Transcript Antiviral Drugs

Antiviral Drugs
General Characteristics of Viruses
• Depending on one's viewpoint, viruses may be
regarded as exceptionally complex aggregations of
nonliving chemicals or as exceptionally simple
living microbes.
• Viruses contain a single type of nucleic acid (DNA
or RNA) and a protein coat, sometimes enclosed
by an envelope composed of lipids, proteins, and
carbohydrates.
• Viruses are obligatory intracellular parasites.
They multiply by using the host cell's synthesizing
machinery to cause the synthesis of specialized
elements that can transfer the viral nucleic acid to
other cells.
Host Range
• Host range refers to the spectrum of host
cells in which a virus can multiply. (narrow
vs. broad)
• Most viruses infect only specific types of cells
in one host species, so they do not generally
cross species barriers.
• Host range is determined by the specific
attachment site on the host cell's surface
and the availability of host cellular factors.
Viral Structure
• A virion is a complete, fully developed viral particle
composed of nucleic acid surrounded by a coat.
• Helical viruses (for example, Ebola virus) resemble long
rods and their capsids are hollow cylinders surrounding
the nucleic acid.
• Polyhedral viruses (for example, adenovirus) are manysided. Usually the capsid is an icosahedron.
• Enveloped viruses are covered by an envelope and are
roughly spherical but highly pleomorphic (for example,
Poxvirus). There are also enveloped helical viruses (for
example, Influenzavirus) and enveloped polyhedral
viruses (for example, Herpesvirus). Pleomorphic: Many-formed. A tumor may
be pleomorphic.
• Complex viruses have complex structures. For example,
many bacteriophages have a polyhedral capsid with a
helical tail attached. Bacteriophage: A virus that infects and lyses certain bacteria.
Schematic of Influenza Virus
Influenza Virus
Nucleic Acid
• Viruses contain either DNA or RNA, never both, and the
nucleic acid may be single- or double-stranded, linear
or circular, or divided into several separate molecules.
• The proportion of nucleic acid in relation to protein in
viruses ranges from about 1% to about 50%.
DNA viruses
• gene expression is much like that of the host cell
• DNA-dependent RNA polymerase synthesizes mRNA
• Host cell ribosomes and tRNAs used to translate
viral mRNA
• Unique viral proteins include structural proteins and
replication enzymes for viral DNA.
• Example-Herpesvirus, Epstein-Barr (mononucleosis)
HBV = Hepatitis B virus, a DNA virus
RNA viruses
• Cells cannot make copies of RNA.
Three kinds of strategies for RNA
viruses:
Positive -strand RNA viruses
• the genome is also a mRNA
• The first task of the virus is to translate
viral-specific proteins including RNAdependent RNA polymerase (viral
transciption/repliction enzyme) from
viral RNA. The enzyme makes more
mRNA and new RNA for viruses.
Positive-stranded RNA: genome is a molecule
of single-stranded "sense" RNA
•
•
•
•
•
•
•
•
•
•
polioviruses
rhinoviruses (frequent cause of the common "cold")
coronaviruses (includes the agent of Severe Acute Respiratory Syndrome
(SARS)
rubella (causes "German" measles)
yellow fever virus
West Nile virus
dengue fever viruses
equine encephalitis viruses
hepatitis A ("infectious hepatitis") and hepatitis C viruses
tobacco mosaic virus (TMV)
Hepatitis C
• Link
• Hepatitis C attacks the liver
• Hepatitis C is transmitted by direct blood
to blood contact
– Injection drug use
– Blood transfusion
– Occupational exposure to blood
Negative-strand RNA viruses
• the genome is the complement of
mRNA
• First task of the virus is to make mRNA.
Therefore, the virus imports RNA
polymerase or transcriptase as a part
of the virus structure.
Negative-stranded RNA viruses: genome consists of one
or more molecules of single-stranded "antisense" RNA
Examples
• measles
• mumps
• respiratory syncytial virus (RSV), parainfluenza
viruses (PIV), and human metapneumovirus. (In
the U.S., these close relatives account for
hundreds of thousands of hospital visits each
year, mostly by children.)
• rabies
• Ebola
• influenza
Retroviruses
• Virus has the enzyme reverse transcriptase as a part
of the viral structure.
• A double-stranded DNA copy of the viral genome is
produced.
• This copy can integrate into the host cell
chromosome.
• Some retroviruses can cause tumors in animals:
oncogenes
• Human immunodeficiency virus (HIV) is a retrovirus.
This is the causative agent of AIDS.
