lipid conjugate of
cidofovir, that uses cellular plasma membrane proteins (flippases) for rapid
entry into cells, that compared to cidofovir, results in higher intracellular
concentrations [203]. Brincidofovir has shown promising clinical effect in a
small number of patients and is currently under development by Chimerix
[204, 205].
Figure 7. Structures of Ganciclovir, Vidarabine and Brincidofovir.
21
Furthermore, adoptive cell therapy has been implicated in treatment of
adenovirus infections in immunocompomised patients. Infusion of
adenovirus-specific T cells from donors to adenovirus infected
immunocompromised patients has shown successful transfer of the
adenovirus immunity, thus potentially inhibiting virus reactivation [206].
Cell Medica has developed Cytovir™ ADV, which is in a phase I/II study that
will be completed in early 2015.
22
3. Herpes Simplex Virus
3.1. History
The first documented cases of Herpes Simplex Virus (HSV) infections can be
traced back to the ancient Greeks. Hippocrates used the term “herpes” to
describe lesions on the skin that appeared to creep or crawl [207]. In 1736,
Jean Astruc discovered that the condition was sexually transmitted among
French prostitutes [208]. In 1921, Gruter isolated HSV and demonstrated
the infectious nature of the virus, by serial transmission of the infection from
rabbit to rabbit [209]. In 1930, Andrews and Carmichael described that
herpes could recur in infected persons and in 1939, Burnet and Williams
defined the concept of latency for herpes simplex infection, i.e. once infected
the infection is persistent throughout life [210, 211]. Moreover, the
introduction of electron microscopy techniques in the 1930’s, was the onset
of virus characterization that has yielded the knowledge we have today about
HSV.
3.2. Taxonomy and classification
HSV is constituted of two types, HSV type 1 and 2 (HSV-1 and HSV-2) and
they belong to the subfamily Alphaherpesviridae, in the family
Herpesviridae. There are three subfamilies in the human Herpesviridae
family, α, β, and γ subfamily, each with different characteristics (Table 3).
Table 3. Classification of human herpesviruses.
Subfamily and characteristics
α - Short growth cycle, rapid spread.
Latent in sensory neurons.
Members
HSV-1 and HSV-2.
Varicella-zoster virus
β - Long growth cycle, slow spread.
Latent in secretory glands and
lymphoreticular cells.
γ - Growth primarily in epithelial
cells and B or T lymphocytes. Latent
in lymphocytes.
Cytomegalovirus
Human herpesvirus
6A, 6B and 7
Epstein-Barr virus
Human herpesvirus 8
Diseases
Labial and genital
lesions.
Chickenpox, zoster
(shingles).
Mononucleosis,
hepatitis, fetal
abnormalities.
Roseola
Mononucleosis,
Burkitt´s lymphoma,
nasopharyngeal
carcinoma.
Kaposi´s sarcoma
!
23
HSV-1 and HSV-2 share homology in approximately 50 % of their genome
sequences [212]. However, the site of infection differs between them. HSV-1
infects the oropharynx and the mucosa of the facial lips while HSV-2 infects
the genital mucosa, even though HSV-1 and HSV-2 may infect at either of
these sites. HSV-1 has been the prototype for all herpes viruses. Hence, the
herpes simplex biology, described below, is exemplified by HSV-1.
3.3. Virion structure
The HSV virion is approximately 120-200 nm in diameter and a linear
double-stranded DNA is located within the core (Fig. 8). The DNA is
encapsidated in an icosahedral capsid (similar to adenovirus) that is
approximately 100-110 nm in diameter. The capsid is composed of 162
capsomeres that mainly contain four proteins, VP5, VP26, VP23 and VP19c.
In addition, the capsid has a cylindrical portal, or tube, formed by the UL – 6
protein that mediates viral DNA entry and exit from the capsid [213]. The
capsid is enclosed in a lipid envelope, in which at least 12 different
glycoproteins are embedded (gB to gM) together with several
nonglycosylated proteins. These envelope proteins are observed as spikes,
with lengths of 10 to 25 nm, and about 600 – 750 spikes cover each virion
[214]. The space between the capsid and the envelope is called tegument [2].
The tegument contains several virus-encoded proteins that are introduced in
the cell upon infection and may play important functions in the viral
replication [215].
Figure 8. The structure of the HSV virion. Figure reprinted with permission
from the Swiss Institute of Bioinformatics.
24
3.4. Genome organization and gene products
The length of the HSV genome is estimated to be approximately 150 kb and
is linear within the capsid, though immediately after release into the cellular
nucleus, the genome circularizes [216]. The genome is generally divided in
unique regions, long (UL) and short (US), according to their relative length,
and is flanked by inverted repeats. The repeats for UL are designated a and b,
whereas for US the repeats are called c (Fig. 9). In total, the genome contains
at least 84 different genes. The gene name defines in what sequence (L or S)
and in what order they are found (1-56 in L and 1-12 in S) in the genome. In
addition, some genes are placed in the b and c segments. The initially
described proteins were named virion protein (VP) or glycoprotein (gp) or, if
found in infected cell extracts, ICP (Infected Cell Protein) [2].
Figure 9. The HSV genome map. Figure reprinted from [217] with
permission from the publisher.
25
3.5. Viral life cycle
3.5.1. Binding and uptake
The glycoproteins, or spikes, of the envelope mediate the initial attachment
to the cells (Fig. 10). The gC, gB and gD glycoproteins are necessary for
attachment to primarily heparan sulfate and potentially also chondroitin
sulfate on the cell surface [218]. Initial attachment is followed by the binding
of gD to specific co-receptors that triggers fusion of the envelope to the
plasma membrane. Four different co-receptors have been characterized:
herpes virus entry mediator (HVEM), nectins (-1 and/or -2), specific sites on
heparan sulfate generated by certain 3-O-sulfotransferases and paired
immunoglobulin-like type 2 receptor alpha (PILRα) [219-221]. The
glycoproteins gH/gL have been suggested to regulate the fusogenic
properties of gB [222]. However, exactly how the virion fuses with the
cellular membrane is not fully understood, although conformational changes
in the gD protein seems to be involved [223]. The cytoplasmic entry of the
virion is pH-independent (different from adenovirus), thus the tegument
proteins that covers the capsid are released into the cytoplasm. As a
consequence the host macromolecular metabolism and DNA synthesis
ceases [2]. Subsequently, the capsid is transported along the microtubule, by
the dynein protein, towards the nuclear pore. Upon arrival to the nucleus,
the viral DNA is introduced in the nucleus where it immediately circularizes
[224].
Figure 10. The receptors and ligands for HSV entry. Reprinted from [225]
with permission from the publisher.
26
3.5.2. DNA replication
Once the viral DNA is in the nucleus, transcription of the viral genome
occurs by the host cell RNA polymerase II. The viral genes can be classified
into immediate early (α), early (β) and late genes (γ1 and γ2). The immediate
early genes are regulatory proteins that, upon expression, activate the early
and late γ1 genes. The γ2 genes are expressed after the onset of the DNA
replication. The early gene products include enzymes (e.g. thymidine kinase
and DNA polymerase) that are essential for virus DNA replication. After the
expression of the required gene products, the HSV DNA replication starts in
a theta replication but then switches to a rolling circle mechanism [226, 227]
(Fig. 11). The UL9 protein binds to the origin sequence, CGTTCGCACTT, and
recruits ICP8, which is a single-stranded DNA binding protein. ICP8 can
unwind the double-stranded DNA and thereby enable binding of both the
helicase-primase complex and the DNA polymerase complex. The helicaseprimase complex further unwinds the double-stranded DNA and
consequently, allows the polymerase complex to replicate the viral DNA
[228].
Figure 11. Illustration of the HSV DNA replication. Figure reprinted from
[229] with permission from the publisher.
27
3.5.3. Assembly and release
The assembly of progeny virions occurs in the nucleus. The major capsid
protein VP5, together with VP26 and several additional proteins form an
immature capsid around a protein scaffold, containing VP21, VP22a and
VP24 (Fig. 12). The viral DNA is then packed inside the capsid. Motor
proteins, UL-6, UL-15 and UL-28, which recognize the a-sequence in the
viral genome, mediate the packaging of the DNA. When they encounter the
next a-sequence, the packaging is aborted. Subsequently, the scaffold
proteins are cleaved by the protease and are ejected from the capsid [230,
231]. In this process, the DNA is enclosed and the capsid undergoes
morphological changes to form a more robust capsid structure [2, 228].
Figure 12. HSV DNA packaging in capsid. Reprinted from Wiley
publications [232] with permission (Wiley publications Copyright © 2011,
John Wiley & Sons, Inc. All rights reserved).
28
The mechanism how the progeny viruses are released from the nucleus,
and finally from the cell, is not clear. One theory is that the nuclear pores
become enlarged thus allows the capsid to exit to the cytoplasm.
Subsequently, Golgi membranes envelop the naked capsids in the cytoplasm
[233]. Another theory is that the capsis are initially enveloped by the nuclear
inner membrane but is de-enveloped upon release from the nuclear
membrane and is the re-enveloped by Golgi membranes in the cytoplasm
[234]. Furthermore, the third and original theory is that the capsids are
enveloped by the nuclear inner membrane and is released from the nucleus
in an enveloped form to the cytoplasm [235]. Common for all theories is that
the enveloped capsids is in, or ends up in, secretory vesicles in the cytoplasm
that later fuse with the plasma membrane to release the enveloped virions
from the cell.
3.6. Latency and reactivation
All today known herpesviruses can establish life-long latent infections. The
precise mechanism for this is however not known. Although, certain events
have been characterized which are described here. Following infection at the
primary site, the progeny virus is spread within the tissue and infects sensory
neurons. This facilitates a retrograde transport of the capsids along the
axons towards the cell bodies of dorsal root neurons that innervate the
primary site of infection [236]. In general, HSV-1 resides in the trigeminal
ganglia, whereas HSV-2 resides in the sacral ganglia. Dynein, the motor
protein, transports the HSV capsids to the neuronal cell bodies [237]. The
viral DNA enters the nucleus and is circularized and associates with
nucleosomes in a chromatin structure [238]. This chromatin structure is
believed to suppress most of the gene expression to keep the infection latent
[239]. The only viral gene products abundantly expressed are specific RNAs,
called latency-associated transcripts (LATs) [240]. The roles of LATs are
controversial, however common themes are that they protect the neurons
from death and that they are involved in suppression of the lytic genes [241,
242].
