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Slide 1
Presented By Dr. Shazzad Hosain Asst. Prof. EECS, NSU The
Protein
Slide 2
Proteins Proteins do the nitty-gritty jobs of every living
cell. Proteins are made of long strings of individual building
blocks known as amino acids.
Slide 3
Connection between DNA and Protein Exon Intron
Slide 4
DNA Transcription and Translation mRNA is read in triplets,
called codon Four different nucleotides, thus 4 3 = 64 possible
codon However, there are only 20 different amino acids Thus genetic
code is degenerate i.e. multiple codon produce same protein mRNA
tRN A
Slide 5
The Genetic Code of RNA The codon AUG for Methionine acts as
start codon
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Protein Functions
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Proteins Make up about 15% of the cell Have many functions in
the cell Enzymes Structural Transport Motor Storage Signaling
Receptors Gene regulation Special functions
Slide 8
Folded proteins are placed into two general categories Fibrous
proteinsglobular proteins
Slide 9
Fibrous proteins have polypeptide chains organized in long
fibers or sheets Water insoluble Very tough physically, may be
stretchy
Slide 10
Functions of fibrous proteins Structural proteins function in
support Insects and spiders use silk fibers to make cocoons and
webs Collagen and elastin are used in animal tendons and ligaments
Keratin is the protein in hairs, horns and feathers
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Functions of fibrous proteins Contractile proteins function in
movement Actin and myosin contract to create the cleavage furrow
and to move muscles Contractile proteins move cilia and
flagella
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Globular proteins have their chains folded into compact,
rounded shapes Easily water soluble
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Functions of globular proteins Storage proteins function in the
storage of amino acids Ovalbumin is the protein in egg whites
Casein is the protein in milk, source of amino acids for baby
mammals
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Functions of globular proteins Transport proteins function in
the movement of other substances Hemoglobin, the iron containing
protein in blood, transport oxygen from lungs to other parts of the
body (C 3032 H 4816 O 872 N 780 S 9 Fe 4 ) Membrane transport
proteins such as channels for potassium and water
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Functions of globular proteins Hormone proteins function as
cellular messenger molecules that help maintain homeostasis
Insulin: sends message allow sugar into cells (when blood glucose
levels are high, cells will transport glucose into the cells for
use or storage) Glucagon: sends message we need more sugar in the
blood (when blood glucose is too low, cells will release
glucose)
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Functions of globular proteins Receptor proteins allow cells to
respond to chemical stimuli Growth factor receptors initiate the
signal transduction pathway when a growth hormone attaches
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Functions of globular proteins Protective proteins function as
protection against disease Antibodies combat bacteria and
viruses
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Functions of globular proteins Enzymes speed up chemical
reactions Amylase and other digestive enzymes hydrolyze polymers in
food Catalase converts hydrogen peroxide H 2 O 2 into water and
oxygen gas during cellular respiration
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Protein Structures
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Peptide Bonds join amino acids Its a condensation reaction
(meaning that H 2 0 (or some other small molecule) is released when
the bond is formed). Two amino acids form a DI-PEPTIDE POLYPEPTIDES
are formed from more than two amino acids bonded together
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Polar R groups make the amino acid hydrophilic Non-polar R
groups make the amino acid hydrophobic
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Ionic R groups make the amino acid hydrophilic
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Polar vs Nonpolar Amino Acids Hydrophilic Hydrophobic
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There are 20 commonly occurring amino acids that are found in
proteins alanine - ala - A arginine - arg - R *** asparagine - asn
- N aspartic acid - asp - D cysteine - cys - C glutamine - gln - Q
glutamic acid - glu - E glycine - gly - G histidine - his - H ***
isoleucine - ile - I leucine - leu - L lysine - lys - K methionine
- met - M phenylalanine - phe - F proline - pro - P serine - ser -
S threonine - thr - T tryptophan - trp - W tyrosine - tyr - Y
valine - val - V Essential Amino Acids are those that must be
ingested in the diet (our body cant make them) *** essential in
certain cases
Slide 32
Proteins have four levels of organization
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Primary structure is the amino acid sequence
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The amino acid sequence is coded for by DNA and is unique for
each kind of protein
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The amino acid sequence determines how the polypeptide will
fold into its 3D shape
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Even a slight change in the amino acid sequence can cause the
protein to malfunction For example, mis-formed hemoglobin causes
sickle cell disease
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Proteins have four levels of organization
