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Rational Drug DesignMalaria
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Chapter 4: Molecular biology in medicine.
Background
The beginning…
• The first link between genetic
inheritance and a human
condition was made in 1902 when
alkaptonuria was identified as an
inherited disorder. (51 years prior
to Watson & Crick discovering DNA)
• Alkaptonuria is a condition in which
urine turns black on exposure to air.
• Sir Archibald E. Garrod (1857–1936)
related this and similar diseases to
a lack of particular enzymes in
the body.
CausesGenetic defects can be due to problems with an entire chromosome, multiple genes, or even a single gene (section of a chromosome).
The human –Globin gene is 626 base pairs long. If a single base substitution mutation occurs in a particular place, the person will develop Sickle Cell Anaemia.Eg: small section of gene
ACT CCT GAG GAG AAG (unaffected person)
ACT CCT GTG GAG AAG (sickle-cell individual)
Inherited diseases
1. Phenylketonuria (PKU) is a disorder characterized by an inability to produce the enzyme phenylalanine hydroxylase, resulting in a potentially fatal or damaging build up of the amino acid phenalalanine, and occurs in one in every 10000 babies.
1. Hypothyroidism is a disorder caused by a small or improperly functioning thyroid gland, or even its complete absence, and occurs in one in every 3500 babies.
2. Galactosaemia is an inherited disorder and occurs in one in every 40,000 babies. Lactose is digested into galactose and glucose. A baby with galactosaemia lacks the enzyme that metabolises galactose and will die if untreated because of the build-up of galactose in the blood.
3. Cystic fibrosis (CF) is an inherited disorder and occurs in one in every 2500 babies. A person with CF produces abnormal secretions that have a serious adverse effect on the function of lungs and digestion. Recent advances in treatment have greatly improved the prognosis for these babies so early diagnosis and treatment are important
The following conditions can be tested by conductiong a simple heel-prick test on a baby 2-3 days after birth,
Early detection
PKU – a case study• The aforementioned conditions are tested for at birth as
early detection is essential in successful treatment.
• Individuals born with PKU can’t produce phenylalanine
hydroxylase (an enzyme).
• Many products contain the amino acid phenylalanine, which
can’t be broken down, the build-up starts to destroy brain
tissue, thereby having a detrimental effect on development.
• Initial detection technique was the “wet nappy” test using
ferric chloride but this often occurred too late
PKU – a case study• An alternative technique was the
Guthrie plate. Blood was collected via
the heel prick test.
• Foetal blood was tested for high levels
of phenalalanine. Using bacteria that
would only grow under certain
concentrations
• Blood is still collected via a heel prick
test but can be tested far more
efficiently via mass spectrometry (a
biochemical analysis of the make-up of
the babie’s blood)
Gene Therapy
Gene therapy• Many genetic diseases are caused by a single gene.
• Eg: PKU
• this single gene defect, could be ‘treated’ by consuming
a special diet.
• This approach is not possible with many conditions.
• Why not try to insert a normal functional gene
into cells that contain a defect?
• Such a procedure is called gene therapy and is a medical
procedure that modifies the genetic material of living cells of
an individual so that a genetic defect is corrected.
Gene therapy
Transferring a piece of DNA into a cell
• The functional piece of DNA inserted into a cell
is specially prepared and is called a cloned
gene.
• How does the cloned gene, get into a patient’s
cells?
• This action is most successful when a vector
is used to carry the gene into a cell.
• The most commonly used vectors for gene
therapy are modified viruses. (Retroviruses
and adenoviruses are the main types used.)
Gene therapy using a retrovirus vector:• Some viruses have DNA, some have
RNA, Retroviruses have RNA that codes
for DNA once it enters a cell
• 1 – remove harmful RNA but leave bits
that code for infection and
transformation to DNA
• 2 – insert RNA version of normal gene in
to virus
• 3 – introduce virus to cells ex-vivo and
allow cells to replicate
• 4 – inject infected cells in to patient
ISSUES
• If inserted in the wrong place could disrupt a
healthy gene
• May cause a severe immune response
Gene therapy using an adenovirus• Adenoviruses contain DNA, in to which a normal copy
of a gene can be integrated
• When the adenovirus enters the host cell, its DNA
enters the nucleus and starts coding for the required
proteins.