Upon local trauma to the innervated primary site of infection, or upon
systemic stress, e.g. UV-light, hormonal imbalance and emotional stress, the
latent virus can be reactivated. It has been shown that induction of
reactivation causes decreased LAT levels and disruption of the chromatin
structure [243, 244]. This triggers the replication of viral DNA and
subsequently the production of progeny virus. The progeny capsids are
transported in an anterograde direction along the axons towards the primary
site of infection. Kinesins are the motor proteins that transport the capsids in
anterograde direction [245]. Upon arrival at the primary site of infection, the
29
tissue is infected and the virus is shed. However, reactivated infections can
shed virus asymptomatically, which have impact on the viral transmissions
in the population [246].
3.7. HSV spread and infections
HSV is highly contagious. It is most commonly spread through direct contact
of infected saliva or secretions from viral ulcerations. However, transmission
from aerosol droplets has been reported [247]. The lipid envelope of the
virion is sensitive to ethanol, heat, chlorine and dry atmosphere. HSV
infections occur worldwide without any specific seasonal distribution.
Geographical location, socioeconomic status and age, influence the
seroprevalence of HSV among populations. The highest incidence of both
HSV-1 and -2 are in developing countries, e.g., in African countries up to 82
% of the women are seropositive for HSV-2 [248]. In USA, 21 % of the
women and 12 % of the men are infected with HSV-2, with higher incidence
among Afro-Americans [249]. In general, women are more susceptible to
HSV-2 infections, because HSV is sexually spread more easily from men to
women than vice versa. Furthermore, 58 % of the Americans are positive for
HSV-1 [250]. The incidence of HSV varies among European countries as
well. The highest incidence of HSV-1 is reported in Bulgaria with 84 %,
whereas in Finland the seroprevalence is 52 %. Moreover, the HSV-2
seroprevalence is 24 % in Bulgaria, compared to 13 % in Finland. The lowest
incidence of HSV-2 is in England and Wales with 4 % [251]. Conclusively,
both HSV-1 and HSV-2 infections are common in societies, albeit HSV-1 is
more common than HSV-2. Socioeconomic status and geographical location
affects the incidence among populations greatly.
3.7.1. HSV infections in immunocompetent persons
In otherwise healthy persons, both HSV-1 and HSV-2 infections are
commonly associated with recurrent ulcerations and blisters. However, HSV
infections have other clinical manifestations as well. Primary infections, if
symptomatic, are generally more severe than recurrent infections and can
cause a more widespread infection with respiratory disease, fever, swollen
tonsils, rash, and more [252]. Most primary HSV-1 infections occur in
children and the infections are most often asymptomatic [2]. However, if
HSV-1 is acquired later in life, pharyngitis and upper respiratory tract
infections are more common [253]. Reactivated HSV-1 is associated with
mucosal ulcerations at the mucocutaneous junction of the lip (known as cold
sores or fever blisters). In addition, HSV-1 can cause generalized
30
dermatologic disease such as primary herpes dermatitis, eczema herpeticum
(primary HSV-1 infection of the skin) and traumatic herpes (infection in
wounds due to burns or lesions). Herpetic whitlow is an occupational hazard
among dentists, hospital personnel and wrestlers, where HSV-1 or HSV-2
infects skin abrasions of the fingers [254, 255]. Furthermore, HSV-1 can
infect the eye and lead to stromal keratitis, which is one of the leading causes
of blindness in the Western world [256]. Herpes simplex encephalitis is a
rare infection of the central nervous system, caused mainly by HSV-1. It is a
severe condition associated with very high mortality rates if untreated.
Moreover, serious neurological sequelae, with memory impairments and
personality disturbances are common in survivors of HSV encephalitis [257].
Primary genital HSV-2 infections are symptomatic in approximately 40 %
of the cases [258]. Primary symptomatic infections are associated with more
severe and prolonged duration of symptoms, compared to recurrent
infection. Genital HSV-2 symptoms include vesicles on the genitals, that
develops to painful ulcers which ultimately crusts. In addition, especially in
females, primary genital HSV infection can be associated with systemic
symptoms such as fever, headache, malaise and muscle pain. HSV-1 is
increasingly recognized as a pathogen causing genital infections, probably
due to alterations in sexual behavior in society. However, genital HSV-1
infections seem to be less severe and not as prone to cause recurrent
infections, compared to HSV-2 [259]. Genital HSV infections and HIV-1
positivity is tightly linked together. One reason for this is that genital
ulcerations can increase the transmission rate of HIV-1 [260]. The risk of
HSV transmission between mother and baby is between 30-50 % if the
mother acquires a primary genital HSV infection in the third trimester [261,
262]. Neonatal HSV infections are associated with high mortality rates and
great morbidity. Hence, caesarian sections are recommended if the mother
has symptomatic genital infection. Furthermore, there is a correlation
between cervical HSV infections and miscarriage [263].
3.7.2. HSV infections in immunocompromised patients
Both HSV and adenovirus (described in section 2.6.2), are problematic
opportunistic pathogens in patients with suppressed immune system, e.g.
BMT-, SCT-, solid organ transplantation recipients. The symptoms of HSV
infection, in these patients, are more severe and generalized. In addition,
suppression of the immune system can potentially trigger the reactivation of
an endogenous latent infection. However, in distinction to adenovirus
infections in the immunocompromised patients, effective antiviral therapies
can suppress HSV disease. The rate of HSV reactivation among SCT
recipients has been reported to be up to 80 %, 4-6 weeks after
31
transplantation when the immunosuppression is the most pronounced
[264]. The most common symptoms are mucocutaneous disease, more in the
orofacial region than the genital tract [265]. Oropharyngeal infections and
esophagitis are also frequently observed, with symptoms as, nausea, chest
pain and vomiting [266]. In addition, HSV caused pneumonitis, hepatitis
and meningitis have been reported in immunocompromised patients [267269]. The golden standard antiviral drug for HSV treatment today, is
acyclovir (see section 3.8). Acyclovir is extensively used for treatment in the
clinic and prophylactically used in immunocompromised patients to prevent
onset of herpesvirus disease. However, in HSV (or other herpesvirus)
seronegative patients, no prophylactic antiviral treatment is necessary due to
that these patients are at low risk of acquiring primary infections. In
seropositive patients, the current recommendation is that acyclovir should
be given intravenously or orally, 3-5 weeks (or longer) after the
transplantation or chemotherapy [270]. The extensive and preemptive
administration of acyclovir, in mainly immunocompromised patients, is
associated with development of acyclovir-resistant HSV that cause more
progressive disease with a prolonged symptomatic phase when reactivated.
Acyclovir-resistant HSV is most commonly isolated from patients with
severe mucocutaneous disease [271, 272]. However, isolation from
pneumonia-, esophagitis- and encephalitis affected patients is also reported
[273-275]. The isolation rate of acyclovir-resistance is higher in
immunocompromised patients than immunocompetent persons. The general
prevalence of HSV with reduced susceptibility to acyclovir, in
immunocompromised patients has been reported to be between 3,5 - 10 %,
compared to 0.1 – 0.7 % in immunocompetent persons [276]. Thus, the risk
of developing acyclovir-resistance is more than ten-fold higher in
immunocompromised patients compared to immunocompetent persons.
Contradictory and a bit surprisingly, a long duration (> 1 year) of acyclovir
therapy, in immunocompromised patients, is associated with low incidence
of acyclovir-resistance [277]. The reason for this is unclear, although based
on my experience the acyclovir-resistant isolates display reduced “fitness”
and have a potentially lower replication rate, compared to acyclovir-sensitive
isolates. In addition, GlaxoSmithKline that markets acyclovir (Zovirax), has
supported the authors of this report which could undermine the credibility
[277]. However, when acyclovir-resistance is observed, alternative antiviral
drugs with differences in their mode-of-action are used. These drugs are
mainly foscarnet and cidofovir that are discussed in section 3.8.
32
3.8. Antiviral drugs used in the clinic
Acyclovir (Zovirax®, Fig. 13) is the golden standard antiviral drug for HSV
treatment today. It is mainly used topically and intravenously and not orally
administrated, due to its low oral absorption. The structure of acyclovir is
based on the discovery of nucleosides from the Caribbean sponge Tethya
crypta [278, 279]. These nucleoside structures led to the synthesis of Ara-A,
or vidarabine, which was primarily aimed for anti-cancer use but was retargeted towards antiviral use after the discovery of its antiviral activity
[280, 281]. Vidarabine is still used in the clinic, mainly as an eye ointment
for HSV keratitis. In addition, one of the first antivirals was trifluridine
(Viroptic®), which also is still used in the clinic as eye drops. Vidarabine was
previously used intravenously but was later replaced by acyclovir, which had
higher selectivity and potency [282-284]. Elion, G., et al. published the
discovery of acyclovir in 1977 and in 1979 a patent application was filed.
Elion, G., was not included among the inventors but she received a shared
Nobel prize in 1988 [285]. In the 1980’s acyclovir was introduced to the
market and since then, several analogs to acyclovir have been developed
(Fig. 13). Valacyclovir (Valtrex®), which is a L-valyl-ester prodrug of
acyclovir, displays better bioavailability after oral administration than
acyclovir and can thus be administrated orally for treatment of various
herpesvirus diseases [286]. Penciclovir display antiviral potency similar to
acyclovir but is, as acyclovir, poorly absorbed when given orally. This led to
the development of famciclovir, which is an oral diacetylated prodrug of
penciclovir [287]
Figure 13. Structures of Acyclovir and Penciclovir, with their respective
prodrugs Valacyclovir and Famciclovir.
33
Since acyclovir and penciclovir have similar mechanisms of antiviral
action (antiviral mode-of-actions are described below in section 4),
acyclovir-resistant HSV isolates are also resistant to penciclovir [288].
