Antiviral agents

Viral diseases constitute a set of difficult-to-treat conditions.

Over the years, many different antiviral agents have been
discovered and synthesized, and these have provided various
means of treatment of viral conditions
Viruses are obligate cellular parasites composed of nucleic acid
core and protein outer shell (envelope)
Viruses take over a host cell to survive and multiply, they depend
completely on host cell for energy and replication.
Responsible for influenza, chicken pox, measles, mumps, viral
pneumonia, rubella and smallpox and many other viral
infections.
Viruses possess only one type of nucleic acids either DNA or
RNA.
 Viruses can be transmitted to human by a variety of ways:
Through air by the infected host sneezing or coughing.
Through arthropods or ticks
Through physical contact (for some viruses which can not
survive outside the host such as:
HIV
Cold sore.
Genital herpes.
Rabies.
Food-borne or water borne viruses such as hepatitis A and E
and viral gastroenteritis.
History:
Smallpox weakened the Roman Empire.
Lethal viruses in Africa such as Ebola, Lassa.
Sever acute respiratory syndrome (SARS) during 2003 in the
Far East.
Recently H1N1
The structure of viruses
Can be classified as:
DNA viruses: contains either single or double strand
DNA
RNA viruses: contains single strand RNA (ssRNA), but
some have double strand RNA.
The nucleic acid is protected within a protein coat
called capsid.
The capsid contains nucleic acid is called
Nucleocapsid.
The whole structure of virus is called virion (the form
that the virus takes when it is outside the host cell)
 Membrane

Nucleic acid Capsid

Viral protein

RNA polymerase

The size of virion can vary

from 10nm to 400nm.
The outer surface of viral cell
CLASSIFICATION OF VIRUSES
Viruses are classified on the basis of several features:
Nucleic acid content (DNA or RNA)
Viral morphology (helical, icosahedral)
Site of replication in cell (cytoplasm or nucleus)
Coating (enveloped or nonenveloped)
Serological typing (antigenic signatures)
Cell types infected (B lymphocytes, T lymphocytes,
monocytes)
Virus life cycle
Viruses are known to infect every form of life.
A typical DNA virus will enter the nucleus of the
host cell, where viral DNA is transcribed into
messenger RNA (mRNA) by host cell RNA polymerase.
mRNA is then translated into virus-specific proteins
that facilitate assembly, maturation, and release of
newly formed virus into surrounding tissues.
RNA viruses are somewhat different, in that their
replication relies on enzymes in the virus itself to
synthesize mRNA.
THE INFECTIOUS PROCESS FOR A
VIRUS
1. Adsorption, attachment of the virus to specific receptors on the surface of the host
cells, a specific recognition process.
2. Entry, penetration of the virus into the cell.
3. Uncoating, release of viral nucleic acid from the protein coat.
4. Transcription, production of viral mRNA from the viral genome.
5. Translation, synthesis of viral proteins (coat proteins and enzymes for replication)
and viral nucleic acid (i.e., the parental genome or complimentary strand). This process
uses the host cell processes to express viral genes, resulting in a few or many viral
proteins involved in the replication process.
The viral proteins modify the host cell and allow the viral genome to replicate by using
host and viral enzymes. The mechanisms by which this occurs are complex.
This is often the stage at which the cell is irreversibly modified and eventually killed.
6. Assembly of the viral particle. New viral coat proteins assemble into capsids (the
protein envelope that surrounds nucleic acid and associated molecules in the core) and
viral genomes.
7. Release of the mature virus from the cell by budding from the cell membrane or
rupture of the cell and repeat of the process, from cell to cell or individual to individual.
Enveloped viruses typically use budding on the plasma membrane, endoplasmic
reticulum, or Golgi membranes.
Non-enveloped viruses typically escape by rupture of the host cell.
Electrostatic attraction