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Secondary structure results from hydrogen bonding between the
oxygen of one amino acid and the hydrogen of another
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Red = oxygen Black = Carbon Blue = Nitrogen Green = R
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The alpha helix is a coiled secondary structure due to a
hydrogen bond every fourth amino acid
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The beta pleated sheet is formed by hydrogen bonds between
parallel parts of the protein
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A single polypeptide may have portions with both types of
secondary structure
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Proteins have four levels of organization
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Tertiary structure depends on the interactions among the R
group side chains
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Types of interactions Hydrophobic interactions: amino acids
with nonpolar side chains cluster in the core of the protein, out
of contact with water = charged = hydrophobic
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Types of interactions Hydrogen bonds between polar side
chains
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Types of interactions Ionic bonds between positively and
negatively charged side chains
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Types of interactions Disulfide bridge (strong covalent bonds)
between sulfur atoms in the amino acid cysteine Link to video
Slide 58
Keratin is a family of fibrous structural proteinsfibrous
structural proteins
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Proteins have four levels of organization
Slide 61
Quaternary structure results from interactions among separate
polypeptide chains into a larger functional cluster
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For example, hemoglobin is composed of 4 polypeptide
chains
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Proteins have four levels of organization
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The folding of proteins is aided by other proteins, called
chaperones Act as temporary braces as proteins fold into their
final conformation Research into chaperones is a area of research
in biology
Slide 67
Denaturation results in disruption of the secondary, tertiary,
or quaternary structure of the protein
Slide 68
Denaturation may be due to changes in pH, temperature or
various chemicals
Slide 69
Protein function is lost during denaturation, which is often
irreversible
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Protein Folding
Slide 71
What is Protein Folding ? Protein folding is the process by
which a protein assumes its functional shape or conformation.
Random Coil Native conformation
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Why is the Protein Folding so important Most of the proteins
should fold in order to function Misfolding cause some diseases.
Cystic Fibrosis, affects lungs and digestive system and cause early
death Alzheimerss and Parkinson's disease It may help us to
understand the structure of proteins which has not been known
Slide 73
LEVINTHAL PARADOX Let have Protein composed of 100 amino acids.
Assume that each amino acid has only 3 possible conformations.
Total number of conformations = 3 100 ~= 5x10 47. If 100 psec
(100x10 -12 sec) were required to convert from a conformation to
another one, a random search of all conformations would require
5x10 47 x 10 -10 sec = 1.6 x 10 30 years. However, folding of
proteins takes place in msec to sec order.
Slide 74
Forces that stabilize protein structure Interactions between
atoms within the protein chain Interactions between the protein and
the solvent Electrostatic Interactions Interaction of charged side
chain with the opposite charged side chain. Hydrogen Bonds &
van der Waals forces Hydrophobic interactions
Slide 75
The kinetic Theory of Protein Folding Folding proceeds through
a definite series of steps or a Pathway. A protein does not try out
all possible rotations of conformational angles, but only enough to
find the pathway.
Slide 76
Energy Landscape
Slide 77
Protein folding models The Framework Model Hydrophobic collapse
Nucleation Model
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The Framework Model Local interactions are main determinants of
protein structures unfolded state Transition state native
state
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Hydrophobic Collapse Hydrophobic core forms first. unfolded
state collapse native state
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Hydrophobic Collapse Formation of hydrophobic globule may
hinder the reorganization of both side chains and whole
protein
Slide 81
Nucleation Model Unites hydrophobic collapse and frame work
model unfolded state formation of a nucleus native state
Slide 82
Nucleation Model Substantial expulsion of water from the burial
of non polar surfaces Good correlation between decrease in
hydrodynamic volume and increase in secondary structure
Slide 83
Disease caused by Protein Mis-folding
Slide 84
Diseases caused by the defect in protein folding: Cystic
fibrosis: Defect in the folding of cystic fibrosis Tran membrane
conductance regulator protein. Diseases caused by misfolding of
Prion proteins: Kuru Disease Creutzfedlt-Jakob Disease Scrapie
Disease in sheep Mad cow disease Misfolded prion protein act as
infectious agents. They act as chaperons which can multiply by
binding to normal PrP and folding it to dangerous form similar to
itself. Mechanisms of the functions of normal prions and the
dangerous ones are still not clear.
