[4] drug discovery-lect-1

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DRUG
DISCOVERY:
FINDING A LEAD

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Drug discovery: Finding a lead
 When a pharmaceutical company or
university research group initiates a new
medicinal chemistry project through to the
identification of a lead compound, they will
consider the following steps in order:
 1-Choosing the disease

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 2-Choosing a drug target
 Drug targets
 Discovering drug targets
 Target specificity and selectivity between
species
 Target specificity and selectivity within
the body
 Targeting drugs to specific organs and
tissues
 Pitfalls

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 3-Identifying a bioassay
 Choice of bioassay
 In vitro test
 In vivo tests
 Test validity
 High-through screening
 Screening by NMR
 Affinity screening
 Surface Plasmon resonance
 Scintillation proximity assay

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 4-Finding a lead compound
 Screening of natural products (the plant
kingdom, the microbial world, the marine
world, animal sources, venoms and
toxins)
 Medical folklore
 Screening synthetic compound “ libraries”
 Existing drugs
 Starting from natural ligand or modulator
(natural ligands for receptors, natural
substrates for enzymes, enzyme
products as lead compounds, natural
modulators as lead compounds)

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 4-Finding a lead compound
 Combinatorial synthesis
 Computer aided design
 Serendipity and prepared mind
 Computerized searching of
structural databases
 Designing lead compounds by NMR

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 5-Isolation and purification
 6-Structural determination
 7-Herbal medicine

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I- Choosing the disease
 Pharmaceutical companies tend to concentrate on
developing drugs for diseases which are prevalent
in developed countries, and aim to produce
compounds with better properties than existing
drugs.
 Pharmaceutical companies have to consider
economic factors as well as medical ones when
they decide which disease to target when
designing a new drug.
 A huge investment has to be made towards the
research and development of a new drug.

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I- Choosing the disease
 Therefore, companies must ensure that they get a good
financial return for their investment.
 As a result, research projects tend to focus on diseases
that are important in the developed world, because it is the
best market for new drugs.
 Thus, research is carried out on ailments such as migraine,
depression, ulcers, obesity, flu, cancer and cardiovascular
disease.
 Less is carried out on the tropical diseases of the developed
world. Only when such diseases start to make an impact in
richer countries, the pharmaceutical companies sit up and
take notice.
 Example: research in antimalarial drugs has increased due to
increase in tourism to more exotic countries and the spread
of malaria into southern states of US.

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II- Choosing a drug target
 Choosing which disease to tackle is usually a
matter for company’s market strategists. The
science becomes important at the next stage.
 A molecular target is chosen which is believed
to influence a particular disease when
affected by a drug.
 The greater the selectivity that can be
achieved, the less chance of side effects.

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1- Drug targets
 Once a therapeutic area has been identified the next stage is to identify
a suitable drug target (e.g. receptor, enzyme or nucleic acid)
 Understanding which biomacromolecules are involved in a particular
disease state is very important.
 This will allow the medicinal chemist whether agonist or antagonist to be
designed for a particular receptor or whether inhibitors should be
designed for a particular enzyme.
 For example, tricyclic antidepressants such as Desipramine are known to
inhibit the uptake of NA from nerve synapses. However, these drugs also
inhibit uptake of serotonin, so the possibility arose that inhibiting
serotonin uptake might be beneficial. A search for selective serotonin
uptake inhibitors has led to the discovery of Fluoxetine, the best selling
antidepressant.

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2- Discovering drug targets
 If a drug or a poison produces a biological effect, there must be a
molecular target for that agent in the body.
 In the past, the discovery of drug targets depends on finding the drug
first. Then, natural chemical messengers started to be discovered.
 But many targets still stay hidden (orphan receptors i.e,novel receptors
whose endogenous ligand is unknown ) and their chemical messengers are
also unknown.
 The challenge is to find a chemical that will interact with these targets in
order to find their function and whether they will be suitable as drug
targets. This is one of the main driving forces behind the rapidly
expanding area of Combinatorial synthesis (synthesis of a large number of
compounds in a short period of time using different reagents and starting
material and are tested for activity.)