Life cycle of virus

Inhibition of RT
would
prevent conversion of
viral RNA genome
into
DNA for
incorporation
HIV protease is required
into the hosts
to process the transcribed
and translated proteins for replicatory
the new virions system
Vaccination
Is the preferred method of protection against viral
diseases.
Extremely successful against childhood diseases such
as polio, measles, mumps, smallpox and yellow fever.
Works by:
introducing the body to foreign material having
molecular similarity to some component of the virus.
Introducing killed or weakened version of the virus.
Or administer fragments of virus having the
characteristics antigen.
 Virus is a hard target
Most of the time the virus spend in the host will be
inside the host cell.
This effectively protect the viral cell from the host
immune system as well as from available circulating
enzymes.
Another problem appears in treating viral infections is
the fact that there are limited number of potential
drug targets since viruses use the host biochemical
mechanisms to multiply.
Antiviral agents
The first effective antiviral agents appear in 1960s and
only three were clinically available:
Idoxuridine and vidarabine for herpes infections.
Amantadine for influenza A infections.
Growing interest in finding effective antiviral agents
after that was due to:
The need to tackle AIDS spread.
The increased understanding of viral genomic sequence
and infectious mechanisms.
Amantadine and Rimantadine
Amantadine and Rimantadine are both drugs that
interfere with penetration of host cells by viruses and
block early stage replication.
Amantadine has been used for years as a treatment for
Parkinson disease
The two differ in pharmacokinetics; Both are well
absorbed and distributed. Amantidine is excreted in
the urine nearly unchanged while Rimantidine is
extensively metabolized.
NEURAMINIDASE INHIBITORS:
ZANAMIVIR AND OSELTAMIVIR
Sheathing the protein coat of the influenza virus is a lipid envelope. Two
macromolecules, surface glycoproteins, are embedded in the lipid envelope:
hemagglutinin and neuraminidase.
These glycoproteins fulfill separate functions in the viral cycle.
Hemagglutinin is important for binding of the virus to the host cell
membrane by a terminal sialic acid residue.
Neuraminidase is an enzyme. It functions in several of the early activation
steps of the virus and occurs in both influenza A and B viruses. Neuraminidase
is believed to be a sialidase, cleaving a bond between a terminal sialic acid unit
and a sugar. This action is important in enhancing the penetration of viruses
into host cells, and hence enhances the infectivity of the virus.
If the sialic acidsugar bond is prevented from being cleaved, the viruses tend
to aggregate and the migration of viruses into host cells is inhibited. Hence,
drugs that inhibit neuraminidase should be useful in interfering with infection
caused by influenza virus type A and B.
Zanamivir
Zanamivir has been shown to form a salt bridge with the
guanidine and Glu-119 and a charge transfer interaction
with Glu-227.
These interactions increase the interaction strength with
the enzyme and create an excellent competitive inhibitor
and an effective antiviral agent for influenza types A and B.
zanamivir is effective when administered before or after
exposure to the influenza virus. If administered before
exposure to the virus, the drug reduced viral propagation,
infectivity, and disease symptoms.
Oseltamivir Phosphate
An important feature of oseltamivir is the ethyl ester,
which makes the drug orally efficacious. This drug is
the first orally active agent for use against influenza A
and B. It is also indicated for the treatment of acute
illness. If administered within 2 days after the onset of
influenza symptoms, the drug is effective.
Oseltamivir is actually a prodrug in its ethyl ester
form. Ester hydrolysis releases the active oseltamivir
molecule.
INTERFERONS: INTERFERON ALFA (INTRON
A, ROFERON A) AND INTERFERON BETA
(BETASERON)
The interferons are a family of small proteins or glycoproteins
of molecular masses ranging from 15,000 to 25,000
Da and 145 to 166 amino acids long. Eukaryotic cells secrete
interferons in response to viral infection. Their mechanism
of action is bimodal. The immediate effect is the recruitment
of natural killer (NK) cells to kill the host cell
harboring the virus (Fig. 4.13). Interferons then induce a
state of viral resistance in cells in the immediate vicinity,
preventing spread of the virus. Additionally, interferons induce
a cascade of antiviral proteins from the target cellIFNs are extremely potent cytokines that possess antiviral,
immunomodulating, and antiproliferative actions. IFNs
are synthesized by infected cells in response to various inducers and, in turn, elicit either an antiviral state in
neighboring cells or a natural killer cell response that destroys the initially infected cell (Fig. 9.3).
There are three classes of human IFNs that possess significant antiviral activity.
These are IFN- (more than 20 subtypes), IFN- (2
subtypes), and IFN-. IFN- is used clinically in a recombinant
form (called interferon alfa). IFN- (Betaseron) is a
recombinant form marketed for the treatment of multiple
sclerosis.
Antiviral agents for DNA viruses
Mainly against herpes viruses such as cold sore, genital
herpes, chicken pox, eye diseases.