• Adenovirus DNA does not integrate with host DNA, so
when mitosis occurs, it does not replicate.ISSUE
• If life span of host cell is
short, positive effects of
this type of gene therapy will be very short-li
ved.
Prenatal testing
Success of gene therapy• Many successful experiments have been conducted with
animals such as mice…
• Still very experimental in humans and can pose risks to
patients’ safety.
• Non-viral gene therapy would be very difficult as this is one of
the few ways of bypassing our immune system
Ultrasound is commonly used to view the uterus and fetus during pregnancy.
Scans are often performed at 18 to 20 weeks into a pregnancy.
Ultrasound maybe performed earlier if there are signs or indications that the fetus is not growing normally.
Ultrasound is also usedto diagnose:
multiple pregnancies(twins, triplets)
gross fetal abnormalitiessuch as trisomy(e.g. Down, Klinefelter,and Turner syndromes)
Screening during Pregnancy
ISSUE
• Not a very useful technique for detecting foetal
abnormalities that have no outward signs
Other types of pre-natal testingCSV (Chorionic Villus Sampling)
Performed at 6-8 weeks
Material gathered can be used for both metabolic and genetic testing
AmniocentesisAmniotic fluid contains both skin cells and urine
Material gathered can be used for both metabolic and genetic testing
Miscarriages are often not random occurrences but actually the result of the foetus dying doue to a genetic abnormality
Thin tube extracts fetal tissue
Amniotic fluid
Genetic Testing
Electrophoresis• DNA collected during screening procedures can be cut in
specific locations using REs (restriction enzymes).
• The DNA can be run through an agarose gel. A current is put
through the gel.
• DNA is negatively charged and will be drawn towards the
positive terminal.
• Smaller fragments will move faster while larger fragment will
take more time to move through the gel
• Every person has 2 copies of every gene and depending on the
abnormality, these can be observed on the gel
• In this case the disease form of the gene is smaller than the
normal version
• The gel shows that both parents were carriers for the condition
and that their first child received each parent’s diseased gene
and therefor developed the condition.
• Fortunately their unborn child received both normal versions of
the gene
OTHER CONDITIONS
The age of onset of many genetic conditions can
vary:
Genetic disease vs predispositionDISEASE
• If you have two affected copies the Huntingtons gene you are
guaranteed to get the disease.
• Onset is usually around the age of 40 and manifests in fairly
rapid brain degeneration. There is no currently available
treatment
PREDISPOSITION
• If you have affected copies of the BRCA1 or BRCA 2 genes for
breast cancer, there is no guarantee that you will get the
disease.
• You have a much higher chance of it developing it, but the
right environmental conditions are required to trigger its
onset
Genetics and DiseaseDown syndrome is often characterized by impairment of cognitive ability and physical growth, as well as some very recognizable facial features
It is caused by an individual being born with a 3rd copy of chromosome 21, due to a separation error during meiosis.Occurrence is estimated at approximately 1:750 births.
Ph
oto
: A
rt T
od
ay
21
Who should be screened
• Anyone with a history of genetic disease
• Two unaffected individuals who have had an affected
child are both carriers of a recessive disease.
• Their chances of having another affected child are 1 in
4
• An affected individual with a dominant disease has one
or both of their alleles carrying the disease
• They have a 1 in 1 or at best a 1 in 2 chance of having
an affected child
Rational Drug Design
Rational drug design• When our immune system fights off the flu it develops a
memory of it, the next time we encounter the same
strain we may not even develop symptoms before it is
fought off.
• The problem is that new strains are constantly
appearing, so the process must be repeated time and
time again.
• Two surface proteins on influenza virus:
• Haemagglutinin is active in gaining entry to a cell.