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3- Target specificity and selectivity between
species
 Target specificity and selectivity is a crucial factor in
modern medicinal chemistry research
 The more the selective a drug is for its target, the less
chance that it will interact with different targets and
have less undesirable side effects.
 For example, penicillin target an enzyme involved in
bacterial cell wall biosynthesis. Mammalian cells does not
have a cell wall, so this enzyme is absent in human cells
and penicillin has few side effects.

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4-Target specificity and selectivity
within the body
 Selectivity is also important for drug acting on targets within the body
 Enzyme inhibitors should only inhibit the target enzyme and not some other
enzyme.
 Receptors agonist/ antagonist should ideally interact with a specific kind of
receptor (adrenergic receptor) rather than a variety of different
receptors, or even a particular receptor type ( such as β- receptor) or even
a particular receptor subtype β2- receptor.
 Ideally, enzyme inhibitors should show selectivity between the various
isozymes of an enzyme.

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5-Targeting drugs to specific organs
and tissues
 Targeting drugs against specific receptor subtypes often allows
drugs to be targeted against specific organ or against specific
areas of brain.
 This is because the various receptor subtypes are not uniformly
distributed around the body, but are often concentrated in
particular tissues. For example, adrenergic receptors in the heart
are predominantly β1 while those in the lungs are β2. If a drug acts on
either, less side effects would be observed.

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6-Pitfalls
 The body is a highly complex system. It is
possible to identify whether a particular enzyme
or receptor plays a role in a particular aliments.
 For any given function, there are usually several
messengers, receptors, and enzymes involved in
the process

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6-Pitfalls
 For example, there is no one simple cause for
hypertension, there are variety of receptors and
enzymes which can be targeted in its treatment. These
include β1-adrenoceptors, calcium ion channels,
angiotessin-converting enzyme (ACE), and potassium ion
channels.
 Sometimes, more than one target may need to be
addressed for a particular ailment. For example, most of
the current therapies for asthma involve a combination
of bronchodilator (β2 agonist) and an anti-inflammatory
agent such as a corticosteroid

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III-Identifying a bioassay
1-Choice of bioassay
 Choosing the right bioassay or test system is crucial to
the success of a drug research program.
 The test should be simple, quick and relevant as there
are usually a large number of compounds to be analyzed.
 Human testing is not possible at such early stage, so
the test has to be done in vitro first. Because in vitro
tests are cheaper, easier to carry out, less
controversial and can be automated than in vivo one.
 In vivo tests needed to check the drugs interaction
with specific target and to monitor their
pharmacokinetics properties.

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2-In vitro tests
 They do not involve live animals. Instead, specific
tissues, cells, or enzymes are isolated and used.
 Enzyme inhibitors can be tested on pure enzyme in
solution.
 Receptor agonist and antagonists can be tested on
isolated tissues or cells.
 Antibacterial drugs are tested in vitro by measuring how
effectively they inhibit or kill bacterial cells in culture

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3-In vivo tests
 In vivo tests on animals often involve inducing a clinical
condition in the animal to produce observable symptoms.
 The animal is then treated to see whether the drug
alleviates the problem by eliminating the observable
symptoms. For example, the development of non-
steroidal inflammatory drugs was carried out by inducing
inflammation on test animals.

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3-In vivo tests
 The animals used may be transgenic i.e,some mouse
genes are replaced by human genes so the mouse
produces the human receptor or enzyme. Or the mouse’s
gene may be altered to be susceptible for some disease
such as breast cancer.

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3-In vivo tests
 There are several problems associated with in vivo
testing. It is slow and it also causes animal suffering.
There are also many problems of pharmacokinetics and
the result obtained may be misleading. For example,
penicillin methyl ester is hydrolyzed in mice into active
penicillin, while it is not hydrolyzed in humans or rabbits.
Also, thalidomide is teratogenic in rabbits and humans
while it is not in mice.

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4-Test validity
 Sometimes the validity of testing procedure is easy and
clear. For example, the antibacterial drug can be tested
by its effect on killing bacteria. Local anaesthetics are
tested by their effect on blocking action potential in
isolated nerve.
 In other cases, the testing procedure is more difficult.
For example, there is no animal model for antipsychotic
drug.
 Thus, validity of the test should be carried out.