Virus
Picorna
Toga
Retro
Orthomyxo
Rhabdo
Genome
RNA
RNA
RNA
RNA
RNA
Polarity
+ss
+ss
+ss
-ss
-ss
Segments
1
1
1+1
6-8
1
Morphology
Icosahedral
Icosahedral
Icosahedral
Helical
Helical
Enveloped
No
Yes
Yes
Yes
Yes
Diseases
Polio, Hepatitis A, Colds
Encephalitis, Rubella
AIDS
Influenza
Rabies
Paramyxo
RNA
-ss
1
Helical
Yes
Parainfluenza, Mumps, Measles
Papova
Adeno
DNA
DNA
ds
ds
1
1
Icosahedral
Icosahedral
No
No
Warts
Respiratory Infections
Herpes
DNA
ds
1
Icosahedral
Yes
HS, VZ, Mononucleosis, Cancer
Pox
Hepatitis B
DNA
DNA
ds
ds
1
1
Complex
Icosahedral
Yes
Yes
Smallpox
Serum Hepatitis
Capsid and Envelope
• The protein coat surrounding the nucleic acid of
a virus is called the capsid.
• The capsid is composed of subunits,
capsomeres, which can be a single type of
protein or several types.
• The capsid of some viruses is enclosed by an envelope
consisting of lipids, proteins, and carbohydrates.
• Some envelopes are covered with carbohydrate-protein
complexes called spikes.
Viruses and Cancer
•
The earliest relationship between cancer and viruses was demonstrated in
the early 1900s, when chicken leukemia and chicken sarcoma were
transferred to healthy animals by cell-free filtrates.
• Transformation of Normal Cells into Tumor Cells:
•
When activated, oncogenes transform normal cells into cancerous cells.
•
Viruses capable of producing tumors are called oncogenic viruses.
•
Several DNA viruses and retroviruses are oncogenic.
•
The genetic material of oncogenic viruses becomes integrated into the host
cell's DNA.
•
Transformed cells lose contact inhibition, contain virus-specific antigens
(TSTA and T antigen), exhibit chromosomal abnormalities, and can produce
tumors when injected into susceptible animals.
Causes of the Common Cold
• More than 200 different viruses are known to cause the symptoms of
the common cold. Some, such as the rhinoviruses, seldom produce
serious illnesses.
• Others, such as parainfluenza and respiratory syncytial virus,
produce mild infections in adults but can precipitate severe lower
respiratory infections in young children.
• Rhinoviruses (from the Greek rhin, meaning "nose")
cause an estimated 30 to 35 percent of all adult colds,
and are most active in early fall, spring, and summer.
More than 110 distinct rhinovirus types have been
identified. These agents grow best at temperatures of
about 91 degrees Fahrenheit, the temperature inside the
human nose.
• Scientists think coronaviruses cause a large percentage of all adult
colds. They bring on colds primarily in the winter and early spring. Of
the more than 30 kinds, three or four infect humans. The importance of
coronaviruses as a cause of colds is hard to assess because, unlike
rhinoviruses, they are difficult to grow in the laboratory.
•
•
Approximately 10 to 15 percent of adult colds are caused by viruses also
responsible for other, more severe illnesses: adenoviruses,
coxsackieviruses, echoviruses, orthomyxoviruses (including influenza A and
B viruses, which cause flu), paramyxoviruses (including several
parainfluenza viruses), respiratory syncytial virus, and enteroviruses.
The causes of 30 to 50 percent of adult colds, presumed to be viral, remain
unidentified. The same viruses that produce colds in adults appear to cause
colds in children. The relative importance of various viruses in pediatric
colds, however, is unclear because it's difficult to isolate the precise cause
of symptoms in studies of children with colds.
http://www.commoncold.org/undrstnd.htm
Influenza
• Influenza is a disease caused by a
member of the Orthomyxoviridae. Many
features are common with those of the
paramyxovirus infections of the
respiratory tract.
CLINICAL FEATURES
• Influenza is characterized by fever,
myalgia, headache and pharyngitis. In
addition there may be cough and in severe
cases, prostration. There is usually not
coryza (runny nose) which characterizes
common cold infections. Infection may be
very mild, even asymptomatic, moderate
or very severe.
• Source The reservoir is acute infection in
other human beings.
• Spread Is rapid via aerial droplets and
fomites with inhalation into the pharynx or
lower respiratory tract.