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Stained section from the cerebral cortex from a patient with
Creutzfedlt-Jakob disease indicating spongiform patahlogy
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Non-local contacts = High contact order contacts between
residues in the primary sequence: NEARBYFAR APART A B B A A B A B
ordering many more residues at once = selecting from more
conformational states -> How is aggregation avoidance
encoded?
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How do high CO structures form co-translationally? in vitro: B
A A B in vivo: A What conformations does A adopt before B appears?
How much native structure can be formed co-translationally?
ribosome ordering many more residues at once = selecting from more
conformational states -> How is aggregation avoidance
encoded?
Slide 88
How are drugs discovered and developed? Drug Discovery
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Choose a disease Choose a drug target Identify a bioassay
bioassay = A test used to determine biological activity.
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Find a lead compound lead compound = structure that has some
activity against the chosen target, but not yet good enough to be
the drug itself. If not known, determine the structure of the lead
compound
Slide 91
Synthesize analogs of the lead Identify
Structure-Activity-Relationships (SARs) Synthesize analogs of the
lead Identify Structure-Activity-Relationships (SARs)
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Identify the pharmacophore pharmacophore = the structural
features directly responsible for activity Optimize structure to
improve interactions with target
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Determine toxicity and efficacy in animal models.
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Determine pharmacodynamics and pharmacokinetics of the drug.
Pharmacodynamics explores what a drug does to the body, whereas
pharmacokinetics explores what the body does to the drug.
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Patent the drug Continue to study drug metabolism Continue to
test for toxicity
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Design a manufacturing process Carry out clinical trials Market
the drug
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Choosing a Disease Pharmaceutical companies are commercial
enterprises Pharmaceutical companies will, therefore, tend to avoid
products with a small market (i.e. a disease which only affects a
small subset of the population)
Slide 98
Choosing a Disease Pharmaceutical companies will also avoid
products that would be consumed by individuals of lower economic
status (i.e. a disease which only affects third world
countries)
Slide 99
Choosing a Disease (cont.) Most research is carried out on
diseases which afflict first world countries: (e.g. cancer,
cardiovascular diseases, depression, diabetes, flu, migraine,
obesity).
Slide 100
The Orphan Drug Act The Orphan Drug Act of 1983 was passed to
encourage pharmaceutical companies to develop drugs to treat
diseases which affect fewer than 200,000 people in the US
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Under this law, companies who develop such a drug are entitled
to market it without competition for seven years. This is
considered a significant benefit, since the standards for patent
protection are much more stringent.
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Identifying a Drug Target Drug Target = specific macromolecule,
or biological system, which the drug will interact with Sometimes
this can happen through incidental observation
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Identifying a Drug Target (cont.) Example: In addition to their
being able to inhibit the uptake of noradrenaline, the older
tricyclic antidepressants were observed to incidentally inhibit
serotonin uptake. Thus, it was decided to prepare molecules which
could specifically inhibit serotonin uptake. It wasn t clear that
this would work, but it eventually resulted in the production of
fluoxetine (Prozac).
Slide 104
The mapping of the human genome should help! In the past, many
medicines (and lead compounds) were isolated from plant sources.
Since plants did not evolve with human beings in mind, the fact
that they posses chemicals which results in effects on humans is
incidental.
Slide 105
Having the genetic code for the production of an enzyme or a
receptor may enable us to over- express that protein and determine
its structure and biological function. If it is deemed important to
the disease process, inhibitors (of enzymes), or antagonists or
agonists of the receptors can be prepared through a process called
rational drug design.