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5-High throughput screening (HTS)
 HTS involves the miniaturization and automation of in
vitro tests such that a large number of tests can be
carried out in a short period of time.
 It involves testing of large number of compounds versus
a large number of targets. The test should produce
easily measurable effect. This effect may be cell
growth, an enzyme catalyzed reaction which produces a
color change (may be a dye) or displacement of
radioactive labelled ligand from its receptors.

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6-Screening by NMR
 NMR was used as a tool for determining the molecular structures of
compounds
 Recently, compounds can be tested or screened for their affinity to
a macromolecular target by NMR spectroscopy. The relaxation
times of ligands bound to a macromolecule are shorter than when
they are unbound (can’t be detected).
 In NMR spectroscopy the compound is radiated with a short pulse
of energy which excites the nuclei of specific atoms (H,N,C)
afterwards, the excited nuclei slowly relax back to the ground state
giving off energy as they so.

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6-Screening by NMR
 There are, several advantages in using NMR as a
detection system:
 1-It is possible to screen 1000 small molecular weight
compounds a day with one machine.
 2-The method can detect weak binding which would be
missed by conventional screening methods.
 3-It can identify the binding of small molecules to
different regions of binding site.
 4-It is complementary to HTS. The later may give false-
positive results, but these can be checked by NMR to
ensure that the compounds concerned are binding in the
correct binding site.

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6-Screening by NMR
 5-The identification of weakly binding molecules allows
the possibility of using them as building blocks for the
construction of larger molecules that bind more
strongly.
 6-Screening can be done on a new protein without
needing to know its function.
 NMR screening also has limitations, the main one being
that at least 200 mg of the protein required.

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7-Surface Plasmon resonance (SPR) &
scintillation proximity assay (SPA)
 SPR (change in refractive index)& SPR (reduction of
emission of light) are two visual methods of detecting
whether ligands bind to macromolecular targets .

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IV-Finding a lead compound
 Once a target and a testing system have been chosen, the next
stage is to find a lead compound. A lead compound is a
compound which shows the desired pharmaceutical activity.
 The level of the activity may not be very great and there may
be undesirable side effects.
 The lead compound provides a start for the drug design and
development process.
 There are various ways in which a lead compound might be
discovered. However, the following are the ways of discovering
the lead compound:
 1-Screening of natural products (the plant kingdom, the
microbial world, the marine world, animal sources, venoms
and toxins)

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 2-Medical folklore
 3-Screening synthetic compound “ libraries”
 4-Existing drugs (Me too drugs & Enhancing the side
effects)
 5-Starting from natural ligand or modulator (natural
ligands for receptors, natural substrates for enzymes,
enzyme products as lead compounds, natural modulators
as lead compounds)
 6-Combinatorial synthesis
 7-Computer aided design
 8-Serendipity and prepared mind
 9-Computerized searching of structural databases
 10-Designing lead compounds by NMR

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1-Screening of natural products
 Natural products are a rich source of biologically active
compounds.
 Many of today’s medicines are either obtained directly
from a natural source or were developed from a lead
compound originally obtained from a natural source.
 The compound responsible for that activity is known as
the active principle.
 Most biologically active natural products are secondary
metabolites with quite complex structures. This has
advantage in that they are extremely novel compounds.

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 But the disadvantage of their complexity makes their synthesis
difficult and the compound needs to be extracted from its
natural source (i.e. costly & inefficient process).
 As a result, there is a need to design simpler analogues of the
lead compounds .
 Natural products can be obtained from different sources such
as:
 1-The plant kingdom: It is rich source of lead compounds
(e.g. morphine, cocaine, digitalis, quinine, tubocurarine, nicotine
and muscarine, paclitaxel (Taxol, recent anticancer), either useful
drugs as morphine or basis for synthetic ).Plants continue to
remain a promising source of new drugs.