• Incubation Is short: 1-3 days. Rapid
spread leads to epidemics
Complications
• Tend to occur in the young, elderly, and persons
with chronic cardio-pulmonary diseases
• Consist of:
• 1. Pneumonia caused by influenza itself; Pneumonia: an
inflammatory condition of the lungs in which they become obstructed with fluid, causing difficult breathing and
possibly suffocation. Pneumonia may be caused by bacteria, viruses, fungi, or chemical agents.
• 2. Pneumonia caused by bacteria- Haemophilus
influenzae- Staphylococcus aureusStreptococcus pneuminiae
• 3. Other viral superinfection, eg.
Adenovirus.Overall death rates increase in times
of influenza epidemics.
The virion is generally rounded but may be long
and filamentous.
A single-stranded RNA genome is closely
associated with a helical nucleoprotein (NP), and
is present in eight separate segments of
ribonucleoprotein (RNP), each of which has to
be present for successful replication. The
segmented genome is enclosed within an outer
lipoprotein envelope. An antigenic protein called
the matrix protein (MP 1) lines the inside of the
envelope and and is chemically bound to the RNP.
The envelope carries two types of protruding
spikes. One is a box-shaped protein, called the
neuraminidase (NA) (pink rectangles on the
surface), of which there are nine major antigenic
types, and which has enzymic properties as the
name implies.
The other type of envelope spike is
a trimeric protein called the haemagglutinin
(HA)
(illustrated on the left)
of which there are 13 major antigenic types.
The haemagglutinin functions during
attachment of the virus particle to the cell
membrane, and can combine with specific
receptors on a variety of cells including red
blood cells.
The lipoprotein envelope makes the virion
rather labile - susceptible to heat, drying,
detergents and solvents.
Haemagglutinin: A substance, such as an antibody, that causes
agglutination of red blood cells.
Agglutination: The clumping together of red blood cells or bacteria.
The Life Cycle of Influenza Virus
Receptor-bound viruses are taken into
the cell by endocytosis. In the low pH
environment of the endosome, RNP is
released from MP1, and the viral
lipoprotein envelope fuses with the lipidbilayer of the vesicle, releasing viral RNP
into the cell cytoplasm, from where it is
transported into the nucleus. New viral
proteins are translated from transcribed
messenger RNA (mRNA). New viral RNA
is encased in the capsid protein, and
together with new matrix protein is then
transported to sites at the cell surface
where envelope haemagglutinin and
neuraminadase components have been
incorporated into the cell membrane.
Progeny virions are formed and released
by budding.
The cell does not die (at least not
initially).
Influenza: Overview
Link
Link
Flu is one of a rare few viruses that has its genome in
separate segments (eight). - This increases the potential
for recombinants to form (by interchange of gene
segments if two different viruses infect the same cell), and
may contribute to the rapid development of new flu strains
in nature - can also be duplicated in the laboratory (used
for making vaccine strains). Avian and human strains
recombining in pigs in the Far East may permit virulent
human strains to evolve.
CLASSIFICATION of virus STRAINS
Is done on the basis of antigenicity of NP (nucleoprotein) and MP (matrix protein)
into three main groups: Influenza A -HA undergoes minor and occasional major
changes.
- NA some variation.
Influenza B) Undergoes relatively slow change in HA with time. Known only in man.
Influenza C) Uncommon strain, known only in man.
Nomenclature of Viruses
A
Singapore
6
86
(H1N1)
Type of
Influenza
Town where
first isolated
Number of
isolates
Year of
isolation
Major Type of
HA and NA
Influenza A virus is essentially an avian virus that has "recently"
crossed into mammals. Birds have the greatest number and range of
influenza strains. Avian haemagglutinins sometimes appear in pig
human and horse influenza strains.
• Every now and then (10 - 15 years) a major new pandemic strain appears in man, with a totally
new HA and sometimes a new NA as well (antigenic shift). This variant causes a major epidemic
around the world (pandemic).
•Over the subsequent years this strain undergoes minor changes (antigenic drift) every two to three
years, probably driven by selective antibody pressure in the populations of humans infected.
Camp Devens is near Boston, and has about 50,000 men, or did have before this
epidemic broke loose. It also has the Base Hospital for the Div. of the N. East. This
epidemic started about four weeks ago, and has developed so rapidly that the
camp is demoralized and all ordinary work is held up till it has passed. All
assembleges of soldiers taboo.
