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Lauren Harrison

BIOS2001

‘The Mechanisms of antiretroviral drugs and how they prevent the development of AIDS'
The Human immunodeficiency virus (HIV) is a ruthless virus that evades the host immune
response and kills an enormous number of people each year
...
6 million
deaths; this is equivalent to the 2004 Indian Ocean earthquake and tsunami occurring seven
times in one year
...
Infecting these cells leads to a reduction in their numbers which
cause the development of the acquired immunodeficiency syndrome (AIDS)
...

Antiretroviral drugs help to prevent replication of HIV, impeding its development into AIDS,
and thereby increasing the life span of those infected
...
The key areas of current drug usage are fusion inhibitors, reverse transcriptase
inhibitors, integrase inhibitors and protease inhibitors, which have all been found to
significantly reduce viral load and hence the symptoms of infection
...
These problems have been considerably
decreased by using combinations of drugs, which is now the main method of treatment in
many countries, called HAART (highly active antiretroviral therapy)
...
Continued drug development is leading to a greater understanding of
this virus and the ability to target steps in its life cycle, with the hope of limiting side effects
and reducing drug resistance
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Lauren Harrison

BIOS2001

How HIV enters immune cells, its replication cycle and how it evades the immune system
HIV must enter host cells in order for the virus to replicate and infect other cells
...
This means the virus affects both
the innate and acquired responses of the immune system
...
These T-cells are cytotoxic which destroy and kill the infection (Delves
et al
...
When the virus infects the CD4 T helper cells it can destroy them causing a large
drop in the numbers of these cells circulating in the blood, preventing signals being sent to
the CD8 T-cells which results in HIV not being eliminated
...
g
...
2013)
...
This spike is a gp120
subunit that is required to adhere to the host CD4 surface molecule
...
2014)
...
Fusion takes place succeeding
the insertion of another viral subunit, gp41 fusion peptide (Craig et al
...
There is
immense diversity of this subunit, except for one common feature; once they are imbedded
in the host cell membrane, they all fold back on themselves into a trimeric hairpin structure
...
The creation of
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BIOS2001

this pore is critical for HIV as it allows entry of its viral nucleocapsid containing the viral RNA,
into the cytosol of the host cell (Melikyan et al
...
This fusion site is a primary target for
antiretroviral (ART) drugs
...
g
...


After entering the cell the next major step is for HIV to have its viral RNA converted into viral
DNA ready to be integrated into the host chromosomes
...
2014)
...
The first function
of the polymerase domain is to transcribe a single-stranded DNA from the viral RNA in the
host cytosol, forming a RNA-DNA helix
...
2009)
...
Integrase, another viral enzyme
also encoded by the pol gene, is then required to bind the newly formed double-stranded
viral DNA and transfer it from the cytosol into the cell nucleus where it can be incorporated
into the host chromosomal DNA (Agapkina et al
...
Viral integrase has three domains
which are all necessary for viral DNA integration
...
These residues bind both the
viral DNA and host DNA into the cleft, consequently allowing the catalytic domain to

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Lauren Harrison

BIOS2001

facilitate nucleotide matching of the two DNA helixes (Esposito & Craigie, 1999)
...
Due to the importance of the
prevention of the virus spreading to infect other immune cells, both RT’s and integrase
enzymes are a main target for ARTs
...
These RNAs then leave the nuclei and some get translated to
viral proteins by ribosomes
...
1997)
...
2012)
...

1999)
...

The protease gets packed with the viral proteins in the envelope and cuts the newly
synthesised proteins
...
Therefore inhibiting the protease enzyme can prevent the formation of the viral
structure, making it a good candidate for drug targeting
...

Normally the replication of a virus inside host cells stimulates interferon secretion, which
stops cells supporting viral replication, and prompts pathogen recognition receptors;
however the virus uses immunosuppressant’s e
...
cyclosporine and subunits called CPSF6 to
hide from the immune system, allowing it to replicate and spread to infect to other cells
(Rasaiyaah et al
...
The control and inhibition of the viral enzymes used in its lifecycle
by ARTs can help prevent the development of the HIV infection into AIDS
...
They inhibit in many different ways,
including mimicking specific substrates, direct binding to the active site making the enzyme
inactive and via targeting sites which stop the formation of correct protein folding
...
Their target is to prevent the synthesis of the viral DNA
through blocking RT and hence the synthesis of doubled stranded viral DNA from the viral
RNA
...
2009)
...
They are competitive
substrate inhibitors as they compete with natural nucleotides and become incorporated
into viral DNA where they cause chain termination
...
The absence of the hydroxyl group on the tail end means that a
phosphodiester bond cannot be made with the 5’ – phosphate of the next nucleotide
...