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 2-The microbial world: microorganisms such as bacteria and fungi
are rich for lead compounds (e.g. Antgimicrobial Drugs: pencillins,
cephalosporines, tetracyclines, aminoglycosides, chloramphenicol,
rifamycins).
 3-The marine world: coral, sponges, fish and marine
microorganisms have biological potent chemicals, with interesting,
anti-inflammatory, antiviral, and anticancer activity. E.g Curacin A
(anti-tumour, from marine cyanobacterium)
 4-Animal sources: antibiotic peptides were extracted from the
skin of African clawed frog.
 Epibatidine (potent Analgasic) was also obtained from Ecuadorian

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 5-Venoms and toxins: from animals, plants,
snakes, spiders, scorpions, insects and
microorganisms. They are potent because they
have specific interaction with a macromolecular
target in the body. Thus, they provide important
tools in studying receptors, ion channels, and
enzymes.
 e.g. Teprotide (from venom of viper) was the lead
compound for the development of
antihypertensive agents Cilazapril & Captopril

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2- Medical folklore
 Berries, leaves and roots used by local healer or shaman
as medicines. Many are useless or dangerous and if they
work this may be due to Palcebo Effect.
 Some of these extracts indeed have a real effect. (e.g.
quinine (cinchona), reserpine (Rauwolfia), atropine
(atropa beladona), morphine (opium poppy), digitalis
(foxglove), emetine (ipeca), cocaine (coca).

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3-Screening synthetic compounds
(libraries)
 Thousands of compounds have been synthesized . The
majority of these compounds are not used or not been in
the market. They have been stored in compound
libraries, and are still available for testing.
 Pharmaceutical companies screen their ‘library’ to study
a new target and find a lead compound

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4-Existing drugs
 A) Me too drugs: Many companies use established drugs
from their competitors as a lead compound in order to
design a drug. By modifying the structure in such way that
avoids the patent restrictions, retain the activity, and
improved the therapeutic properties.
 For example i) Captopril (Anti-hypertension) used as lead
compound by different companies to produce their own
anti-hypertension drugs.
 ii) Modern penicillins are more selective, more potent and
more stable than original penicillins

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4-Existing drugs
 B) Enhancing a side effect: An existing drug may have a
minor or undesirable side effect, which might be used in
another area of medicine. And such compound could be a
lead compound on the basis of its side effects.
 The aim is to enhance the desired side effect and to
eliminate the major biological activity.
 e.g. Sulfonamides are Antibacterial agents but some
sulfonamides has convulsive side effect due to
hypoglycaemia effect. This, undesirable side effect was
useful in the development sulfonamides drugs for treatment
of diabetes (e.g.antidiabetic sulfonyl urea, Tolbutamine).

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5- Starting from the natural ligands or
modulator
 A) Natural ligands for receptors:
 Natural ligands of a target has been sometime
used as the lead compound.
 E.g. Adrenaline and noradrenaline (natural
neurotransmitters) were used for
developement adrenergic β-agonists such as
Salbutamol, dobutamine, xamoterol, H2
antagonists as cimetidine, and morphine(led to
opiate receptors, and endogenous
opiates:endorphins and enkephalins.

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5- Starting from the natural ligands or modulator
 B) Natural substrates for
enzymes:
 The natural substrate for an enzyme
can be used as the lead compound in
the design of enzyme inhibitors.
 e.g. enkephalines used as a lead
compound to design an inhibitor of
enkephalinases.

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5- Starting from the natural ligands or
modulator
 C) Enzyme products as lead compounds: enzymes catalyze a
reaction in both directions ,so enzyme products can be used as a lead
compound for an enzyme inhibitor e.g. L-benzyl succinic acid inhibit
enzyme catalyzed carboxy peptidase hydrolysis of peptides.
 D) Natural modulators as lead compounds: the natural or
endogenous chemicals that exert allosteric control of receptor or
enzymes called Modulators and can be also as lead compounds.
 e.g. Benzodiazepines: were discovered to modulate the receptor γ-
aminobutyric acid (GABA) by binding to allosteric binding site then
endogenous endozepines were discovered.

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6-Combinatorial synthesis
 Combinatorial synthesis is automated solid-phase
procedure aimed at produce as many as different
structures as possible in short time as possible.
 The reactions are carried out on very small scale, often
in a way that will produce mixtures of compounds.
 Combinatorial synthesis aims to mimic what plants do, i.e.
produce a pool of chemicals.
 One of these compounds may be prove to be a useful lead
compound.

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7-Computer –aided design
 Knowledge of target binding site aids in design of novel
compounds intended to bind with that target.
 The enzyme and receptors can be crystallized and it is
possible to determine their structure (structure of
protein & binding site) by X-ray crystallography.
 Molecular modelling software programs can be used to
study the binding site and to design drugs.

