PRE-CLINICAL BIOLOGICAL TESTING OF ACTIVE COMPOUNDS

Dr Charles FOKUNANG

Strategy for HTS Screens

Run HTS Decided which compounds are active and which are inactive Cluster the actives to put them into series Visualize clusters of actives (showing 2D structures) and pick series of interest
Identify scaffold for each series Use similarity or substructure search on inactives to find inactives related to these series Use SAR techniques to discover differences between actives and inactives in a series

Information generated at different points in the Drug Design process

Gene chip experiments Protein structures Project selection decisions Assay protocols HTS results Series selection decisions SAR studies Combinatorial Expts. Pharmacophores ADME studies Lead cmpd decisions Toxicology studies Scaleup reactions

Clinical Trials data Doctor/patient studies Marketing, surveys, etc

Information storage breakdowns

Large amounts of information generated: Some is not kept at all Some is kept but loses its meaning Often data is kept, but not semantics or decisions e.g. keep the HVX2 assay result for this compound was 3.2, but not what the assay protocol was, whether the compound was considered active, nor whether it was followed up on. Bigger picture or derivative information is usually not stored E.g. all the compounds with a tri-methyl group seemed to have much lower activity for this project

Information access breakdowns

Some information is only available in one physical location Some information is only available within one part of the discovery process Often information is not contextualized for use outside a particular domain When someone is clear about a piece of information they need; that piece of information exists, but they dont know how to access it. E.g. What system to use, what Oracle table its in, or even the knowledge of whether that piece of information does exist!

Missed opportunities

Not a specific breakdown, but if the right piece of information had been available at the right time, better decisions could have been made E.g. A group of compounds is being followed up as potential drugs, but a rival company just applied for a patent on the compounds A large amount of money is being spent developing an HTS assay for a target, but marketing research shows any drug is unlikely to be a success A group of compounds is selected from an HTS as good candidates for follow up, but 20 years ago they were followed up for a similar project and had severe solubility problems

Information use breakdowns

The meaning of data is incorrectly interpreted A single piece of information is used, whilst using a wider range of information would lead to different conclusions Lessons learned from one project are incorrectly applied to another Fuzzy information is taken as concrete information

Genomics & Proteomics Information Handling

Understanding the link between diseases, genetic makeup and expression of proteins

Genomics our understanding of how DNA, genes, Genomics is fast-forwarding

proteins and protein function are related, in both normal and disease conditions Human genome project has mapped the genes in human DNA Hope is that this understanding will provide many more potential protein targets Allows potential personalization of therapies

ATACGGAT TATGCCTA

functions

Geneallow us to Chips Gene chips

look for changes in protein expression for different people with a variety of conditions, and to see if the presence of compounds administered drugs changes that expression Makes possible the design of drugs to target different phenotypes expression profile
(screen for 35,000 genes)

people / conditions e.g. obese, cancer, caucasian

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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Drug discovery: Finding a lead

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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Drug discovery: Finding a lead

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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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 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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Drug discovery: Finding a lead

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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Drug discovery: Finding a lead

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 companys 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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II- Choosing a drug target 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
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uptake inhibitors has led to the discovery of Fluoxetine, the best selling

antidepressant.

II- Choosing a drug target 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 22 and are tested for activity.)

II- Choosing a drug target

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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II- Choosing a drug target 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 - receptor) or even a particular receptor subtype 2- receptor.

Ideally, enzyme inhibitors should show selectivity between the various isozymes of an enzyme.
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II- Choosing a drug target

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 1 while those in the lungs are 2. If a drug acts on

either, less side effects would be observed.

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II- Choosing a drug target

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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II- Choosing a drug target

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 1-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 (2 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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III-Identifying a bioassay 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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III-Identifying a bioassay

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 nonsteroidal inflammatory drugs was carried out by inducing inflammation on test animals.
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III-Identifying a bioassay

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 mouses gene may be altered to be susceptible for some disease such as breast cancer.

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III-Identifying a bioassay

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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III-Identifying a bioassay 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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III-Identifying a bioassay 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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III-Identifying a bioassay 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 (cant 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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III-Identifying a bioassay 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 falsepositive 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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III-Identifying a bioassay 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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III-Identifying a bioassay 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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IV-Finding a lead compound

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 40

IV-Finding a lead compound 1-Screening of natural products

Natural products are a rich source of biologically active compounds. Many of todays 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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IV-Finding a lead compound 1-Screening of natural products

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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IV-Finding a lead compound 1-Screening of natural products

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

frog.

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IV-Finding a lead compound 1-Screening of natural products

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 44

IV-Finding a lead compound

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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IV-Finding a lead compound 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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IV-Finding a lead compound

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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IV-Finding a lead compound

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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IV-Finding a lead compound 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 -agonists such as Salbutamol, dobutamine, xamoterol, H2

antagonists as cimetidine, and morphine(led to opiate receptors, and endogenous opiates:endorphins and enkephalins. 49

IV-Finding a lead compound

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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IV-Finding a lead compound 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 -

aminobutyric acid (GABA) by binding to allosteric binding site then

endogenous endozepines were discovered.
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IV-Finding a lead compound

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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IV-Finding a lead compound

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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IV-Finding a lead compound 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 (-blocking) have unexpected give a benefit of discover Practolol. Propanolol is a -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 1 inhibitor with fewer side effects on CNS. Sulfonamides and tolbutamide
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IV-Finding a lead compound 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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IV-Finding a lead compound 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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IV-Finding a lead compound 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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Summary

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 61 a different therapeutic area.

Preclinical Studies
ADME/PK

Objective: To study the effects of test materials with respect to absorption, distribution, metabolism, and excretion Duration: hours to days Animals Required: typically 2 species (rodent and non-rodent)

Safety Pharmacology

Objective: To investigate undesirable pharmacological effects of the test material Duration: Usually single dose Animals Required: 2 species (rodent and nonrodent) Core battery: Cardiovascular, Respiratory, CNS

Preclinical Studies
Acute Toxicity

Objective: To determine Maximum Tolerated Dose (MTD) and No Observable Effect Level (NOEL) Duration: Typically 14 days after single dose Animals Required: 2 species (rodent and nonrodent) Parameters:

Mortality Weight change

Clinical pathology Gross necropsy Clinical observations

Points to consider:

Dose selection for repeat dose studies Choice of Species (Fialuridine)

Preclinical Studies
Sub Acute Toxicity Objective: To determine toxicity after repeated administration of the test material Duration: 14 28 days Animals Required: 2 species (rodent and nonrodent) Parameters: Clinical pathology Urinalysis Mortality Weight change Clinical obs Histology Points to consider: Dosing regimen similar to clinical Recovery period Duration of clinical trials (Phase I, II, III) Toxicokinetics Immunotoxicity

Preclinical Studies
Subchronic/Chronic Toxicity

Objective: In support of products used to treat chronic conditions Duration: 30 days to 2 years Animals Required: 2 species (rodent and non-rodent) Parameters:

Mortality Clinical obs Histology

Clinical pathology Behavioral Assessment Weight change

Points to consider:

Clinical Trials (EU)

Preclinical Studies
Carcinogenicity Objective: To evaluate the tumorigenic potential in animals and risk to humans Duration: 12 months + Species: Mouse or Rat Parameters: Clinical pathology Tumor development Clinical observations and assessment Points to consider: Considerations from: Pharmacology, Pharmacokinetic or Toxicology (mechanistic in vitro and in vivo) data Structure-activity relationships Compound accumulation over long-term use Continuous use in humans for 6 months +

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 drugs 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

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.

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