Drugs in this class include zidovudine, abacavir, lamivudine, zalcitabine, stavudine and
emtricitabine
...
Instead of a hydroxyl group on the tail end,
zidovudine has an azide group (N=N +=N-), resulting in chain termination
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Lauren Harrison

BIOS2001

Figure 2 – shows the difference
in tail groups between the
natural nucleoside Thymidine,
with a hydroxyl group, and the
ART Zidovudine with an azide
group
...


2009)
...
It is a Nucleotide
reverse-transcriptase inhibitor (NtRTI), which is in the same class of ARTs as NRTIs
...
NtRTIs
already have one phosphate group and so require only two chemical steps opposed to three
(requires double phosphorylation opposed to triple phosphorylation) to become active in
the cell
...
2002)
...
This factor combined with
tenofovir’s longer half-life means that a lower dose can be administered; normally one dose
a day
...
Tenofovir has a higher concentration in resting infected cells
than NRTIs, increasing its effectiveness (Kearney, Flaherty & Shah 2004)
...
It is also being used as a
pre-exposure prophylaxis for preventative measures in individuals who are at high risk of
getting the HIV infection
...
2012)
...


With Tenofovir being a first line HIV treatment, its long term effects are being monitored
closely and there are concerns regarding severe Kidney tubular dysfunction (KTD)
...
Other studies say that although this is a
significant percentage, in the wider population the clinical effect is not large enough to
cause enough concern to limit tenofovir usage (Cooper et al
...
What is clear though is
that a large proportion of patients do experience some kidney abnormalities after long term
use, often involved with the transporter proteins that are required to remove tenofovir
from the body (Rodriguez-Novoa, Labarga & Soriano, 2009)
...
They are small hydrophobic compounds that
require no metabolic action in order to become active
...
2004)
...
RTs have two subunits, p51 and
p66
...
The NNRTIs bind in
the p66 subunit at an allosteric site that is located in the palm region at the base of the
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BIOS2001

thumb (Sarafianos et al
...
This is a hydrophobic site that is 10 angstroms away from the
polymerase catalytic domain and 60 angstroms away from the RNase H domain of the RT
...
When NNRTIs bind at the base of the thumb they lock the
thumb into a distorted open position that prevents transcription occurring (Hsiou et
al
...
This NNRTI binding causes interactions with amino acids which prevents them
from forming in the correct sequence and therefore blocks the correct conformational
changes necessary for transcription to occur
...
2007)
...
The different
regions of the p66 subunit are
all in different colours, with
the NNRTI bound at the base
of the thumb region, holding it
open
...
2004)
...

Protease enzymes are activated as budding occurs
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The NNRTI, efavirenz, is found to be able to
bind to this RT leading to an early activation of the protease enzymes
...


Another class of ART drugs are the protease inhibitors (PIs) which specifically obstruct
cleavage of the HIV precursor polyproteins by binding the viral enzyme protease
...
The first FDA approved PIs was saquinavir in 1995, and the most recent being
darunavir in 2006
...
They are also associated with lipodystrophy which
is the re-distribution of fat around the body, often settling on the upper back and gut
...
Many also
have a low half-life as they are quickly metabolised, hence require multiple dosing around
every eight hours
...


Protease inhibitors have also had a particular problem with HIV drug resistance, which can
leave them ineffective
...
This gives drugs like the secondgeneration darunavir (which is often boosted with low doses of ritonavir) (Dobroszycki et
al
...
The
binding between darunavir and the protease enzyme is a highly exothermic reaction, which
contributes to it having such a high affinity (Velazquez-campoy et al
...
This PIs is able
to not only bind very strongly to susceptible HIV strains but it can also bind with resistant
HIV strains with the same level of affinity
...
Darunavir also has some decreased side effects for example reduced
diarrhoea and lipodystrophy, and it requires only a singular dosage a day when boosted with
ritonavir (Mckeage, Perry & Keam, 2009)
...
By boosting with a dose of ritonavir the half-life is increased
to approximately 15 hours, hence the need for only one dose
...
The first FDA approved drug in this class was
raltegravir in 2007, followed by recent approval of elvitegravir in 2012 and dolutegravir in
2013
...
IIs prevent this bond formation hence prevent integration (Hicks & Gulick, 2009)
...
This is the addition of glucuronic acid, with assistance
from the enzyme UGT1A1, which makes the IIs water soluble and hence allows for their
excretion (Brainard et al
...
This means that IIs can be given to patients with either
hepatic or renal problems and drug accumulation will not occur
...
This is a key goal
in ART drug development along with decreased drug-drug interactions
...