These men start with what appears to be an ordinary attack of LaGrippe or
Influenza, and when brought to the Hosp. they very rapidly develop the most
viscous type of Pneumonia that has ever been seen. Two hours after admission
they have the Mahogany spots over the cheek bones, and a few hours later you
can begin to see the Cyanosis extending from their ears and spreading all over the
face, until it is hard to distinguish the coloured men from the white. It is only a
matter of a few hours then until death comes, and it is simply a struggle for air until
they suffocate. It is horrible. One can stand it to see one, two or twenty men die,
but to see these poor devils dropping like flies sort of gets on your nerves. We have
been averaging about 100 deaths per day, and still keeping it up. There is no doubt
in my mind that there is a new mixed infection here, but what I dont know.
Copy of original letter found in Detroit in 1959
Camp Devens, Mass.
Surgical Ward No 16
29 September 1918
(Base Hospital)
• When viruses infect the body, the frequently result in the production of large
amounts of cytokines
• Cytokines signal immune cells, like T cells and macrophages to respond to the
infection, but can also result in the production of still more cytokines.
• If this immune system loop becomes uncontrolled, it can lead to a cytokine storm,
resulting in extremely large amounts of inflammation and fluid production.
http://www.cytokinestorm.com/cytokine_storm.html
This constant antigenic
change down the years
means that new vaccines
have to be made on a
regular basis.
Anti-Viral Medications
The relative simplicity of the virus machinery,
coupled with the rapid mutation of strains, makes
it challenging to design effective anti-viral
medications
Anti-influenza Agents
Amantadine · Oseltamivir · Peramivir · Rimantadine · Zanamivir
Anti-herpesvirus agents
Aciclovir · Cidofovir · Docosanol · Famciclovir · Foscarnet · Fomivirsen ·
Ganciclovir · Idoxuridine · Penciclovir · Trifluridine · Tromantadine ·
Valaciclovir · Valganciclovir · Vidarabine
Antiretroviral Agents
NRTIsZidovudine · Didanosine · Stavudine · Zalcitabine · Lamivudine ·
Abacavir · Tenofovir
NNTI’s
Nevirapine · Efavirenz · Delavirdine
PIsSaquinavir · Indinavir · Atazanavir · Ritonavir · Nelfinavir · Amprenavir ·
Lopinavir · Tipranavir
Other antiviral agents
Fomivirsen · Enfuvirtide · Imiquimod · Interferon · Ribavirin · Viramidine
The final stage in the life cycle of a virus is the release of completed viruses
from the host cell, and this step has also been targeted by antiviral drug
developers. Two drugs named zanamivir and oseltamivir that have been
recently introduced to treat influenza prevent the release of viral particles by
blocking a molecule named neuraminidase that is found on the surface of flu
viruses, and also seems to be constant across a wide range of flu strains.
Both these drugs are effective against the known strains of H5N1 in mouse
models.
Tamiflu has been disappointing in recent real world use in human H5N1
infection due to
1. delays in treatment and
2. the emergence of resistance. Relenza has not yet been tried in
human H5N1 infection.
• Most attention has been given to oseltamivir (Tamiflu) because it is a tablet,
which is easy to administer.
• Zanamavir (relenza) is administered as a dry powder inhaler much like
some asthma inhalers. An intravenous version of Relenza has been
administered to volunteers under study conditions but it is not yet approved
or in production.
• Both drugs can be used to treat influenza; they are also both approved for
the prevention of influenza.
• These drugs are also effective against all strains of influenza A, unlike
vaccines which are specific only to the strain for which they were designed.
•
Both medications are well tolerated with few side effects, although there is
concern over the possibility of psychological effects of Tamiflu and there
may be occasional problems with asthmatics who use Relenza.
How do these drugs work?
Current anti-influenza drugs target an enzyme used in
release of the virus from the cell.
What is Sialic Acid?
Negatively charged at neutral pH
R = Carbohydrate
HO
OH
CO2H
HO
O
H
OR
AcNH
OH
Sialic acid-rich oligosaccharides on the glycoconjugates found on
surface membranes help keep water at the surface of cells. The sialic
acid-rich regions contribute to creating a negative charge on the cells
surface. Since water is a polar molecule, it has a partial positive charge
on both hydrogen molecules, it is attracted to cell surfaces and
membranes. This also contribues to cellular fluid uptake.
It seems that the Hemagluttinin subtypes in avian influenza strains bind
preferentially to a2,3 linked sialic acid residues, while the human influenza
isolates prefer a2,6 linked sialic acid residues.
