Drug Discovery- A Brief History

Question 0

What did Ehrlich discover when experimenting with azo dyes?

Answer 0

Trypan red can cure trypanosome infected mice.

Consequently the dye became the first man made chemotherapeutic agent.

Question 1

What is Paul Ehrlich’s modern notion of chemotherapy?

Answer 1

Use of drugs to injure an invading organism without injury to the host.

Question 2

What was the problem with the Trypan red dye?

Answer 2

It was found to be inactive to humans.

Question 3

What was arsenious acid found to cure?

Answer 3

Trypanosomiasis in mice, however all the mice died. But they no longer had the disease

Question 4

What is the simplest process of drug discovery?

Answer 4

Target identification > screening > pharmaceutical

E.g. In the case of aspirin,
The target: pain
The screening: someone chewing on bark was relieved of pain
The pharmaceutical: and handful of willow bark.

Question 5

What is the active ingredient of aspirin?

Answer 5

Salicin (a glycoside) when this is injected it is hydrolysed to give glucose and salicyl alcohol.
The salicyl alcohol is then oxidised to form salicylic acid, the true drug

Question 6

Why is salicylic acid not suitable as a drug?

Answer 6

It is very bitter and extremely irritating to the stomach

Question 7

What are the two important points to note from the discovery of aspirin?

Answer 7

The concept of a pro drug. This is a medicine administered which the body metabolises to the active form.

The chemical modification of salicylic acid to form acetyl salicylic acid was one of the first examples of structural modification in medicinal chemistry

Question 8

What is a second example of the simple model of drug design?

Answer 8

The discovery of penicillin by Alexander Fleming in 1928, London

Question 9

Why is there a need for new classes of antibiotics?

Answer 9

There are many strains of MRSA present in hospitals

Question 10

What soon became a problem with penicillin?

Answer 10

Strains of staph aureus located in long one hospitals were resistant to penicillin G.

However it was also found that chemical modifications of the amide side chain could lead to the production of new classes of semisynthetic beta lactam antibiotics with improved activity towards resistant organisms. E.g. Ampicillin and methicillin.

Question 11

What are two important points to note from the discovery of penicillin?

Answer 11

Penicillin is unique as it is a classic example of serendipitous nature of drug discovery

The use of purified naturally produced penicillins that are then converted into more active semi synthetic pharmaceuticals. This is not a relatively common practice especially when the synthesis of the main fragment is complicated or expensive

Question 12

What is another example of semi synthesis?

Answer 12

Zocor:

Mevastatin is a natural compound discovered in penicillin citrinum. It is inhibits HMG CoA reductase but was found to have many side effects.

Mevastatin is chemically converted into Zocor which has very few side effects. So we obtain Mevastatin in huge amounts through cell culture of penicillin and then chemically convert this to Zocor

Question 13

Is Zocor a prodrug?

Answer 13

Yes. In the body, the cyclic ester lactone is hydrolysed to give a poly hydroxy acid.v

Question 14

What is a bioassay?

Answer 14

A test to determine the effect of a compound on a specified biological target. This can be done in vitro or in vivo

Question 15

What is a screen?

Answer 15

An optimised bioassay capable of testing a large number of samples against a specific target.

High throughput screening can involve the generation of 500,000+ data points from tested samples. A data point can be as simple as active or inactive

Question 16

What is a hit?

Answer 16

This is a molecule with a good dose-response activity in the primary screen and known confirmed structure

Question 17

What is a progressive hit?

Answer 17

This is a representative compound of. Series with activity via an acceptable mechanism of action and some limited structure activity relationship info

Question 18

What is a lead?

Answer 18

A representative compound of a series with sufficient potential, as measured by potency, selectivity, pharmacokinetics, physicochemical properties, absence of toxicity, novelty of structure to progress to a full drug development programme

Question 19

What is target identification?

Answer 19

Extremely important aspect of drug discovery. This involves picking the therapeutic target with which the pharmaceutical will interact.

Question 20

How have target identification changed throughout the years?

Answer 20

Until recently, the target identification was done by observing the results of compounds by acting upon the cells.
Recent developments in molecular genetics have presented a whole new method for identifying drug targets.

Question 21

How were anticancer agents discovered?

Answer 21

By testing ability of compounds to kill cancer cells in test tubes.

Question 22

Why is understanding cellular processes important?

Answer 22

The more we understand cellular processes, the more likely it is that we can identify a specific cellular process that is peculiar to an invading microbe or cancer cell. This means a target that is not present in normal healthy cells of the human.
This would be specific and represents a good target for a pharmaceutical

Question 23

What is one method we can use to measure the effect of compounds on the target ?