The class of ARTs called entry or fusion inhibitors (FIs) only have one FDA approved FI in
2003 called enfuvirtide
...
Enfuvirtide is an extracellular, competitive inhibitor that disrupts the
mechanisms of the gp41 subunit (Hardy & Skolnik, 2004)
...
HRs are structural motifs that contain seven amino acids that get repeated,
and it is these repeats on the HR1 and HR2 domains that interact with each other in order to
form the trimeric hairpin motif that is essential for membrane fusion and pore formation
...
Without the formation of the pore, the viral RNA cannot
be injected into the host cell; therefore FIs can block viral replication and HIV spread,
helping to prevent its development into AIDS
...
The FI has to be subcutaneously
injected twice a day into either the upper arm, inner thigh or the abdomen (Mirza &
Turiansky, 2012)
...
The side effects range from fatigue to diarrhoea and also cysts at the site of injection
...
Some cases of amyloidosis have
appeared due to the subcutaneous injection of FIs (D’Souza et al
...
This is where there
is a build-up of insoluble proteins under the skin at the site of injection
...


Small molecule FIs are being tested that target another area of the entry mechanism, the
crucial conformational change of the gp120 subunit on the viral envelope which allows
binding to the host cell e
...
NBD-556
...
The NBD-556 mimics the CD4 receptor by inserting its phenyl ring into the
hydrophobic cavity and binding to the Phe43 (Madani et al
...
This inhibits entry of HIV
into CD4+ immune cells; however this unintentionally allows binding of the gp120 to host
cells that lack the CD4 molecule
...
Studies have shown
that by binding to both of the gp120 sites, it can inhibit entry into CD4- host cells (Lalonde et
al
...


Figure 5 – Shows the viral
gp120 subunit which has the
FIs phenyl ring inserted into
the hydrophobic cavity
containing Phe43, and the
electrostatic interaction
which is between the Arg59
residue on the FI and the
Asp368 residue on the
gp120
...


2012)
...
This has been overcome by using small molecules with a higher
affinity for the gp120 than the NBD-556
...
2014)
...
These target the co-receptors, CCR5 and CXCR4, in the entry mechanism
...
It binds in an allosteric site away from
the CCR5-gp120 binding site
...
Being a
comparatively new ART, the long-term side effects of maraviroc are not fully understood,
but so far the studies have been hopeful with no serious adverse effects recorded (Hardy et
al
...
The CCR5 co-receptors have been found to have many different conformations
and this gives them all different sensitivities
...


The variety of ways ART drugs can inhibit HIV replication and spread is key to their success
...


Use of HAART and its benefits
Highly active antiretroviral therapy (HAART) is the combination of ART drugs which are used
in order to further decrease the viral load of HIV, which helps avert the destruction of host
immune cells, and aids to reduce the number of opportunist infections
...
Particular ARTs can be avoided if their side effects will directly
aggravate an already pre-existing condition of the patient or if the patient has HIV drug
resistance to certain ARTs
...
The HIV genome is approximately 10,000 nucleotides in length and for each replicated
HIV virion there will be roughly 1-10 mutations (Arts & Hazuda, 2012)
...
Many of these mutations involve a simple
switch of a singular amino acid for another, for example the mutation M184V, where one
methionine is replaced by a valine
...
There are also many mutations that cause
resistance to zidovudine, the main six mutations involving the amino acid thymidine
...

Treatment is usually a combination of three different ART drugs, from two different classes,
so different viral enzymes are targeted in the same treatment
...


The use of drugs to treat HIV is necessary, but ARTs can also cause an advantage to the HIV
resistance strains, hence the need for the constant development of new drugs or new
combinations
...
The
resistant HIV is given an advantage by drug use, creating a never ending cycle of fighting
resistance and creating resistance
...
There have been clinical
studies and there are statistical methods of predicting which combinations to use in HAART
to reduce acquired resistance
...
It has been shown
that patients who have previously been on four or more ART drugs that have failed are
more at risk and likely to develop other mutations, hence it is important to understand the
mutations enough to select the correct combinations of drugs first time round (Lawyer et al
...
A common combinational therapy is the use of one NNRTI and two NRTI’s
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2013)
...
A different
commonly used combination is two NRTIs and a PI
...
2009)
...
Recent studies have shown
that by also introducing IIs into the HAART therapy, for example raltegravir, the risks of
these side effects could be dramatically reduced
...
2014)
...
The frequency of mutations means that drugs can become ineffective as soon as
they appear
...


Conclusion

The mechanisms of HIV infection are extremely complicated and not as yet fully understood
...
The ongoing balancing act to provide drugs which stem the virus’s progress
have to be set against the many side-effects these new drugs can have
...
Both require long term management via medication and
lifestyle; but if managed well, can offer patients a long life
...
The HAART cocktail of drugs attempts
to disrupt different stages in the life cycle of the virus which represents the current best
outcome for those infected with HIV
...
Inflammation due to enduring immune activation is also a
key issue that contributes to complications and increased mortality e
...
cardiovascular
disease
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More long
term HIV therapies will therefore need to include inflammation management in order to
reduce this major problem
...
A
potentially good area to look into may possibly be in the few individuals that are able to
control HIV infection in the absence of therapy
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(Word count 5000)
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