Sialic acid binding site of human influenza A hemagglutinin subtype H3. Sialic acid
bound in the pocket appears in gray. The listing below shows the binding affinities for
sialic acid when particular amino acids are changed experimentally by site-directed
mutagenesis (Martín et al. 1998). The number on the left defines the amino acid site in
HA1, x → y shows the original and new amino acid, and the number on the right is the
binding affinity of the mutant as a percentage of the affinity of the wild type. Dashes
show cases in which the receptor site is not properly expressed.
What is neuraminidase?
Negatively charged at neutral pH
HO
R = Carbohydrate
HO
OH
OH
CO2H
HO
O
H
OR
HO
Neuraminidase
CO2H
OH
+
H2O
AcNH
H
O
ROH
AcNH
OH
OH
Sialic Acid
(also: N-acetylneuraminic acid)
• Neuraminidase has functions that aid in the efficiency of virus release from cells.
• Neuraminidase cleaves terminal sialic acid residues from carbohydrate moieties on
the surfaces of infected cells.
• This promotes the release of progeny viruses from infected cells.
• Neuraminidase also cleaves sialic acid residues from viral proteins, preventing
aggregation of viruses.
• Inhibiting neuraminidase slows the release of the virus from the host cell and
commercial drugs target this enzyme
Currently utilized inhibitors of neuraminidase
HO
HO
OH
HO
H
O
OH
CO2H
AcNH
OH
HO
H
Sialic Acid
CO2H
H
AcNH
OH
O
CO2H
O
AcNH
HN
NH2
NH
Zanamivir
(Relenza, GSK)
(inhalation)
NH2
Oseltamivir
(Tamiflu)
(oral)
How were these drugs designed?
Question: Knowing the reactant and the product of an enzyme-catalyzed
Reaction, how do you design an inhibitor to bind more tightly than either one?
Recall: An enzyme speeds a reaction by lowering the energy of
The transition state.
Therefore: the overall DG at the transition state must be greater
(I.e. more negative) than at either starting material or product.
Therefore: If you design an inhibitor to resemble the transition state,
It should be very tightly bound at the active site.
Let’s look at the reaction mechanistically
sp3 hybridized
HO
sp2 hybridized
sp3 hybridized
OH
HO
H
HO
CO2H
OH
HO
OR
OH
:
O
HO
CO2-
O
H
+
HO
+
H
O
AcNH
AcNH
AcNH
OH
OH
OH
HO
OH
HO
Neuraminidase
H
O
CO2H
OH
+
H2O
ROH
AcNH
OH
Sialic Acid
(also: N-acetylneuraminic acid)
CO2-
So it is logical that the inhibitors were designed to be
sp2 hybridized ‘flat’ at the carbon adjacent to the
CO2H group
sp2 hybridized
sp3 hybridized
HO
HO
sp3
OH
HO
hybridized
CO2H
O
OH
CO2-
O
H
OR
H
+
HO
AcNH
AcNH
OH
OH
sp2 hybridized
HO
OH
HO
CO2H
O
H
O
CO2H
H
sp2 hybridized
AcNH
HN
NH2
AcNH
NH
NH2
Oseltamivir
(Tamiflu)
(oral)
Zanamivir
(Relenza, GSK)
(inhalation)
Assigned Reading
Wang, Taia T.; Palese, Peter. Unraveling the mystery of swine influenza virus.
Cell (Cambridge, MA, United States) (2009), 137(6), 983-985.
Salomon, Rachelle; Webster, Robert G. The influenza virus enigma.
(Cambridge, MA, United States) (2009), 136(3), 402-410
Cell
Tscherne, D. M.; Garcia-Sastre, A. Virulence determinants of pandemic
influenza viruses. The Journal of Clinical Investigation (2011), 121 (1): 6-13.
Optional Reading
Nelson, Martha I.; Holmes, Edward C. The evolution of epidemic influenza.
Nature Reviews Genetics (2007), 8(3), 196-205.
Lehmann, F.; Tiralongo, E.; Tiralongo, J. Sialic acid-specific lectins:
occurrence, specificity and function. Cellular and Molecular Life Sciences
(2006), 63(12), 1331-1354.
Homework Questions
1) Describe three markers of viral virulence (pathogenicity).
2) Summarize the characteristics of both the 2009 H1N1 virus and the avian
H5N1 virus with respect to these markers of pathogenicity.
3) What species is believed to be the primary reservoir for the influenza
virus?
4) The haemaglutannin surface glycoprotein of human and avian influenza
virus isolates seem to have slightly different binding affinities for
structurally different forms of sialic acid linked to galactose. Explain.
5) Since resistance to current antiviral arises rapidly, it is important to find
new therapeutic targets. List a few such targets.