Hence, alternative antiviral drugs with different mode-of-action are needed
for treatment of acyclovir/penciclovir-resistant HSV. Foscarnet (Foscavir®)
is the second in line drug used (Fig 14). Foscarnet is given intravenously for
7-21 days, or until complete healing is observed [270]. However, foscarnet
usage can be associated with nephrotoxicity, hemoglobin disturbances and
seizures [289]. If foscarnet therapy was unsuccessful, the third in line drug is
cidofovir (Fig. 6). As in the case when used to treat severe adenovirus
infections, Cidofovir is given intravenously in combination with probenicid
to reduce potential nephrotoxicity.
Figure 14. Structure of Foscarnet.
A new class of anti-HSV drugs that are in the pipeline is the helicaseprimase inhibitors, with Pritelivir (BAY571293, AIC316) in the lead (Fig. 15).
Pritelivir is developed by AiCuris and has shown promising reduction of
HSV-2 symptoms with low toxicity, both in animal models and in phase II
clinical trials [290, 291]. Unfortunately, the food and drug administration
(FDA) has recently halted the development of Pritelivir due to skin and
haemotological toxicity in monkeys [292].
Figure 15. Structure of Pritelivir.
34
4. Antiviral strategies and drug modes-ofaction
Today there are over 200 different virus species that are known to infect
humans and three to four additional species are discovered each year [293].
Each one of these viruses has different ways to infect the eukaryotic cell and
secure progeny spread. However, all human viruses share general steps in
their infection cycle (Fig. 16). Consequently, antiviral drugs that target each
one of these steps have been developed.
Figure 16. General steps and antiviral drug targets in viral infection cycle.
This section will cover the stages where antiviral drugs have been
developed to combat adenovirus and HSV infection. Currently, the main
antiviral group for adenovirus and HSV treatment is the nucelsoside analogs.
In addition, recent focus on entry and protease (maturation) inhibitors has
yielded interesting compounds. Hence, these groups are discussed here.
Moreover, since both adenovirus and HSV are DNA viruses and share
common molecular features, especially in the DNA replication machinery, an
overlap of antiviral drugs inhibiting both adenovirus and HSV is expected.
35
Figure 17 displays a generalized infection cycle for both adenovirus and HSV.
Steps in the infection cycle that have been targeted for antiviral therapy are
marked red.
Figure 17. A generalized infection cycle for adenovirus and HSV. Currently,
most antivirals against adenovirus and HSV target the DNA replication,
binding and protease (maturation) steps, as marked in red.
4.1. Binding inhibitors
Inhibition of the initial step of the infection cycle is an effective approach to
block viral replication and to inhibit viral spread within the tissue.
Attachment of the virus to host cell receptors can be blocked by compounds
that either bind to the virion or on the cellular receptor. Focus has been on
development of soluble compounds that mimic part of the cellular receptors
that interact with the virion. One example is sialylated compounds for
treatment of adenovirus EKC [294]. By attachment of the cellular receptor
sialic acid on carrier molecules, the viral spread is reduced [94]. Adenovir
Pharma AB develops these sialyated compounds and is currently in a phase
36
II clinical study with an eye drop administration [295]. Separately, NovaBay
Pharmaceuticals is currently conducting clinical trials for treatment of
adenovirus-caused EKC with their NVC-422 drug, a N-chlorotaurine
analogue. NVC-422 has broad antimicrobial activity, and acts as a virucidal
agent for adenovirus type 5 by oxidizing amino acids in the hexon and fiber
proteins [296]. In addition, virucidal activity of NVC-422 was observed on
HSV-1, coxsackievirus A24, enterovirus 70, adenovirus types 8, 19, 37, which
all are ocular pathogens [297].
Adenovirus type 5 manifests a strong liver tropism, which impairs the
efficacy of type 5 as a vector in gene therapy. Hence, small molecules have
been identified that reduce the strong liver tropism [298]. In the blood,
factor X, a central component of the coagulation system, binds to the virion
and mediates virus entry in, specifically, liver cells [299]. Consequently,
inhibition of the specific liver tropism may aid the gene therapy vector to be
distributed to other tissues in the patient.
In the HSV field, sulfated polysaccharides that mimic the HSV receptor
heparane sulfate, are under development [300]. In addition, several peptides
and proteins have displayed antiviral activity by blocking the viral entry.
Example of this is lactoferrin/lactoferricin that can block both HSV and
adenovirus entry by either interacting with glycosaminoglycan receptors on
the host cell or by direct binding to the virion [301-304].
4.2. Inhibitors of DNA replication – Nucleoside/Nucleotide
analogues
The most successful approach for antiviral drug development has been the
nucleoside and nucleotide analogues. However, the initial development of
nucleoside analogues was aimed at treatment of cancer, and it was later
discovered that some compounds were more suited to inhibit the viral
replication. Today, nucleoside analogues are used as therapeutic drugs for
the treatment of both cancer and viral diseases. Nucleosides and nucleotides
are endogenous molecules and are the building blocks of DNA and RNA.
They are inserted in the growing DNA strand by the DNA polymerase.
Nucleotides have a phosphonate group attached to the sugar moiety of the
nucleoside structure. However, the term nucleoside analogues may cover
both nucleosides and nucleotides. Currently, there are over 25 approved
antiviral nucleoside analogues [305]. The general mode-of-action features
are
similar
among
them.
Hence,
the
mode-of-action
for
nucleoside/nucleotide analogues is exemplified here by cidofovir and
acyclovir that are used against adenovirus and HSV.
Cidofovir, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine or
HPMPC, is an acyclic nucleoside (cytidine) phosphonate with broad antiviral
37
activity against several DNA viruses (Fig. 6 and 18). The phosphonate
mimicks a phosphate group but is protected against cleavage by cellular
esterases [306]. Nucleotides need three phosphates to be incorporated in the
DNA. Since the phosphonate group functions as a phosphate, cidofovir needs
to be phosphorylated twice. Once inside the cell, cidofovir is phosphorylated
by pyrimidine nucleoside monophosphate kinase. The second and last
phosphorylation is performed by nucleoside diphosphate kinase, pyruvate
kinase or creatine kinase [307] (Fig 17). Both these phosphorylation steps
can occur in both infected and uninfected cells, though is more efficacious in
infected cells [308]. When cidofovir is di-phosphorylated, at the
phosphonate group, it is a more specific substrate (or competitive inhibitor)
for viral DNA polymerases than cellular DNA polymerases, hence the
antiviral specificity [309, 310]. Integration of the mono-phosphate cidofovir
molecule into the growing DNA strand terminates further chain elongation
(Fig. 18). However, two consecutive incorporations of cidofovir is needed for
chain elongation termination, at least in cytomegalovirus inhibition [311].
Figure 18. Mode-of-action for cidofovir. Figure reprinted from [312] with
permission.
38
Acyclovir, 9-[(2-hydroxyethoxy)methyl]guanine, is an acyclic nucleoside
(guanosine) analogue specifically active against herpesviruses (Fig. 13 and
19). The herpesvirus specificity is due to the initial phosphorylation of
acyclovir, conducted by the virus-encoded thymidine kinase (TK) [285, 313].
Acyclovir monophosphate is successively phosphorylated twice by cellular
kinases [314] (Fig. 18). Similarly to cidofovir, the triphosphate structure of
acyclovir is a substrate for the viral DNA polymerase, which incorporates the
mono-phosphate structure in the DNA strand and further chain elongation is
inhibited. Resistance to acyclovir is most commonly due to mutations in the
viral TK gene that inhibits the initial phosphorylation of acyclovir [315].
However, mutations in the viral DNA polymerase, with or without TK
mutations have been reported as well [274, 316].
Figure 19. Mode-of-action for acyclovir. Figure reprinted from [312] with
permission.
Foscarnet, phosphonoformic acid (Fig. 14), is used on acyclovir-resistant
HSV and is active against all herpesviruses. It inhibits the viral DNA
polymerases in a different way compared to acyclovir. The structure of
foscarnet mimics pyrophosphate, which is produced during DNA chain
elongation [317]. Consequently, foscarnet binds to pyrophosphate sites on
the viral DNA polymerase and inhibits the release of pyrophosphates formed
when tri-phosphorylated nucleotides are incorporated in the DNA [276]. It
has been stated that foscarnet is about 100-fold more selective for herpes
39
DNA polymerase than cellular DNA polymerases [318]. Foscarnet is not
active against adenoviruses [319].
Ribavirin, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Fig. 6),
has previously been used for treatment of adenovirus disease but is currently
not recommended as antiviral alternative [194]. The mode-of-action is
controversial since several contradictory observations have been reported.
Ribavirin is similarly to cidofovir, tri-phosphorylated by cellular kinases and
it has been observed that ribavirin depletes nucleotide pools, causes lethal
mutagenesis of viral RNA genomes, potentiates interferon action, reduces
viral transcription, and more [320-323].
4.3. Inhibitors of viral maturation - Protease inhibitors
Targeting viral proteases is a promising antiviral strategy since they are
virus-encoded and most often have essential roles in the infection cycle
[324]. Thus, selective inhibition of the viral protease is less likely to have
cellular side effects. Development of protease inhibitors has been focused on
HIV-1 and Hepatit C Virus (HCV). However, the adenovirus and HSV
protease have recently been assessed as potential antiviral targets. Both the
adenovirus protease and the HSV protease are essential for infective progeny
virions and they are involved in the late virion maturation process (See
section 2.5.5. and 3.5.3, respectively). Broad cysteine protease inhibitors
have antiviral activity against adenovirus [325]. Additionally, by utilizing a
virtual docking approach, several small-molecule inhibitors of the
adenovirus protease have been identified [43]. Characterization of these
compounds is needed to determine their in vivo potential. In the herpesvirus
field, protease inhibitors have been identified although mainly focused on
the CMV protease [326, 327]. More general cysteine protease inhibitors have
been reported to have activity on HSV protease [328]. In addition, the
clinically used HIV-1 protease inhibitor Nelfinavir, has been reported to
inhibit the maturation and export of HSV-1 [329].