Three major mechanisms of actions:

1. Inhibition of viral DNA polymerase.
2. Inhibition of tubulin polymerization.
3. Antisense therapy: which blocks the translation of
viral RNA.
Nucleoside antimetabolite

Trifluridine
Idoxuridine

Iodine atom and triflouro carbon are similar in in size to

the methyl group of the thymidine (this is called isosteric
replacement)
Nucleoside antimetabolite
DNA polymerase inhibitors (Iodoxuridine and Trifluridin)
They are active against DNA viruses such as herpesviruses
Prodrugs that need to be phosphorylated by viral
thymidylate phosphate, further phosphorylation is carried
out by cellular enzymes to give the active triphosphate.
The triphosphate inhibit and act as a substrate for DNA-
polymerase, producing DNA that contain iodinated or
trifluronated pyrimidines which is prone to strand
breakage and miscodes in RNA synthesis.
Iodine atom and triflouro carbon are similar in in size to
the methyl group of the thymidine (this is called isosteric
replacement)
 Nucleoside antimetabolite

Are phosphorylated equally by viral and cellular

thymidine kinase, so they are more toxic than
acyclovir. (we will talk about it later)
The first nucleoside-based antiviral agents (inhibit
both viral DNA polymerase and thymidylate
synthetase).
 Acyclovir: is the most effective of a series of acyclic nucleosides that possess antiviral
activity. In contrast with true nucleosides that have a ribose or a deoxyribose sugar
attached to a purine or a pyrimidine base, the group attached to the base in acyclovir is
similar to an open chain sugar, albeit lacking in hydroxyl groups.

Acyclovir triphosphate prevents DNA replication

By two ways:
Can bind to DNA polymerase instead of
deoxyguanosine triphosphate.
Can be incorporated into the growing DNA
chain discontinuation of chain extension due to
the absence of 3 hydroxyl group.
 Acyclovir selective toxicity
Why Acyclovir does not inhibit DNA polymerase in
normal, uninfected cells:

The first step in phosphorylation requires the viral version of

kinase (100 times more active than the host enzyme).

There is a selective uptake of acyclovir by infected cells.

Acyclovir triphosphate is 50 times more selective on viral DNA

polymerase compared to the cellular polymerase.
 Acyclovir analogues
Were synthesized mainly to overcome the poor water
solubility and to improve oral availability.
 Valaciclovir
Is an L-valyl ester prodrug of acyclovir.
Absorbed more effectively
from the gut than acyclovir
although it has the same polarity.
That fact that the D-valyl prodrug has poor absorption
suggesting that there is a special transport system
(intestinal oligopeptide and di/tripeptide transporter)
required for valaciclovir absorption.
After absorption, valaciclovir will be hydrolyzed to
acyclovir.
 Cidofovir
Some viruses do not have thymidine kinase, so
resist the action of acyclovir.

Cidofovir is already phosphorylated, so no

Mimic
need for the kinase, then this will be
phosphorylated by cellular thymidine kinase to
give the active Cidofovir triphosphate.

Could be more toxic than acyclovir (why?).

 AIDS
Acquired Immune Deficiency Syndrome caused by
human immunodeficiency virus (HIV virus).
Immune deficiency because the virus attacking the T-
cells which are crucial to the immune system.
Acquired because with weakened immune system, the
patient will be more susceptible for opportunistic
secondary diseases.
infection by opportunistic pathogens (e.g. pneumonia,
TB) ultimately kills the host not the virus itself in most
of the cases.
 HIV virus
Is one of the RNA retroviruses.
Two types:
HIV-1: is responsible for AIDS in America, Europe, and
Asia.
HIV-2: prevalent in Western Africa.
Most clinically useful antiviral agents against AIDS are
either:
Reverse transcriptase inhibitors.
HIV protease inhibitors.
Life cycle of HIV virus

Inhibition of RT
would
prevent conversion of
viral RNA genome
into
DNA for
incorporation
HIV protease is required
into the hosts
to process the transcribed
and translated proteins for replicatory
the new virions system
Antiviral agents against HIV
Until 1987, no anti-HIV drug was available.
Extensive studies carried out on the life cycle of HIV
have led to identifying possible drug targets within the
viral cell:
Reverse transcriptase.
Protease.
Unfortunately, HIV undergoes mutation extremely
easily, which results in rapid development of
resistance.
For that, current therapy depends on the use of
combination of reverse transcriptase inhibitors and
protease inhibitors.
The ideal anti-HIV agent
Must have high affinity for its target.
Activity range in picomolar.
Be effective in preventing the virus multiplying and
spreading.
Show low activity against host enzymes.
Safe and well tolerated.
Has a broad antiviral activity.
It must be inexpensive since it will be used for the life
time of the patient.
Nucleoside Reverse Transcriptase
Inhibitors (NRTIs)
This enzyme is unique to the virus so it is an ideal
target.
However, it still a DNA polymerase like, so there is a
possibility that its inhibitor might affect the cellular
DNA polymerase.
Nucleoside-like drugs have been proved as useful anti-
viral agents:
Nitrogen base + Deoxyribose sugar.
Should be phosphorylated three times (by cellular
kinases) to form the active nucleotide triphosphate.
Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
O O