Slide 106
Simultaneously, Chemistry is Improving! This is necessary,
since, ultimately, plants and natural sources are not likely to
provide the cures to all diseases. In a process called
combinatorial chemistry large numbers of compounds can be prepared
at one time. The efficiency of synthetic chemical transformations
is improving.
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Selectivity is Important! e.g. targeting a bacterial enzyme,
which is not present in mammals, or which has significant
structural differences from the corresponding enzyme in
mammals
Slide 108
The Standards are Being Raised More is known about the
biological chemistry of living systems For example: Targeting one
subtype of receptor may enable the pharmaceutical chemist to avoid
potentially troublesome side effects.
Slide 109
Problems can arise Example: The chosen target, may over time,
lose its sensitivity to the drug Example: The
penicillin-binding-protein (PBP) known to the the primary target of
penicillin in the bacterial species Staphylococcus aureus has
evolved a mutant form that no longer recognizes penicillin.
Slide 110
Choosing the Bioassay Definitions: In vitro: In an artificial
environment, as in a test tube or culture media In vivo: In the
living body, referring to tests conductedin living animals Ex vivo:
Usually refers to doing the test on a tissue taken from a living
organism.
Slide 111
Choosing the Bioassay (cont.) In vitro testing Has advantages
in terms of speed and requires relatively small amounts of compound
Speed may be increased to the point where it is possible to analyze
several hundred compounds in a single day (high throughput
screening) Results may not translate to living animals
Slide 112
Choosing the Bioassay (cont.) In vivo tests More expensive May
cause suffering to animals Results may be clouded by interference
with other biological systems
Slide 113
Finding the Lead Screening Natural Products Plants, microbes,
the marine world, and animals, all provide a rich source of
structurally complex natural products.
Slide 114
It is necessary to have a quick assay for the desired
biological activity and to be able to separate the bioactive
compound from the other inactive substances Lastly, a structural
determination will need to be made
Slide 115
Finding the Lead (cont.) Screening synthetic banks
Pharmaceutical companies have prepared thousands of compounds These
are stored (in the freezer!), cataloged and screened on new targets
as these new targets are identified
Slide 116
Finding the Lead (cont.) Using Someone Elses Lead Design
structure which is similar to existing lead, but different enough
to avoid patent restrictions. Sometimes this can lead to dramatic
improvements in biological activity and pharmacokinetic profile.
(e.g. modern penicillins are much better drugs than original
discovery).
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Finding the Lead (cont.) Enhance a side effect
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Use structural similarity to a natural ligand
Slide 119
Computer-Assisted Drug Design If one knows the precise
molecular structure of the target (enzyme or receptor), then one
can use a computer to design a perfectly-fitting ligand. Drawbacks:
Most commercially available programs do not allow conformational
movement in the target (as the ligand is being designed and/or
docked into the active site). Thus, most programs are somewhat
inaccurate representations of reality.
Slide 120
Serendipity: a chance occurrence Must be accompanied by an
experimentalist who understands the big picture (and is not solely
focused on his/her immediate research goal), who has an open mind
toward unexpected results, and who has the ability to use deductive
logic in the explanation of such results. Example: Penicillin
discovery Example: development of Viagra to treat erectile
dysfunction
Slide 121
Finding a Lead (cont.) Sildenafil (compound UK-92,480) was
synthesized by a group of pharmaceutical chemists working at
Pfizer's Sandwich, Kent research facility in England. It was
initially studied for use in hypertension (high blood pressure) and
angina pectoris (a form of ischaemic cardiovascular disease). Phase
I clinical trials under the direction of Ian Osterloh suggested
that the drug had little effect on angina, but that it could induce
marked penile erections.
Slide 122
Pfizer therefore decided to market it for erectile dysfunction,
rather than for angina. The drug was patented in 1996, approved for
use in erectile dysfunction by the Food and Drug Administration on
March 27, 1998, becoming the first pill approved to treat erectile
dysfunction in the United States, and offered for sale in the
United States later that year. It soon became a great success:
annual sales of Viagra in the period 19992001 exceeded $1
billion.