4.4. Antiviral prospects
A recent aspect in antiviral drug development is to target the host cell
instead of the virus, since there are several events in the viral infection cycle
that are dependent on cellular proteins. Advantages of this approach may be
that virus is less prone to develop resistance to cellular proteins and that
these antiviral drugs will be active against viral mutants that already have
developed resistance to conventional antivirals. Antiviral effects mediated
via cellular targets would also be expected to have a broad antiviral activity
since several viruses require mutual cellular proteins. In addition, a
combination therapy with conventional antiviral drugs and cellular antiviral
40
drugs would have beneficial effects. Unfortunately, targeting cellular
proteins may be associated with more cytotoxicity and side effects, although
several drugs on the market today targets cellular proteins without severe
toxicity. Identification and inhibition of a cellular protein that is upregulated
during viral infection would be a desirable target.
Cyclin-dependent kinases (CDK) are essential for replication of several
viruses and have been expoited as antiviral targets. CDK inhibitors such as,
roscovitine with selectivity for CDK2, CDK7 and CDK9, and the ATP
competitor olomoucine, with selectivity for CDK2/cyclin interaction, have
displayed antiviral activity against HSV-1 and CMV, respectively [330, 331].
Since then, several purine containing CDK inhibitors have been reported to
have antiviral activity against HSV-2, HIV, Varicella-zoster virus (VZV) and
Epstein-Barr virus (EBV) [332-334]. Roscovitine has displayed both positive
and negative results in mouse models. In SCID mice with skin VZV
xenografts, viral reduction was observed, whereas in a HSV-1 keratitis model
no significant reduction was seen with roscovitine treatment [335, 336].
Interestingly, cidofovir in combination with the CDK inhibitor olomoucine II
have been reported to almost completely eliminate the spread of adenovirus
type 4 spread from infected cells [337]. However, to truly address CDK
inhibitors as antiviral agents, further developments in vivo are needed.
Moreover, with the increasing acceptance of cellular targets as antiviral
agent, several reviews with focus on cellular targets as antivirals have been
published. These include influenza virus, HIV (entry inhibitors) and
arenavirus [338-340].
Another evolving aspect in antivirals, that is not drug-related, is adoptive
T-cell transfer. The first report of adoptive transfer of immunity dates back
to 1955 [341]. However, with the recent expansion of molecular techniques
and knowledge the idea is more and more clinically used. Briefly, in this
case, adoptive T-cell transfer is collection of virus-specific T-cells that, after
in vitro expansion, are transferred to the patient. The virus-specific T-cells
are collected from seropositive donors and transferred to seronegative
recipients. This approach has proven successful for a number of viral
infections (as well as cancers). In immunocompromised patients, the
depletion of virus specific T-cells is a prominent risk for disseminated
infection of opportunistic viruses, such as adenovirus. Hence, preemptive
treatment with adenovirus specific T-cells infusion has been reported to
reduce the rate of primary infections and reactivations [206, 342]. Due to the
effective therapy with acyclovir and its analogs, adoptive T-cell transfer is
not emphasized for recurrent HSV infections. However, for other more latent
species of herpesvirus, such as CMV and EBV that also may have major
complications in the immunocompromised patient, adoptive T-cell transfer
is implicated [343-345].
41
5. Drug discovery and development
The driving force for each drug discovery project is to find cures for diseases
or conditions that lack satisfactory therapies. Knowledge of the underlying
mechanisms for the disease is critical to identify possible drug targets and to
develop assays that can distinguish active compounds (hits) from non-active.
Basic research within the academic, is most often the foundation for the drug
discovery process. The drug discovery process is divided in several phases
and in general each phase must be completed before proceeding to the next
(Fig. 20). However, based on the outcome, reassessments within the process
are frequent.
Figure 20. The drug discovery and development process.
5.1. Targets and assays
Most often, the target is a protein essential for manifestation of the disease.
Enzymes and receptors are attractive drug targets since their activity easily
can be measured [346]. Target identification is an important, if not the most
important, step in drug discovery since all of the succeeding steps are
dependent on a good target. A good target has to fulfill many criteria such as,
i) the activity of the target is specific for the disease; ii) it is safe to modify the
function of the target; iii) the target is “druggable”. Druggable targets are
prone to interact with compounds and upon compound interaction, the
function of the target may be altered to benefit disease treatment. In
addition, the function of the target can easily be measured in vitro and in
vivo. When screening for compounds that directly affect the function of a
target protein, the assay is called biochemical or target-based assay.
However, when assessing the impact of compounds in whole cells, e.g. using
a GFP-expressing viral vector, the assay is called cell-based or phenotypic.
Prior to screening, the assay needs to be optimized and quality assured. It is
fundamental to have a significant separation between positive and negative
42
signals to be able to select hit compounds. By calculating the Z´-factor of the
assay, the separation and ultimately the quality of the assay can be
determined [347].
5.2. Compound Libraries
Today there are numerous commercially available compound libraries. The
size varies between a few hundred compounds to several hundred thousands
compounds. Initially, the philosophy was the bigger the better, i.e. the aim
was to screen as large library as possible. And still in the pharmaceutical
industry, the compound collections can be one million compounds and
beyond. However, more and more are now aiming for more focused libraries.
These smaller libraries can be diverse and still represent the chemical space,
although with less number of compounds, and more focused towards
interaction with known target families, e.g. kinases, ion-channels, hormone
receptors etc. In addition, NMR-guided fragment-based library screening is
common. Fragment-based libraries consist of smaller compound entities
that potentially can interact with different parts of the target. Hence, by
combining the segments a larger chemical space can be covered compared to
conventional compound libraries [348]. The compound source is mainly
from synthetic chemistry, e.g. fragment-based, diverse and targeted libraries,
however commercial natural product libraries are also available that include
samples from e.g. plants, invertebrates and microorganisms. In addition,
library collections with already developed drugs are available. These
compounds may have been used in the clinic or have passed phase 1 clinical
trials. Screening these drugs against new targets may lead to that approved
drugs can be re-targeted for other indications.
5.3. Screening and lead identification
High-throughput screening (HTS) is widely used in the pharmaceutical
industry and it enables large numbers of compounds to be screened for
biological activity in a short period of time. Using fully integrated robots for
liquid handling and pipetting, 100,000, or more, compounds can be assessed
against a target each day [349]. In general, HTS is mainly used for targetbased screening, whereas for cell-based screening, High-content screening
(HCS) is mainly used. The end analysis for HTS is measurement of a proteins
activity using, e.g. microplate readers, whereas for HCS, the cellular
phenotype is analyzed using, e.g. automated microscopy. In an academic
setting, screening may be directed against diseases but can also be directed
to identify chemical probes that alter specific biological pathways. These
chemical probes are used to understand basic biological processes.
Additionally, academic screens usually analyze smaller sets of compound
43
libraries and are not fully automated but most often semi-automated, e.g.
with pipetting robots that aid manual laboratory work.
When the screening assay is developed and quality assured, the primary
screen is usually performed at one concentration in a single experiment.
Based on the Z´-factor and preferably the activity of positive control
compounds, the criteria for hit selection are determined. Hit selection rate
describes how many compounds of the compound library that fulfill the
criteria. The activity of the hits needs to be confirmed, primarily by re-testing
the compounds under the same conditions and generation of dose-response
curves. In addition, the activity of the hit compound is validated, e.g. to
ensure that the signal is truly due to interaction with the target and not
general toxicity or assay interference. Subsequently, the hit compounds are
tested in orthogonal assays, i.e. different assays that quantify the activity of
the compounds. Both the efficacy and the toxicity of the compound are
addressed. Based on the outcome, compounds are selected for medicinal
chemistry programs to optimize the efficacy and cytotoxicity of the
compounds. Structure-activity relationship (SAR) analysis assesses the
chemical space around a compound with the intention to find structural
analogues with improved activity. Most often the analogues are synthesized
in generations and the activity of each generation guides the synthesis of the
next. The SAR analysis narrows down the number of hits to a few
compounds with improved activity. These optimized hits are called leads.
5.4. Lead optimization
Lead optimization involves both in vitro and in vivo assessment and
includes a variety of parameters. The overall aim is to determine the
pharmacological aspects and to ascertain that the lead compounds have
potential to be used as drugs in man. Exploratory pharmacokinetic (PK)
analysis of the leads is performed, both in cell culture and in animal models,
which mainly are mouse models at this phase. PK parameters include the
ADME steps (Absorption, Distribution, Metabolism and Excretion) that
describe behavior of the compounds in an organism (here, most often in
mouse). If a pharmaceutical formulation of the compound is needed, the
compound liberation rate from the formulation, among other parameters, is
analyzed as well. Thus, L may be added to the ADME parameters to form
LADME. In addition, preclinical proof-of-concept studies in vivo are
performed to establish the activity of the compound in an organism. With
medicinal chemistry the compounds are optimized and modified if any
unfavorable results are observed. Based on the outcome for each lead
compound, one, or possibly two to three compounds are selected for further
development.
44
5.5. Drug development
Drug development is the later stage when candidate drugs will be assessed
with a clear aim to reach the market and the patients. Drug development
includes thorough preclinical analysis in two or more species of rodents and
non-rodent animal models. Here, a full ADME-tox analysis is performed to
determine the absorbance rate of the drug to the blood, how the drug is
distributed in the body, how the drug is metabolized, the complete toxicity of
the drug with its metabolites and the mechanism when drug metabolites are
excreted. The drug and its formulation, needs to be produced according to
good manufacturing practice (GMP) to secure high quality. Once the
preclinical process is complete, a regulatory approval is needed for candidate
drugs to enter the phase I-III clinical assessment in humans. Briefly, in
phase I trials the drug is administrated to a limited number of healthy
volunteers to assure that the drug is tolerated without apparent side-effects.
Phase II trials includes the efficacy of the drug against the target in a larger
group of patients with the disease/condition. In addition, the intervals of the
dosage of the drug are analyzed. Phase III trials includes more patients with
the disease, which are closely clinically monitored to detect any potential
side effects of the drug. When the drug is approved, by regulatory
authorities, it can be marketed and become accessible for the patients.