NH NH

N O N O

HO HO

O O
H H
H H
H H
H H N3 H
OH H
Non-nucleophilic
Deoxythymidine Azido group Zidovidine (AZT)
 GROWING GROWING
CHAIN OF NEW CHAIN OF NEW
STRAND STRAND

GUANINE GUANINE
O O
O O
O P O P O
O REVERSE O
TRANSCRIPTASE
O H H O H H
NH
H H H
OH H O H
O
P N O
O O
O
NH O
H H
N O H
O N3 H
O
O O P
O O
P O H H
P
O O H
O N3 H
O
NON-NUCLEOPHILIC
RT uses zidovidine triphosphate FUNCTIONAL GROUP
in place of thymidine triphosphate
as complementary base to Adenine
In template strand
 3' 5'
-
O
O -
O
P O O-
O O P O
O O P
O O
O
O
O O
O
O
A G
C
A
T
C
G
O T
O
O
O
O
O O
P O
OH
O N3
O- O

P O
No further
-
O
O
nucleic acid
extension
- P O
O
O

P O
-
O
O-

5'

3'
Other NRTIs
 Non-nucleoside reverse
transcriptase inhibitors (NNRTIs).
They are hydrophobic molecules bind to the allosteric
binding site which is hydrophobic in nature (non-
competitive reversible inhibitors).
Rapid resistance emerges due to mutation in the
NNRTI binding site.

First generation Second generation

Allosteric site vs. catalytic site
Nevirapine binding to the NNRTI allosteric site

NNRTI binding transmits a

conformational change
through the RT protein

Relative orientation of nucleophilic 3-OH from

growing strand and electrophilic 5-phosphate from
nucleotide triphosphate substrate altered and can
no longer form a bond elongation stopped
 Viral protease
Has a broad substrate specificity, can cleave variety of
peptide bonds in viral polypeptides.
Mostly it can cleave the peptide bond next to proline
residue and an aromatic residue (phenylalanine and
tyrosine).
This cleavage (next to proline) is not common with
mammalian proteases such as renin and pepsin.. This
results in better selectivity against HIV protease over the
mammalian proteases.
HIV-1 protease is 50% similar to HIV-2 homologue, the
differences is far from the active site so high possibility to
get inhibitors against both a the same time
The role of the Proline residue
Tertiary amide

Induces turn
in the backbone
Mechanism of hydrolysis by
protease enzyme
 Targeting HIV protease
HIV protease is a much smaller enzyme than the
equivalent host aspartate proteases.
Cleaves substrates at the N-terminal to proline
residues unlike mammalian proteases.
Peptides from infected cells suggested that Tyr-Pro
sites were the likely cleavage sites.
Rationale for inhibitor design was based on Phe-
Pro or Tyr-Pro motif.
 HIV Protease Inhibitors (PIs)
Are not prodrugs and do not need to be activated.
So can be tested in-vitro to test activity (IC50.the
concentration of drug required to inhibit the enzyme by
50%) especially after knowing that viral protease can
easily isolated.
Low IC50 does not mean a good antiviral activity
(Why?).
Most of them are derived from peptide lead compounds.
 HIV Protease Inhibitors (PIs)
They are oligopeptides in general:

Less well absorbed.

Susceptible to first pass metabolism by cytochrome P-
450, which may results in drug-drug interactions with
many other drugs taken by AIDS patients such as
ketoconazole, rifampicin and astemizole.
Rapidly excreted (Why?).
High plasma protein binding (Why?).
 The Lead PI N and C-terminal
protecting groups
HO
prevent hydrolysis
O
by exopeptidases
O OH

HN Asn
NH O NH2
Pro
O

HN O O
H
NH N N
O
O
O
O

Tyr
OH
Possible structural modification on
the lead PI
The size of the compound: di, tri, tetra, or
pentapeptide.

The amino acid motif: tyr-pro or phe-pro.

The nature of C and N-terminal protecting group.

The use of proline analogues

Improve binding
To the active site

Better water
Solubility and
availabiliy