Documente Academic
Documente Profesional
Documente Cultură
Dr Charles FOKUNANG
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
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
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
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
Understanding the link between diseases, genetic makeup and expression of proteins
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
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)
11
12
Drug targets
Discovering drug targets Target specificity and selectivity between species Target specificity and selectivity within the body
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
14
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)
15
Combinatorial synthesis Computer aided design Serendipity and prepared mind Computerized searching of structural databases Designing lead compounds by NMR
16
17
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.
18
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.
19
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.
20
Once a therapeutic area has been identified the next stage is to identify
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
21
uptake inhibitors has led to the discovery of Fluoxetine, the best selling
antidepressant.
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
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.)
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.
23
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.
Ideally, enzyme inhibitors should show selectivity between the various isozymes of an enzyme.
24
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
25
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
26
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
27
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.
28
Enzyme inhibitors can be tested on pure enzyme in solution. Receptor agonist and antagonists can be tested on isolated tissues or cells.
III-Identifying a bioassay
III-Identifying a bioassay
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.
31
III-Identifying a bioassay
penicillin methyl ester is hydrolyzed in mice into active penicillin, while it is not hydrolyzed in humans or rabbits.
by its effect on killing bacteria. Local anaesthetics are tested by their effect on blocking action potential in isolated nerve.
33
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.
34
compounds
Recently, compounds can be tested or screened for their affinity to a macromolecular target by NMR spectroscopy. The relaxation
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.
36
NMR screening also has limitations, the main one being that at least 200 mg of the protein required.
37
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 .
38
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
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)
39
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
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.
41
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 .
(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.
42
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.
43
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
Some of these extracts indeed have a real effect. (e.g. quinine (cinchona), reserpine (Rauwolfia), atropine
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.
4-Existing drugs
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
47
4-Existing drugs
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).
48
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
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.
50
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.
52
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.
53
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
54
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.
55
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.
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.
57
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.
58
6-Structure determination
59
7-Herbal medicines
Herbal medicines may be interacting with prescribed medicines and there is no regulations or control of this
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:
Points to consider:
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:
Points to consider:
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.
67
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.
68