In summary, it takes between 10-15 years to develop a new drug, including
the drug discovery phases. To develop a drug that reaches the market
generally costs more than 1 billion US dollars. However, this number is
increasing for each year and currently, big pharmas can spend up to 5 billion
US dollar per drug [350].
5.6. Orphan drugs
Adenovirus infections in immunocompromised patients are associated with
morbidity and high mortality rates, especially in children (see section 2.6.2).
Fortunately, the number of patients affected by life-threatening adenovirus
disease is low and the condition is considered to be rare. Rare conditions are
seldom attractive for larger pharmaceutical companies, mainly due the
massive costs when developing a drug contra the small market for rare
conditions. However, it has been estimated that between 6 – 8 % of the total
EU population suffer from rare diseases [351]. Hence, to stimulate
development of drugs for rare conditions, the FDA implicated an orphan
drug act in 1983 [352]. This legislation was successively adopted by
Singapore in 1991, Japan in 1993, Australia in 1997 and in 2000 by Europe
[353]. The orphan drug legislation includes stimulatory incentives if the
development of a drug is intended for the treatment of a rare disease.
45
However, the disease needs to fulfill certain criteria to be classified as rare.
These criteria differ between countries. In EU and US the number of persons
affected by a rare disease should not exceed 5 in 10,000 citizens. In Japan,
any disease with fewer than 50,000 prevalent cases (0.4 %) is defined as a
rare disease. In Australia, if a drug used to treat a disease that affects fewer
than 2,000 individuals, the disease is considered to be rare [354]. In
addition, no satisfactory treatment for the diseases should be available. If the
disease meets these criteria, the inventors, or sponsors, can apply to
regulatory authorities for an orphan drug status. However, the European
commission is the decisive body in consultation with the Committee for
Orphan Drug Medicinal Products (COMP), which handle the applications. In
the US, the FDA Office of Orphan Products Development (OOPD) is
responsible for the decisions of orphan drug status. If the applicaton is
approved, the drug is designated with an orphan drug status and the
development of the drug may be stimulated under the orphan drug
legislation. The legislation includes several incentives. Perhaps the most
appealing incentive for pharmaceutical companies is the ten-year, or seven
years in US, market exclusivity of the drug [355]. The additional incentives
are reduced charge for scientific advice and reduced fees for regulatory
activities, e.g. application inspections and support. In addition, in the EU,
the marketing authorization is central and valid in all EU member countries.
Importantly, other drugs intended for the same rare disease must be proven
superior to the current orphan drug, for orphan drug status approval and
access to the stimulatory incentives [356]. However, if the second applicant
of the orphan drug status has the consent from the original orphan drug
applicant, two orphan drug designations are possible for the same disease
[355].
The European Medicines Agency (EMA) has classified adenovirus
infection in the immunocompromised host, as a rare disease at least twice.
In 2001 they stated that 0.6-3 in 10,000 persons in EU are affected and in
2014, that 0.2 in 10,000 persons in the EU were affected [189, 190]. Hence,
orphan drug status could possible be achieved. To my knowledge, Cell
Medica Ltd. is the only applicant with approved orphan drug status for
treatment of adenovirus infection in allogeneic haematopoetic stem cell
transplant recipient [357]. Cell Medica Ltd. develops immunotherapeutics
and adenovirus specific T cell transfer (see section 4.5). The promising drug
Brincidofovir (see section 2.7), is a broad-acting antiviral that is in phase III
for treatment of severe adenovirus disease but is not (officially) designated
as an orphan drug. However, Brincidofovir was recently approved for
compassionate use in Josh Hardy, a 7-year old boy suffering from a
dissiminated adenovirus disease. This case has recently been spread in social
media worldwide [358, 359].
46
5.7. Natural products in drug discovery
Natural products include all compounds produced by living organisms.
However, from a pharmaceutical perspective, natural products mainly
include secondary metabolites that are produced by an organism in response
to external stimuli. These stimuli can be nutritional changes, infection or
competition between organisms. Hence, for the organism, secondary
metabolites may function as differentiation effectors, competitive weapons,
sexual hormones, spore formation stimulators, quorum sensing signals and
more [360, 361]. In addition, secondary metabolites are not essential for the
growth, development and reproduction of the producing organism. The
production of secondary metabolites does not follow the growth phase
(trophophase) of the organism but is produced in a subsequent stage
(idiophase) [362]. Many organisms have the potential to produce secondary
metabolites, including plants, microbes, fungi and animals. However, this
section will mainly focus on secondary metabolites derived from microbes.
Secondary metabolites have been a rich source for the identification of
new compounds in drug discovery. The most well known drugs that originate
from secondary metabolites may be the antibiotics, including the first
discovered antibiotic penicillin. However, numerous secondary metabolitederived drugs are currently used for treatment of a variety of disease areas,
e.g.
paclitaxel
and
doxorubicin
(anti-cancer),
rapamycin
(immunosuppressant),
amphotericin
B
(anti-fungal),
avermectin
(antihelmetic), acarbose (anti-diabetes) and more [363-367]. In addition,
approximately 50 % of the approved new chemical entities between 19812006 are secondary metabolites or derivates thereof [368]. However, with
the introduction of HTS in the 1990´s, the focus on secondary metabolites as
a source for new drug leads was shifted towards combinatorial chemistry and
synthetic compound libraries [369]. The advantages of combinatorial
chemistry are the production of enormous amounts of new compound
structures, which biological activity easily can be assessed using HTS and
future favorable intellectual property aspects. Nonetheless, the
combinatorial chemistry approach has resulted in relatively few new
approved drugs on the market. Consequently, the secondary metabolites
have recently regained focus as a source for discovery of new drug leads,
particularly in infection research. Secondary metabolites are generally more
complex in their structure, compared to synthetic compounds. This
complexity has been selected during evolution to interact with biological
targets. Hence, a combinatorial approach, designated diversity-oriented
synthesis (DOS), has been utilized to combine the complexity of secondary
metabolite structures with synthetic chemistry for the production of more
natural product-like compounds [369]. DOS, applied for natural products,
assesses the chemical space around a privileged structure of a bioactive
47
secondary metabolite and considers secondary metabolites as starting points
for further structural optimization. This may result in numerous analogues
that may have more potent and more targeted activity than the original
structure. In addition, by structural classification of scaffolds for the to-date
known secondary metabolites, the chemical space explored by nature can be
visualized [370].
Plants are the major producers of secondary metabolites followed by
microbes. Among the microbes, actinomycetes are known to produce a
variety of bioactive secondary metabolites and are responsible for about half
of the to-date known microbial secondary metabolites [371]. Actinomycetes,
or actinobacteria, are gram-positive bacteria abundantly found in soil where
they play important roles in the decomposition of organic matter. In the
phylum of actinobacteria, the genus Streptomycetes is the major producer of
secondary metabolites that have been developed to clinically important
drugs, mainly antibiotics, e.g. streptomycin, chloramphenicol and
tetracycline [372]. The high productivity of secondary metabolites among
actinobacteria is associated with their large genomes, which harbors several
gene clusters for secondary metabolism. The genus Streptomyces has in
general a genome, between 8-10 Mb, which contains over 20 secondary
metabolic gene clusters for biosynthesis of polyketides, peptides, terpenoids,
shikimate-derived metabolites and more [373]. Thus, actinobacteria, and
especially the genus Streptomycetes, which both are easily accessible in soil,
have been extensively studied for their capacity to produce bioactive
compounds with drug development potential. Consequently, the isolation
rate of new actinobacteria-derived secondary metabolites has declined while
the rate of re-isolation for previously known compounds with already
characterized bioactivity has increased [374]. With the emerging drugresistance worldwide, it is eminently crucial to find new compounds that
have drug development potential. This has lead to a quest to identify new
sources and environments that may harbor uncharacterized actinobacteria,
which may produce new types of secondary metabolites. The oceans cover
more than 70 % of the surface of the earth and its microbial diversity is still
relatively unexploited. It has recently been recognized that marine
actinobacteria can produce secondary metabolites with unusual structures
and properties [375, 376]. Hence, marine actinobacteria are an unexploited
source for new secondary metabolites. Isolation and characterization of these
compounds will hopefully lead to new therapeutic agents.
48
Aims
The overall aim of this thesis was to identify and characterize compounds
with antiviral activity against adenovirus. The specific aim for each paper
was:
I - To develop a whole cell-based screening assay utilizing a replication
competent GFP-expressing adenovirus vector. Furthermore, to screen a
diverse set of 9800 small molecules for anti-adenoviral activity and select
potential hit compounds for further characterization.
II - To perform SAR-analysis of a hit compound class found in the screening,
for evaluation of the chemical space around the structure and for structure
optimization.
III - To determine the antiviral specificity for the optimized compound class,
by evaluation of the antiviral activity against HSV and clinical acyclovirresistant HSV isolates.
IV - To establish a library of secondary metabolites derived from marine
actinobacteria and to screen the library for anti-adenoviral activity.
Furthermore, to identify and characterize potential hit compounds.
49
Methods
Screening assay
This thesis is based on the screening of compounds for anti-adenoviral
activity using a robust screening assay based on a unique replicationcompetent adenovirus type 11 GFP-expressing vector (RCAd11GFP).
RCAd11GFP has the GFP-gene inserted upstream of the E1-region of the
adenovirus genome without hampering the infectivity of the virus [377]. In
addition, RCAd11GFP manifests a strong GFP expression that initially can be
visualized 4 h post infection (unpublished data). However, 24 h post
infection the GFP expression reaches its maximal plateu and the GFP signal
can easily be observed in microscope or be quantified using a plate reader
[378]. We used this vector as a tool for detection of compounds that reduced
the GFP expression without killing the cells. In the first paper, we assessed a
diverse collection of 9800 small drug-like molecules using suspensional
human erythroleukemic K562 cells that are highly susceptible for adenovirus
type 11 [379]. Compounds and RCAd11GFP were added simultaneously to
the wells, to achieve 50 µM compound concentrations and 1 pg per cell of the
vector. The GFP expression was measured in a plate reader, 24 h post
infection. In parallel, the cytotoxicity of the compounds was quantified using
an MTT-based cytotoxicity assay. This assay measures the potential of
mitochondrial enzymes to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) to a coloured formazan, which is
quantified [380]. Thus, the viability of the cells can be determined. We
defined criteria for selecting hit compounds as, reduction of 80 % or more of
the GFP-signal and 50 % or more of viable cells. These criteria yielded a hit
rate of approximately 4 %. These compounds were re-tested in a doseresponse analysis and the most potent compounds were selected for further
hit evaluation.
