DRUG DISCOVERY

A lead compound
(i.e. the "leading" compound, not lead metal) in drug
discovery is a chemical or protype compound that
has desire pharmacological or biological activity and but
may have other undesirable characteristics eg. High
toxicity, insolubility or metabolism problems,
whose chemical structure is used as a starting point
for chemical modifications in order to improve potency
,selectivity or pharmacokinetic parameter, Lead
compounds are often found in high throughput
screenings ("hits") or are secondary metabolites from
natural sources.
High-throughput screening (HTS)

Is a method for scientific experimentation especially

used in drug discovery. Using data processing and
control software and sensitive detectors, High-
throughput Screening allows a researcher to quickly
conduct millions of chemical, genetic or
pharmacological tests.
Hit

Identification of novel active chemical

compounds called "hits“, hits also routinely
originate from structural observations of small
molecule "fragments" bound to therapeutic
targets (enzymes, receptors, etc.), where the
fragments serve as starting points to develop
more chemically complex forms by synthesis.
 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

5
 Drug discovery: Finding a lead

• 1-Choosing the disease

• 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

6
 Drug discovery: Finding a lead
• 3-Identifying a bioassay
– Choice of bioassay
– In vitro test
– In vivo tests
– Test validity
– High-through screening(HTS)
– Screening by NMR
– Affinity screening(AS)
– Surface Plasmon resonance(SPR)
– Scintillation proximity assay(SPA)
– Isothermal titration calorimetry(ITC)
– Virtual screening 7
 Drug discovery: Finding a lead
• 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 or

banks”

– Existing drugs
8
– Starting from natural ligand or modulator (natural
ligands for receptors, natural substrates for
enzymes, enzyme products as lead compounds,
natural modulators as lead compounds)

– Combinatorial synthesis

– Computer aided design

– Serendipity and prepared mind

– Computerized searching of structural databases

– Designing lead compounds by NMR

9
 Drug discovery: Finding a lead
5-Isolation and purification
6-Structural determination
7-Structure-activity relationships
8-Identification of Pharmacophore
9-Target-Oriented drug design
10-Pharmacokinetic drug design
11-Drug metabolism
12-Manufacture-synthetic issues
13- Toxicity testing
14-Clinical trials
15- Patents 10
Overall different Stages
1) To identify target disease
2) To identify drug target
3) To establish testing procedures
4) To find a lead compound
5) Structure Activity Relationship (SAR)
6) To identify a pharmacophore
7) Drug design- optimising target interactions
8) Drug design - optimising pharmacokinetic properties
9) Toxicological and safety tests
10) Chemical development and production
11) Patenting and regulatory affairs
12) Clinical trials
 1- To Identify the target 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.
12
• 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. 13
• 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.

14
The following were the drug target
A) Lipids
Cell membrane lipids

B) Proteins
Receptors
Enzymes
Carrier proteins
Structural proteins (tubulin)

C) Nucleic acids
DNA
RNA

D) Carbohydrates
Cell surface carbohydrates
Antigens and recognition molecules
 2.1- 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 bio-macromolecules 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.
16
For example, tricyclic antidepressants such as
as Desipramine are known to inhibit the
uptake of NA(noradrenaline) 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.
 2.2- 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.
18
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.)
 2.3- 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. 20
 2.4-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 other enzyme.

• Receptors agonist/ antagonist should ideally interact

with a specific kind of receptor (adrenergic receptor)
rather than a variety of different receptors,

21
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.
 2.5-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. 23
2.6-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

24
• 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
25
 3-Identifying a bioassay
3.1-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.

26
 3.2- 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.

• Antibacterial drugs are tested in vitro by measuring

how effectively they inhibit or kill bacterial cells in
culture

27
• For example: HIV Protease has been cloned
and expressed in the bacterium Escherichia
coli(E.Coli)
• A variety of experiment can be carrried out on
this enzyme to determine whether an enzyme
inhibitor is competitive or non-competitive
• Receptor agonist and antagonists can be
tested on isolated tissues or cells, sometime
these tissues can be used to test drugs for
physiological effects
• For example: bronchodilator activity can be
tested by observing how well compounds
inhibit contraction of isolated tracheal smooth
muscle.
• The affinity of drugs for receptors(how
strongly they bind) can be measured by radio-
ligand studies.
 3.3-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.
30
• The animals used may be transgenic (these are animals

whose genetic code has been altered) i.e,It is possible to

replace some mouse genes with human genes so the

mouse produces the human receptor or enzyme and this

allows in vivo testing against that target.

31
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.
33
 3.4-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.

34
 3.5-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. 35
• High throughput screening (HTS) involves the
screening of the entire compound library
directly against the drug target or in a more
complex assay system, such as a cell-based
assay, whose activity is dependent upon the
target but which would then also require
secondary assays to confirm the site of action
of compounds (Fox et al., 2006).
• a hit as being a compound which has the
desired activity in a compound screen and
whose activity is confirmed upon retesting.
progressible hit

targeted synthetic design

Lead
3.6-Screening by NMR

• NMR was used as a tool for determining the

molecular structures of compounds

• 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).
38
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.
 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.
40
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.

41
 3.7-Surface Plasmon resonance (SPR) & scintillation
proximity assay (SPA)

• Are two optical method to detecting whether a legand

binds to its macromolecular targets.

42