Slide 123
Finding a Lead (cont.)
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Structure-Activity-Relationships (SARs) Once a lead has been
discovered, it is important to understand precisely which
structural features are responsible for its biological activity
(i.e. to identify the pharmacophore)
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The pharmacophore is the precise section of the molecule that
is responsible for biological activity
Slide 128
This may enable one to prepare a more active molecule This may
allow the elimination of excessive functionality, thus reducing the
toxicity and cost of production of the active material This can be
done through synthetic modifications Example: R-OH can be converted
to R-OCH3 to see if O-H is involved in an important interaction
Example: R-NH2 can be converted to R-NH-COR to see if interaction
with positive charge on protonated amine is an important
interaction
Slide 129
Link
Slide 130
Next step: Improve Pharmacokinetic Properties Improve
pharmacokinetic properties. pharmacokinetic = The study of
absorption, distribution, metabolism and excretion of a drug
(ADME). Video
exercise=MedicationDistribution&title=Medication%20
Absorption,%20Distribution,%20Metabolism%20and%
20Excretion%20Animation&publication_ID=2450
Slide 131
Metabolism of Drugs The body regards drugs as foreign
substances, not produced naturally. Sometimes such substances are
referred to as xenobiotics Body has goal of removing such
xenobiotics from system by excretion in the urine The kidney is set
up to allow polar substances to escape in the urine, so the body
tries to chemically transform the drugs into more polar
structures.
Slide 132
Metabolism of Drugs (cont.) Phase 1 Metabolism involves the
conversion of nonpolar bonds (eg C-H bonds) to more polar bonds (eg
C-OH bonds). A key enzyme is the cytochrome P450 system, which
catalyzes this reaction: RH + O 2 + 2H + + 2e ROH + H 2 O
Slide 133
Mechanism of Cytochrome P450
Slide 134
Phase I metabolism may either detoxify or toxify. Phase I
reactions produce a more polar molecule that is easier to
eliminate. Phase I reactions can sometimes result in a substance
more toxic than the originally ingested substance. An example is
the Phase I metabolism of acetonitrile
Slide 135
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The Liver Oral administration frequently brings the drugs (via
the portal system) to the liver
Slide 138
Metabolism of Drugs (cont.) Phase II metabolism links the drug
to still more polar molecules to render them even more easy to
excrete
Slide 139
Metabolism of Drugs (cont.) Another Phase II reaction is
sulfation (shown below)
Slide 140
Phase II Metabolism Phase II reactions most commonly detoxify
Phase II reactions usually occur at polar sites, like COOH, OH,
etc.
Slide 141
Manufacture of Drugs Pharmaceutical companies must make a
profit to continue to exist Therefore, drugs must be sold at a
profit One must have readily available, inexpensive starting
materials One must have an efficient synthetic route to the
compound As few steps as possible Inexpensive reagents
Slide 142
The route must be suitable to the scale up needed for the
production of at least tens of kilograms of final product This may
limit the structural complexity and/or ultimate size (i.e. mw) of
the final product In some cases, it may be useful to design
microbial processes which produce highly functional, advanced
intermediates. This type of process usually is more efficient than
trying to prepare the same intermediate using synthetic
methodology.
Slide 143
Toxicity Toxicity standards are continually becoming tougher
Must use in vivo (i.e. animal) testing to screen for toxicity Each
animal is slightly different, with different metabolic systems,
etc. Thus a drug may be toxic to one species and not to
another
Slide 144
Example: Thalidomide Thalidomide was developed by German
pharmaceutical company Grnenthal. It was sold from 1957 to 1961 in
almost 50 countries under at least 40 names. Thalidomide was
chiefly sold and prescribed during the late 1950s and early 1960s
to pregnant women, as an antiemetic to combat morning sickness and
as an aid to help them sleep. Before its release, inadequate tests
were performed to assess the drug's safety, with catastrophic
results for the children of women who had taken thalidomide during
their pregnancies. Antiemetic = a medication that helps prevent and
control nausea and vomiting
Slide 145
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Birth defects caused by use of thalidomide
Slide 147
Example: Thalidomide From 1956 to 1962, approximately 10,000
children were born with severe malformities, including phocomelia,
because their mothers had taken thalidomide during pregnancy. In
1962, in reaction to the tragedy, the United States Congress
enacted laws requiring tests for safety during pregnancy before a
drug can receive approval for sale in the U.S. Phocomelia presents
at birth very short or absent long bones and flipper-like
appearance of hands and sometimes feet.