In the second screening campaign (paper 4) the parameters in the
screening assay were slightly different. Here, we utilized a High Content
Screening (HCS) automated microscope for measuring the RCAd11GFP
derived GFP-expression. In addition, this automated microscope facilitated
an initial evaluation of the cytotoxicity of the compounds since the
morphology of the Hoschst-stained nuclei easily could be assessed. Adherent
human lung carcinoma, A549, cells were used in the screening and we
lowered the amount of RCAd11GFP to 0.025 pg per cell due to the high
sensitivity of the HCS automated microscope. Our established extract library
of secondary metabolites from marine actinobacteria contained unknown
compounds at unknown concentrations. Hence, the compound
concentrations in the wells were unknown. However, based on previous
50
biological assessments of the extracts (unpublished data), we diluted the
extracts to 1:200 in the wells. As previously, RCAd11GFP and the compounds
(extracts) were added simultaneously and the microplates were analyzed 24
h post infection. Hoeschst was added to the wells 20 min prior to analysis.
After analysis in the HCS automated microscope, the cytotoxicity was
measured using an XTT-based toxicity assay. This assay measures the
potential of mitochondrial enzymes to reduce 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) to a coloured formazan,
which is similar to the MTT assay, albeit with fewer steps and higher
sensitivity [381]. Extracts that decreased the GFP-signal without apparent
toxicity were selected for further dose-response analysis. Potent extracts
were fractionalized using HPLC and each fraction was re-tested in the
screening assay for identification of active fractions. The compounds in the
active fractions were structurally determined. Database and literature
searches revealed that three of our hit compounds were previously known
with assessed antiviral activity (Pentalenolactone, Nonactin and
Cycloheximide). However, the fourth compound was a known butenolide
compound with previously uncharacterized antiviral activity.
Due to the lack of effective antivirals that targets adenovirus infections,
the Z´factor for the two screening campaigns could not be determined. The
Z´factor requires both negative and positive controls to define the quality of
the screening assay (see section 5.1).
Hit expansion
The synthesis of compounds followed conventional chemistry using typical
methods as liquid chromatography-mass spectrometry (LC-MS), highperformance liquid chromatography (HPLC) and nuclear magnetic
resonance (NMR). The purity of the synthesized compounds was above 95 %.
Assays for hit evaluation
To characterize the antiviral activity of the hit compounds and their analogs,
we have utilized several assays that each analyse different stages in the
infection cycle. In chronological order; to assess if the compounds inhibited
the binding of the virus to the cells we used a binding assay with 35S labelled
adenovirus virions. This facilitates the quantification of the amount of
virions that are bound to the cell surface at 4°C (used in paper I and IV).
Furthermore, to analyse if the compounds inhibited the uptake and the
intracellular transportation to the nucleus, we labelled adenovirus type 5
51
virions with the fluorphore Alexa fluor 555. Using confocal microscope and
inverted fluorescence microscope, individual adenovirus particles could be
traced intracellulary along the microtubules and to the nuclear pore (data
not published). To assess the replication of viral DNA, during which most
antiviral compounds have inhibitory effect, we performed quantitative PCR
(qPCR). The number of newly synthezised viral genomes was determined
using specific primers against the genes of structural proteins of both
adenovirus and HSV [382-384]. In parallel, the number of analysed cells was
determined by the use of primers for the housekeeping gene RNAseP. Hence,
the number of viral genomes per cell can be expressed. The qPCR assay is a
very sensitive assay that can distinguish small variations in the number of
genomes. Consequently, the qPCR-assay was the most frequently used assay
for assessment of the antiviral activity of the compounds. In addition, doseresponse analysis and EC50 determination of the compounds were performed
using qPCR. The later steps in the infection cycle was analysed using flow
cytometry. Using a monoclonal antibody (mAB8052), directed against newly
synthezised adenoviral hexon proteins, the potential antiviral effect on the
viral protein translation could be evaluated. However, if the antiviral effect is
due to inhibition of a prior step of the infection cycle, the antiviral effect will
be observed in the later steps as well.
Assessment of the toxicity of compounds was performed with several
assays. The predominant assay used, was the XTT-assay (described in
section screening assay above). Additionally, the DNA intercalating agent,
propidium iodide (PI) was used to stain the DNA of dead cells, using flow
cytometry (used in paper I). Moreover, to determine any cell membrane
disruption, the extracellular release of lactose dehydrogenase (LDH) was
analyzed using a LDH cytotoxicity kit (unpublished data).
52
Results and Discussion
The discussed figures and tables herein refer to each paper, unless otherwise
stated.
Paper I
Small-molecule screening using a whole-cell viral replication
reporter gene assay identifies 2-[2-(benzoylamino)benzoylamino]
benzoic acid as a novel antiadenoviral compound.
Andersson EK, Strand M, Edlund K, Lindman K, Enquist PA, Spjut S, Allard
A, Elofsson M, Mei YF, Wadell G.
Antimicrob. Agents Chemother., 2010, 54 (9): 3871-3877.
There is a need for new antiviral drugs, especially for the treatment of severe
adenovirus infections. Today there are no approved anti-adenoviral drugs
available, although adenovirus infections are associated with morbidity and
high mortality rates in, particularly immunocompromised children. With
this in mind, we have developed a whole-cell based screening assay using a
unique replication competent GFP-expressing adenovirus vector
(RCAd11GFP), to screen a diverse chemical library of 9800 small molecules.
This library was selected to cover a large chemical space. The main
advantages of cell-based screening assays are the discovery of potential novel
targets and the knowledge that the compounds have potential to pass the cell
membrane. In addition, in cell-based assays the preliminary cytotoxicity of
the compounds can be assessed. In target-based assays, non-target
interactions that may result in toxicity, is not observed. Additionally, potent
compounds that interact with the intracellular target may not sufficiently
pass the cell membrane. The major disadvantage of cell-based assays is that
the study design will not provide any information on the mode-of-action of
the compounds. However, when considering the need for novel antiviral
compounds with, preferably, new modes-of-action, a cell-based assay is to
prefer.
The screening campaign resulted in 408 hit compounds that fulfilled our
hit selection criteria (see previous section Methods – Screening assay). These
408 compounds were re-tested in dose-response analysis and we selected 24
of these compounds for further analysis. The antiviral activity of these 24
compounds was assessed in a number of assays, each reflecting a different
step in adenovirus infection cycle (see previous section Methods – assays for
hit evaluation). Based on the overall results, we selected 2-[2(benzoylamino)benzoylamino]-benzoic acid. This compound was designated
as A02 in paper I and as 1 in paper II. In paper III we designated the
53
compound class as Benzavir and 2-[2-(benzoylamino)benzoylamino]benzoic acid as Benzavir-1. Hence, 2-[2-(benzoylamino)benzoylamino]benzoic acid will hereinafter be designated as Benzavir-1. Benzavir-1 was
active against representative adenovirus types from several species with a
EC50 value between 2.4 - 4.7 µM. In addition, Benzavir-1 was about 5 times
more potent than cidofovir (Table 2). Benzavir-1 did not display any
inhibitory effect on the binding of the virus to the cells (Fig. 5a). However,
moderate antiviral activity was observed when assessing the GFP-expression
from the RCAd11GFP vector (Fig. 1). Surprisingly, when re-assessing
Benzavir-1 in conditions similar to the original screening, the percentage
inhibition does not reach 80 %, which was one of the hit selection criteria for
the screening (Fig. 1a). However, when the GFP signal was measured in
A549 cells and analyzed in flow cytometry, the percentage inhibition reached
80 % (Fig. 1b). Assessment of the effect of Benzavir-1 on the replication of
adenovirus DNA revealed prominent inhibition, using the more sensitive
qPCR assay (Fig. 4). In the step subsequent to DNA replication, the
expression of structural hexon proteins, the antiviral potency is moderate
(Fig. 5b). The reason for this is unknown but this could be due to the fact
that transcription and expression of hexon proteins still can occur from a
reduced number of viral DNA templates.
The toxicity of Benzavir-1 is low. The CC50 value was determined to 199
µM on A549 cells and approximately 1 % of the A549 cells were non-viable
according to PI toxicity assay (Fig. 4 and Fig. 5c).
To our surprise, the purchased batch of Benzavir-1 turned out to contain
two additional compounds, one two-ring structure and one four-ring
structure (Fig. 2). These compounds are most probable remnants during
synthesis. Fortunately, the three-ring Benzavir-1 structure was determined
to be the active structure (Fig. 3). However, this represented an initial
structure-activity relationships (SAR) analysis of Benzavir that was
expanded upon in paper II.
54
Paper II
Synthesis,
biological
evaluation,
and
structure-activity
relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid
analogues as inhibitors of adenovirus replication.
Öberg CT, Strand M, Andersson EK, Edlund K, Tran NP, Mei YF, Wadell G,
Elofsson M.
J. Med. Chem., 2012, 55 (7): 3170-3181.
To assess the chemical space around Benzavir-1 and to potentially discover
analogues with improved potency, we performed SAR analysis of Benzavir-1.