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7- Serendipity and the prepared mind
 Lead compounds are found as a result of serendipity (i.e.
chance)
 e.g. i) Cisplatin (Anti-cancer) & peniciilins
 ii) Development of propanolol (β-blocking) have
unexpected give a benefit of discover Practolol.
 Propanolol is a β-blocker but it is a lipophilic drug and can
enter CNS and cause side effect, by introducing
hydrophilic amide group inhibit passage the blood-brain
barrier and Practolol produced more selective
cardioselective β1 inhibitor with fewer side effects on
CNS.
 Sulfonamides and tolbutamide

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7- Serendipity and the prepared mind
 Workers in TNT factories always complained from
headache due to dilatation of brain blood vessels.
TNT was the basis to prepare nitro derivatives
which were used in angina to dilate coronary blood
vessels and alleviate pain.
 Mustard gas tanks used in second world war
exploded in italian harbor. They discovered that
persons who survived and inhaled this gas lost their
defense against microorganisms due to destruction
of white blood cells.
This led to the discovery of mustard like drugs
which were used in leukemia to inhibit excessive
proliferation of white blood cells.

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9-Computerized searching of
structural databases
 New lead compounds can be found by carrying out
computerized searches of structural databases.
 In order to carry out such search, it is necessary to know
the desired pharmacophore.
 Data base searching is known as database mining.

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10-Designing lead compounds by NMR
 Recently NMR spectroscopy has been used to design a
lead compound rather than to discover one.
 The method sets out to find small molecules
(epitopes) which can bind to specific binding site.
 Lead discovering by NMR can be applied to proteins
of known structure which are labeled with N15.

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V-Isolation and purification
 If a lead compound is present in a mixture of other compounds it
has to be isolated and purified.
 The isolation and purification depends upon structure, stability,
and quantity of the compound.
 e.g. Fleming recognized penicillin, qualities & non-toxic to human
but could not use it clinically because he was unable to purify it.
He could isolate it in aqueous solution, but when he tried to
remove water the drug was destroyed.
 Purification and isolation of penicillins were possible until
development of new experimental procedure such as freeze-drying
and chromatography.

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6-Structure determination
 X-ray crystallography, NMR spectroscopy, mass,
and IR are important in structure
deterimination.

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7-Herbal medicines
 Herbal medicines contain a large variety of different
compounds.
 Several of these may have biological activity, but there
is a significant risk of side effects and toxicity. The
active principle present in small amount, so herbals are
expected to be less active than pure compound.
 Herbal medicines may be interacting with prescribed
medicines and there is no regulations or control of this
matter and their uses.
 But it is an important lead to discover and design new
drugs.

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 A lead compound is a structure which shows a useful pharmacological
activity and can act as the starting point for drug design.
 Natural products are a rich source of lead compounds. The agent
responsible for biological activity of a natural extract is known as
the active principle.
 Lead compound have been isolated from plants, trees,
microorganisms, animals, venoms, and toxin. A study of medical
folklore indicates plants and herbs which may contain novel lead
compounds.
 Lead compounds can be found by screening synthetic compounds
obtained from combinatorial syntheses and other sources.
 Existing drugs can be used as a lead compounds for design of novel
structures in the same therapeutic area. Alternatively, the side
effects of an existing drug can be enhanced to design novel drugs in
a different therapeutic area.
Summary

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Summary
 The natural ligand, substrate, product, or modulator for a
particular target can act as a lead compound.
 The ability to crystallize a molecular target allows the use
of X-ray crystallography and molecular modeling to design
lead compounds which will fit the relevant binding site.
 Serendipity has played a role in the discovery of new lead
compounds.
 Knowledge of an existing drug’s pharmacophore allows the
computerized searching of structural databases to
identify possible new lead compounds which share the
pharmacophore.
 NMR spectroscopy can be used to identify whether small
molecules (epitopes) bind to specific region of a binding
site. Epitopes can be optimized then linked together to
give a lead compound.

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Summary
 If a lead compound is present in a natural extract or a
combinatorial synthetic mixture, it has to be isolated and
purified such that its structure can be determined. X- ray
crystallography and NMR spectroscopy are particular
important in structure determination.

[4] drug discovery-lect-1

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