• Neuraminidase allows the exit of new virus
particles from a cell, freeing them to infect other
cells.
Rational drug design
• Neuraminidase is an enzyme that varies in
structure from strain to strain.
• Examination of strains of the virus from past years
demonstrated that although most of the
molecule changed dramatically, one small
part remained constant.
• Fortunately, this non-variable part is the active
site of the enzyme.
Using the active site
• If a drug was to be designed to
inhibit the active site of
neuraminidase,
• the molecular structure of the
site had to be worked out.
• This was done by computer
modelling so that the active site’s
exact shape and the spatial
arrangements of the atoms
surrounding it became known.
Computer representation of the anti-flu drug in the
active site of neuraminidase.
Designing the drug• Once the detailed structure of the active
site was known
• a molecule could be designed to fit and
bind to the active site,
• Then an anti-flu drug was created!
• This technique, in which the active site
of a molecule is determined and a
second molecule (the drug) is
constructed to fit into that active site to
inhibit the activity of the first molecule,
is called rational drug design.Ph
oto
: C
DC
How does it work?
Developing Vaccines
Types of VaccineThere are two basic types of vaccine:subunit vaccines and whole-agent vaccines.
Recombinant vaccines
Toxoids
Conjugated vaccinesAcellular vaccines
Attenuated(weakened)
Inactivated(killed) ie. just antigen
Subunit Vaccine
Contains some part or product of micro-organisms that can produce
an immune response
Whole-Agent
VaccineContains whole,
nonvirulent microorganisms
Case study: Malaria
MalariaThere are a number of strains, all carried by the Anopheles mosquito1/3 of the world’s population is at risk. Every year there are 4 million new cases and 2 million deaths
Involves a very complex life cycle
Illness mainly due to:Capillaries becoming blocked by “sticky protein” on red blood cells
Release of the toxin GPL
Healthy blood cells being overtaken by the parasite
Only Plasmodium Faliciparum curable as in other strains parasites lay dormant in gut
ResistanceMosquitoes becoming resistant to DDT
Plasmodium becoming resistant to Chloroquine
Developing VaccinesMALARIA
Developing a vaccine is difficult due to Malarial DNA constantly changing and therefore its recognizable protein coat aswell.
A potential vaccine could pre-expose people to a microscopic quantity of the GPL toxin
Another option is to create an inhibitor to block the receptor site on the “sticky protein” so that it is unable to bind to tissue and block capillaries
MEASELSStill a major cause of death in developing countries due to unavailability of vaccine and difficulty in storing it
Current trials have spliced Measles antigen into a strain of lettuce
Lettuce is freeze-dried in to powder and then packaged in to tablets
Cheap and does not require refrigeration
Manufacturing Biological Molecules
Manufacturing biological molecules
Insulin:
• produced by the pancreas, a hormone that
controls the level of glucose in the blood by
controlling its uptake from the blood by
cells…
• A deficiency of this hormone results in an
abnormally high level of glucose in the blood
- diabetes.
• Because the amino acid sequence of the
active molecule of insulin is known, a piece
of DNA carrying the instructions (code) for
insulin can be built and inserted into a
plasmid vector.
Manufacturing biological molecules
Manufacturing biological moleculesApplication for manufactured molecules
Insulin (for diabetics)
Growth hormone
Factor VIII (blood clotting agent missing in hemophiliacs)
AdvantagesCan be produced in very large quantities
If viral antigen can be copied, can be produced using no materials of human biological origin – minimises risk of associated disease
Eg. in the past haemophiliacs receiving transfusions of Factor VIII from blood risked HIV
Eg. patients receiving growth hormone from cadavers risked the brain disease CJD
Nanoparticles
Manufacturing biological moleculesVery small human-made particle (0.1-100 nM in
diameter)
Being used to deliver drugs directly to the cells requiring them
In cancer trials with mice, survival chances increased by 30%
Made up of 3 parts
Fluorescent stain – to follow progress
Methotrexate – a drug that destroys cancerous cells
Folic Acid – a vitamin required by rapidly reproducing cells