Paper I displayed that only the trimeric Benzavir-1 structure was active,
hence, only trimeric analogues were analyzed. The SAR-analysis was
performed in three generations of studies where the biological activity of the
analogues in each generation guided the synthesis of the next. A total set of
42 compounds was analyzed. In brief, the aim of the first generation of
analogues was to determine the importance of the ortho-ortho substituent
pattern of Benzavir-1 (designated as 1 in paper II). The carboxylic acid on the
right-hand ring and the substituents of the central ring was moved to meta
and para positions. In addition, the importance of the carboxylic acid was
determined. The general conclusion of generation 1, was that the ortho-ortho
substituent pattern and the presence of the carboxylic acid in the ortho
position was of importance for the antiviral activity (Table 1 and 2). The
second generation focused on the N-terminus, left-hand side, of the
structure. By modifications of the ring-structure and by substituent
attachments at various positions, we concluded that the overall antiviral
activity of these compounds were moderate. However, by attachment of an
electron-withdrawing substituent, fluoride (F), in the ortho position in the
left-hand ring the activity was slightly increased compared to the activity of
Benzavir-1 (compound 17h, Table 3). The third and last generation of
analogues was a continuation of compound 17h, where electron-donating
substituents, methoxy (OMe) or electron-withdrawing substituents, F or
chloride (Cl), was added to the central or right-hand ring of the structure
(Table 4). Substituation of the middle ring was favorable, whereas on the
right-hand ring substituation resulted in a decreased activity. Attachment of
Cl and OMe on meta and para positions on the middle-ring was beneficial.
However, two F on the middle-ring yielded the greatest antiviral activity
(compound 35j in Table 4). Cl is larger and more lipophilic than F, thus
increasing the size and lipophilicity of the molecule. The size of F is
comparable with hydrogen (H), thus little change in the overall steric bulk of
the molecule is expected. We excluded dual substituation with OMe and Cl
on the middle-ring due to steric hinderence. Further evaluation of compound
35j displayed an improved EC50 value compared to Benzavir-1, from 3.7 µM
55
for Benzavir-1 to 0.58 µM. In addition, the toxicity at two fixed
concentrations, 30 µM and 60 µM, was reduced (Table 5). We decided to
assess the toxicity at two fixed concentrations below 100 µM to ascertain that
no solubility issues would interfere with the toxicity read out. The improved
analogue to Benzavir-1 is hereinafter designated as Benzavir-2. Benzavir-2 is
produced in a five-step synthesis and is currently our lead candidate,
however synthesis and biological assessments of additional Benzavir-2
analogues may result in further improved compounds.
Paper III
2-[4,5-Difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic
acid, an antiviral compound with activity against acyclovirresistant isolates of herpes simplex virus types 1 and 2.
Strand M, Islam K, Edlund K, Öberg CT, Allard A, Bergström T, Mei YF,
Elofsson M, Wadell G.
Antimicrob. Agents Chemother., 2012, 56 (11): 5735-5743.
The discovery of Benzavir-1 as a novel anti-adenoviral compound, in paper I,
was followed by paper II, in which the exploration and evaluation of
Benzavir-1 analogues was described. Paper II yielded the improved
compound Benzavir-2. Here, in paper III, the antiviral activity of both
Benzavir-1 and Benzavir-2 were assessed on another virus of medical
importance, namely HSV. In addition, the activity on clinical acyclovirresistant isolates of HSV was determined. Since Benzavir-1 is active against
representative types from all adenovirus species (Table 2 in paper I), the
initial aim of this paper was to evaluate the antiviral specificity of this
compound class. HSV and adenovirus are both DNA viruses, and they share
general steps in their replication cycle. However, HSV and adenovirus
utilizes and express different proteins, all of which each may serve as an
antiviral target. Hence, to determine if the antiviral activity of Benzavir-1 and
-2, is specific for adenovirus or if the antiviral activity would also include
HSV, we assessed the number of HSV genomes by the qPCR assay and
determined the antiviral activity on both types of HSV. For initial analysis we
assessed the anti-HSV activity of both Benzavir-1 and -2, at two fixed
concentrations, 15 and 5 µM (Fig. 2). Acyclovir was included as positive
control and as observed, all three compounds almost completely inhibited
the HSV infection at 15 µM. Additionally, at 5 µM, Benzavir-2 and acyclovir
were the most potent compounds with approximately 80 %, or more,
inhibition whereas Benzavir-1 displayed around 70 % inhibition. Further
characterization concluded that the EC50 values of Benzavir-2 and acyclovir
on both HSV types were similar, ranging from 1.5 to 1.6 µM (Fig. 3).
56
However, as concluded in Fig. 2, Benzavir-1 was less potent with an EC50 of 3
and 4.3 µM, respectively. In addition, the clinically used antiviral drug
cidofovir was assessed (Fig. 3b). Cidofovir displayed an EC50 value of 11 µM
on HSV type 1, which indicates that our improved compound, Benzavir-2, is
approximately seven times more potent than cidofovir. The profile of the
dose-response curves differs between our Benzavir compounds and, most
notably, acyclovir. The reason for this is unclear, however acyclovir and
cidofovir inhibits the viral DNA replication in a two-step process, with
essential phosphorylation steps prior to insertion to the growing viral DNA
strand. The initial phosphorylation step of acyclovir, by the viral TK, is a
rate-limiting step. Hence, a 1:1 stoichiometry and a straighter dose-response
slope can be expected for acyclovir. However, cidofovir does not require the
initial phosphorylation, due to its phosphonate group, thus the doseresponse slope is not as straight as for acyclovir. The mode-of-action for
Benzavir-1 and -2 is currently not known, consequently the impact of the
steep dose-response curve can only be speculated upon. As in paper II, we
determined the toxicity of the compounds at two fixed concentrations, 30
and 60 µM. Benzavir-2 has reduced toxicity compared to Benzavir-1,
however no toxicity was observed for acyclovir (Table 4). Nonetheless,
acyclovir is developed to be active only in virus-infected cells that produce
the viral TK protein. Thus, acyclovir in uninfected cells is considered to be
inactive.
In collaboration with professor Tomas Bergström at Sahlgrenska
University Hospital in Gothenburg, we obtained five acyclovir-resistant
clinical isolates of both HSV types. Four isolates were identified as HSV type
2 and one was HSV type 1. These isolates were collected at various lesion
sites from patients aged 52 to 83 years (Table 1). In comparison to the age of
the patients with acyclovir-sensitive HSV isolates, which were collected from
a 22- and a 24-year-old patient, the age of the patients suffering from
acyclovir-resistant HSV is substantially higher. In general, acyclovirresistant HSV is more frequently isolated from elderly patients than younger
patients. The reason for this is probably due to conditions that are associated
with a compromised immune system, which is more common in elderly
persons. As expected, acyclovir had low or moderate effect on these isolates.
However, both Benzavir-1 and -2 was active in the initial antiviral
assessment at 15 and 5 µM (Table 2). Although as previously, Benzavir-2
displayed improved potency compared to Benzavir-1. Thus, Benzavir-2 was
selected for thorough dose-response analysis (Fig. 4). Here, Benzavir-2
displayed similar EC50 values ranging from 1.1 to 1.6 µM, against all
acyclovir-resistant isolates. Consequently, Benzavir-2 is active against both
acyclovir-sensitive and acyclovir-resistant HSV isolates of both types, with
similar potency. Hence, it can be concluded that Benzavir-2 and acyclovir
does not share exactly the same mode-of-action. In addition, Benzavir-2 is a
57
promising antiviral compound that deserves further characterization as a
drug candidate.
Paper IV
Isolation and characterization of anti-adenoviral secondary
metabolites from marine actinobacteria.
Strand M*, Carlsson M*, Uvell H, Islam K, Edlund K, Cullman I, Altermark
B, Mei YF, Elofsson M, Willassen NP, Wadell G, Almqvist F.
Mar. Drugs, 2014, 12(2): 799-821. (*equally contributing authors).
The previous three papers originated from a screening campaign where
9800 small molecules were screened for anti-adenoviral activity, using a cellbased assay. In this paper, we expanded our screening-based approach and
screen ethyl acetate extracts derived from marine actinobacteria that were
isolated from sediments of the Arctic sea. Since actinobacteria is a major
source for a variety of bioactive secondary metabolites, we wanted to assess
the potential to discover compounds with antiviral activity. In addition, there
are 1 million, or more, virus particles per ml of seawater [385]. Hence,
organisms living in the sea must have evolved defense mechanisms against
the persistent attack of viruses. This defense mechanism may have a broad
antiviral activity and be active against other types of viruses, i.e. human
adenovirus.
The collaboration partners at University of Tromsö, Norway, performed
the collection of sediment samples. The research vessel Jan Mayen (depicted
on the back cover of this thesis), collected sediment samples from various
depths outside the coast of Norway. From these sediment samples,
actinobacteria were selected for their potential to produce diverse bioactive
secondary metabolites (see section 5.7). In total, 57 actinobacterial strains
were isolated. Furthermore, these actinobacteria were grown in different
media compositions to form an ethyl acetate extract library containing 912
extracts. These extracts were screened using the RCAd11GFP vector and
analyzed in an automated microscope (see previous section Methods –
Screening assay). After bioactivity-guided fractionation of active compounds,
one fractionation contained a diastereomeric mixture of butenolide
compounds, previously known to the literature but with previously
uncharacterized antiviral capacity (Fig 2). The bacterial strain that produced
these compounds was isolated at GPS coordinates N67 52.11512 E16
22.97003 at a depth of 417 m. However, when optimizing the production of
these butenolide compounds, three additional analogues were discovered
(Fig. 2). We proposed biosynthetic pathway for these compounds based on
their relative time of appearance during cultivation and their structure (Fig.
4). All isolated butenolide compounds were assessed in a dose-response
58
analysis, where potency differences among the compounds were observed
(Fig. 5). In addition, the two most potent butenolide compounds, an
analogue with keton side-chain (3) and an analogue with a nonfunctionalized side-chain (4), had similar EC50 values around 90 µM but
displayed substantial differences in toxicity (Table 3 and Fig. 6). The toxicity
was determined on two different cell types, human lung carcinoma (A549)
cells and primary foreskin fibroblast (FSU) to assess any cell type specificity
of the toxicity. However, the toxicity of compound 4 did not differ between
the cell types. Hence, the most potent and non-toxic butenolide analogue 3
was selected for further characterization. Time-of-addition analysis revealed
that cellular pre-treatment with compound 3, completely inhibited the
adenovirus infection (Fig. 7A). However, no inhibitory effect of the viral
binding was observed, or on the integrity of the viral particle (Fig. 7B and C).