Slide 148
Example: Thalidomide Researchers, however, continued to work
with the drug. Soon after its banishment, an Israeli doctor
discovered anti-inflammatory effects of thalidomide and began to
look for uses of the medication despite its teratogenic effects. He
found that patients with erythema nodosum leprosum, a painful skin
condition associated with leprosy, experienced relief of their pain
by taking thalidomide. Teratogenic = Causing malformations in a
fetus
Slide 149
Thalidomide Further work conducted in 1991 by Dr. Gilla Kaplan
at Rockefeller University in New York City showed that thalidomide
worked in leprosy by inhibiting tumor necrosis factor alpha. Kaplan
partnered with Celgene Corporation to further develop the potential
for thalidomide. Subsequent research has shown that it is effective
in multiple myeloma, and it is now approved by the FDA for use in
this malignancy. There are studies underway to determine the drug's
effects on arachnoiditis, Crohn's disease, and several types of
cancers.
Slide 150
Clinical Trials Phase I: Drug is tested on healthy volunteers
to determine toxicity relative to dose and to screen for unexpected
side effects
Slide 151
Clinical Trials Phase II:Drug is tested on small group of
patients to see if drug has any beneficial effect and to determine
the dose level needed for this effect.
Slide 152
Clinical Trials Phase III: Drug is tested on much larger group
of patients and compared with existing treatments and with a
placebo
Slide 153
Clinical Trials Phase IV: Drug is placed on the market and
patients are monitored for side effects
Slide 154
Assigned Reading Haffner Marlene E; Whitley Janet; Moses Marie
Two decades of orphan product development. Nature reviews. Drug
discovery (2002), 1(10), 821-5. LinkLink Franks Michael E;
Macpherson Gordon R; Figg William D Thalidomide. Lancet (2004),
363(9423), 1802-11. LinkLink Abou-Gharbia, Magid. Discovery of
innovative small molecule therapeutics. Journal of Medicinal
Chemistry (2009), 52(1), 2-9. LinkLink Paul, S. M. et al. How to
improve R&D productivity: the pharmaceutical industrys grand
challenge. Nature Reviews Drug Discovery (2010), 9: 203-214.
Jorgensen, W. L. The many roles of computation in drug discovery.
Science (2004) 303: 1813-1818. Butcher, E. C. et al. Systems
biology in drug discovery. Nature biotechnology (2004) 22(10):
1253-1259.
Slide 155
Optional Additional Reading Bartlett J Blake; Dredge Keith;
Dalgleish Angus G The evolution of thalidomide and its IMiD
derivatives as anticancer agents. Nature reviews. Cancer (2004),
4(4), 314-22. LinkLink Cragg, G. M.; Newman, D. J. Nature: a vital
source of leads for anticancer drug development. Phytochemistry
Reviews (2009), 8(2), 313-331. LinkLink Betz, U. A. K. et al.
Genomics: success or failure to deliver drug targets? Current
Opinion in Chemical Biology (2005), 9: 387-391 Sams-Dodd, F.
Target-based drug discovery: is something wrong? Drug Discovery
Today (2005) 10: 139-147.
Slide 156
Homework Questions What is an orphan drug. Why has the Orphan
Drug Act been successful? Thalidomide is actually a mixture of two
compounds. Draw their structures and list the physiological effects
of each. What does ADMET stand for? List several possible reasons
for poor efficacy of drug candidates in in vivo models. Explain how
structure-based design was used to develop an inhibitor with
improved selectivity for TACE over MMP-1 and MMP-9. How can the
pharmaceutical industry increase the probability of technical
success (p(TS))? What are the major causes of Phase II and III
attrition?