Interestingly, reduction of the 2-furanone moiety in the butenolide structure,
eliminated the antiviral activity (Fig. 8B). In addition, the toxic potential of
the analogue with a non-functionalized side-chain, 4, was slightly reduced
(Fig. 8C). This suggests that both the side-chain and the 2-furanone moiety
are involved in the bioactivity. It could be speculated that, the more
lipophilic nature of 4 may increase the transport over the cell membrane,
thus the intracellular concentration of 4 would be higher than 3. However,
this is a bit contradictory since the antiviral potency for 3 and 4 is similar.
Hence, the mode-of-action for these compounds needs to be determined to
assess the importance of side-chain substituents.
In sum, the butenolide compound 3 is an anti-adenoviral non-toxic
compound that is small and amendable for further optimization of the antiadenoviral activity.
Concluding remarks and future prospects
The overall aim of this thesis was to discover and characterize novel antiadenoviral compounds that may meet the unmet clinical need among
patients affected by severe adenovirus infections. Hence, the discoveries
included in this thesis may lead to that new antiviral drugs will be evaluated
for clinical use. Our most well characterized compound, Benzavir-2, has low
toxicity and is active against adenovirus with an EC50 value below 1 µM. In
addition, similar potency is displayed against HSV and clinical acyclovirresistant HSV isolates. Thus, Benzavir-2 is a promising new antiviral
compound. However, the mode-of-action for Benzavir-2 is currently not
known. We intend to determine the mode-of-action for Benzavir-2. It is most
likely that Benzavir-1 and -2 share the same mode-of-action. We have
performed attempts to determine the mode-of-action, so far with
inconclusive results. A biotinylated Benzavir-1 compound was synthesized
59
and was used as a bait to “fish-out” potential target proteins in infected and
uninfected cell lysates. However, no specific target interactions could be
determined. Thus, our next approach will be to develop Benzavir-2 resistant adenovirus mutants. This approach has been used to identify
mutations responsible for cidofovir-resistance in adenovirus [386].
Adenovirus will be propagated in the presence of increasing concentrations
of Benzavir-2, which will be expected to trigger the development of escape
mutants that are resistant to Benzavir-2. Subsequently, the mutant virus
isolates will be sequenced and their genome composition analyzed for
mutations. However, if Benzavir-2 interacts with a cellular protein(s) or a
virus-induced cellular protein(s), the interpretation of the developed
mutations will be a challenge. An alternative approach is to assess the
antiviral activity on other types of viruses, including RNA-viruses, to expand
assessment of antiviral specificity of Benzavir-2. Hopefully, we would
identify a virus type that is not inhibited by Benzavir-2, which in comparison
to other Benzavir-2 sensitive virus types, may aid the mode-of-action
assessment by reducing the number of potential Benzavir-2 targets.
Additionally, we can exclude steps in the adenovirus infection cycle that we
know are not involved in the antiviral activity of Benzavir-1 and -2. We know
that Benzavir-1 does not affect the integrity of the adenovirus virion
(unpublished data) or inhibit the binding of the virion to the cell (Fig. 5a in
paper I). In addition, we know that Benzavir-1 inhibits neither the uptake
nor the intracellular transport of the viral particles to the nucleus, analyzed
by fluorophore-labeled adenovirus virions (unpublished data). We also know
that Benzavir-2 is not a general DNA intercalating agent (unpublished data).
However, the major antiviral activity is observed in conjunction with the
viral DNA replication. Hence, it is tempting to assume that Benzavir-2
affects the initiation or progress phase of the viral DNA replication. This
hypothesis may also be supported by the requirement of Benzavir-2 presence
either prior to, or during, the production and initiation of the viral DNA
replication machinery that occurs 6-8 h post infection (unpublished data). If
Benzavir-2 is added 8 h post infection no, or moderate, antiviral activity is
observed. However, the molecular mechanisms that are involved in the
antiviral activity for Benzavir-2 needs to be determined.
So far, Benzavir-2 seems to be a promising antiviral compound in vitro.
However, the in vivo situation may differ from the in vitro situation. Hence,
we have initiated in vivo assessments of Benzavir-2 in mouse models. At
present, Benzavir-2 is assessed in a vaginal HSV-2 mouse model in
collaboration with professor Kristina Eriksson at Sahlgrenska University
hospital in Gothenburg. In addition, we plan to develop an adenovirus
mouse model, using a luciferase-expressing adenovirus vector and a noninvasive in vivo imagining platform, to quantify the potential anti-adenoviral
activity of Benzavir-2 in the mouse.
60
To conclude paper I - III, we discovered Benzavir-1 as a novel antiadenoviral compound, which potential was enhanced by the development of
Benzavir-2. Both the antiviral activity and the toxic potential of Benzavir-2
have been thoroughly characterized. We aim to continue the characterization
of Benzavir-2, to assess its potential as a new antiviral drug. Consequently, a
Umeå Biotech Incubator (UBI) project was initiated that has resulted in a
spin-off company, Eirium AB. Eirium AB has the intention to continue the
characterization of Benzavir-2 towards the pharmaceutical market. Eirium
AB has applied for patent that covers Benzavir-2 and additional chemical
space, for both EU and US, and will perform PK analysis of Benzavir-2 both
in vitro and in vivo. In addition, Eirium AB will assess the anti-adenoviral
efficacy in vivo and the mode-of-action for Benzavir-2.
Paper IV was a new endeavor. Here, we established an extract library
originated from actinobacteria isolated from the depths of Vestfjorden in
Norway. We identified a butenolide compound class that inhibited the
adenovirus infection and we proposed a biosynthetic pathway for the
production of the isolated butenolide compounds. The two most potent
butenolide compounds had similar potency but differed widely in their toxic
potential. One of these butenolides, compound 3, had no prominent toxicity
at 2 mM, which was the highest assessed concentration, and an EC50 value of
91 µM. This potency may not be that appealing, especially when comparing it
to e.g. Benzavir-2, which has an EC50 value of 0.58 µM. However, when
considering the low toxicity of 3, a favorable therapeutic window is expected
for this compound. The concentration range between the efficacy and
toxicity is always to be considered in drug discovery and especially in
antiviral drug discovery, since the virus require a viable host cell. Thus,
cellular toxicity may be interpreted as antiviral effect.
The antiviral mode-of-action for these butenolide compounds is currently
unknown. However, the time-of-addition assay may indicate that these
compounds target a cellular mechanism, since pre-treatment of the cells had
improved antiviral activity (Fig. 7A). Interestingly, if this is the case,
targeting this cellular mechanism does not result in any prominent toxicity.
The reduction of the 2-furanone moiety of the butenolide structure
resulted in elimination of the antiviral activity. Hence, this structure is
responsible for the inhibition of the adenovirus infection but is not solely
responsible for the toxic effect (Fig. 8B and C). This enables the usage of the
2-furanone moiety as a bait to fish out target proteins. In addition, the fatty
tail may be used as a linker that could be covalently attached to a surface, e.g.
on magnetic beads. Subsequently, separation and characterization of 2furanone moiety interacting proteins will potentially reveal the mode-ofaction. Another future prospective is to identify the enzyme(s) responsible
for the oxidation of compound 4, as proposed in the biosynthetic pathway
61
(Fig. 4). This unknown enzyme oxidizes compound 4 to form compound 1
and one of the two possible diastereomers of compound 2. However, the
stereochemistry of compound 2 has not been determined. Hence,
identification of the enzyme would enable the cloning and expression of the
enzyme. Ultimately it would lead to the determination of the stereochemistry
of the butenolide compounds and support our proposed biosynthetic
pathway.
To conclude paper IV, we have identified anti-adenoviral secondary
metabolites derived from actinobacteria isolated from the sediment of
Vestfjorden, Norway. The compounds display interesting differences in their
bioactivity and further characterization may identify the antiviral target(s) of
the active 2-furanone moiety. In addition, the interaction between the 2furanone moiety and the target is not associated with prominent toxicity in
our assays. Consequently, this moiety could serve as a starting point for
further characterization and optimization of antiviral activity.
62
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Acknowledgements
So this was it. The acknowledgements should really be on the frontpage
instead of the back, since there are a lot of people that have been involved in
this thesis to whom I´m very grateful. However, I would like to acknowledge
following people that have been of particular importance for the completion
of this thesis and the included papers.
My main supervisor, Göran Wadell, for guidance in the scientific world
and for that you have shared your knowledge, enthusiasm and experience in
the field of virology (and beyond) with me. Mikael Elofsson, my cosupervisor, for your inspiring drive and effectiveness. Also for your brilliant
inputs and comments on various documents, including this thesis, and for
the one and only (so far) Grit-session. Ya-fang Mei, my second cosupervisor, for constructing the RCAd11GFP-vector, without the vector this
thesis would not be what it is today. Karin Edlund, for all your support
over the years and for always taking time to help and answering questions
(when you’re actually busy). Katrine Isaksson-Nyström for all your help
with everything, and for fixing all the things that only you can fix. Kristina
“Kickan” Lindman, for always being able to help with lab issues. You and
Karin are, as many have said before me, the cornerstones of the virology lab.
Koushikul Islam, for all your qPCR runs and screening assistance. Emma
Andersson, for teaching me the fantastic qPCR-assay. Nitte and Marko
for the hard-hitting and exhausting squash matches and sorry Lars that I
broke your arm. The poker-gang for letting me win your money. Johan
Skog for introducing me to virology during my degree work and thanks to
all present and former lab members of the virology unit for creating a
pleasant work atmosphere.
Marcus Carlsson and Hanna Uvell, for your collaboration and
positive attitude. Producing paper IV was fun with you two. Fredrik
“Frippe” Almqvist for the trip to Tromsö and for teaching me basic organic
chemistry when I was a student. Christopher Öberg and Michael Saleeb
for your synthesis of Benzavir, among other compounds. The people at
LCBU for help and assistance when screening.
UBI for fruitful seminars and discussions and in particular, Pia Keyser,
for your coaching and organization of meetings. With your support, let us
now take Eirium AB to the next level.
The collaboration partners at Sahlgrenska University hospital, Tomas
Bergström, for the HSV isolates and Kristina Eriksson, for assessing
Benzavir-2 in your in vivo model.
Friends and family
My beloved Jenny, for cheerful “you can do it” support when writing.
Let´s now sail the Swedish archipelagos (when we have bought a boat).
104