Answer 23

Bioassay

Question 24

What form of test would we need to test 500,000+ compounds?

Answer 24

High throughout screening

Question 25

What is the aim of HTS?

Answer 25

To allow as many compounds as possible to be tested against a particular target in the shortest possible time scale.

This requires special robotics for handling the compounds for performing the bioassay and for reading results. These require a easily measureable biological effect which can be detected and measured automatically

Question 26

What is the effect of the compound usually related to?

Answer 26

Its ability to
Kill cells
Inhibit the function of an enzyme catalysed reaction
Displace natural endogenous ligands from receptors

Question 27

How is a bioassay conducted?

Answer 27

Adding cancer cells in buffer solution to each well.
Add test compounds to each well, and test 8 compounds with tenfold serial dilution from high concentration to low concentration

Wait for 2-3 days, this gives enough time to for the cancer cells to multiple if the compound does not kill them.

Determine which compounds which have killed cancer cells. This is done by attaching a yellow dye MTT to each well

Question 28

What colour is the yellow dye metabolised into to indicate a biologically active compound?

Answer 28

Blue/purple formazan product this means that the compound did not work.

Question 29

How is the formazan product determined?

Answer 29

By measuring the light absorbance at 540nm
. The intensity of the formazan colour is directly related to the number of healthy living cells in the well.

If the appropriate control experiments are run then you can compare the intensity to work out the relative activity of the compounds.

Question 30

What happens if the drug is active?

Answer 30

The cells will look yellow.

Question 31

What parameter are we looking for when plotting a dose response curve?

Answer 31

The concentration of the drug required to kill 50% of cells.

This is the inhibitory concentration for 50%

Question 32

What happens if the compound is inactive?

Answer 32

The MTT will remain yellow

Question 33

How is the amount of formazan product in each well determined?

Answer 33

By measuring the light absorbance at 540nm

Question 34

Why is absorbance measured at 540nm for a bioassay?

Answer 34

This is the wavelength that formazan absorbs and MTT doesn’t

Question 35

How would we use a bioassay to find a pharmaceutical compound which inhibited enzyme function?

Answer 35

We need a way of measuring if the enzyme works or not. We can do this by modifying the substrate so that if reacted by an enzyme, the product formed will be a coloured compound

Question 36

What is a clinical example of a pharmaceutical compound discovered using enzyme bioassay?

Answer 36

The discovery of new antithrombolytic agents which are useful in the treatment of heart attack patients

Question 37

What does thrombin do?

Answer 37

It is an enzyme which normally binds a polypeptide substrate and hydrolyses an amide bond to form smaller peptide products

Question 38

What is the viable substrate for thrombin?

Answer 38

The synthetic peptide L-Alanyl-L-arginine-p-nitroanilide

Question 39

What is the process of the enzyme inhibition assay?

Answer 39

1. Start with sample of the enzyme (thrombin) and add in substrate (L-Alanyl-L-arginine-p-nitroanilide) and test the compound.
2. Analyse via UV absorbance to determine if substrate is being hydrolysed.

Question 40

How are the results of an enzyme inhibition bioassay interpreted?

Answer 40

If p-nitroaniline is produced, our test compound has not inhibited enzyme, hence a negative result.

If UV analysis indicates no p-nitroaniline is being produced then our test compound has inhibited the function of the enzyme. Hence a positive result. Our compound is active

Question 41

How are receptors used in the bioassay screen?

Answer 41

This involves adding a radiolabelled ligand that is known to bind to the receptor and the test compound. An active compound will bind to the receptor and will not allow the ligand to bind.

The radioactivity if the receptor can be measured automatically, and dose response curves and calculation of the IC50 can be done.

Question 42

What is a clinical example of pharmaceutical discovery using receptor bioassays?

Answer 42

Anti-anxiety agents like Valium which target the GABA-benzodiazepine membrane receptor

Question 43

How was the bioassay for the discovery of Valium conducted?

Answer 43

Flunitrazepam is used as a ligand and radiolabelled with tritium on the N-methyl group.
We add in the test compound and receptor and allow it to react.
We then filter the receptor out of the reaction mixture and measure the radioactivity of the receptor

Question 44

How are the results of the receptor bioassay of anti-anxiety agents interpreted?

Answer 44

Radioactive receptor = test compound has not bound to receptor = negative result

Non-radioactive receptor = test compound has displaced labelled flunitrazepam from receptor = compound is active = positive result

Hence the less radioactivity the more potent the drug

Question 45

What are the typical radioactive markers for receptor bioassay?

Answer 45

3H, 32P, or 128I typically incorporated into its structure

Question 46

How can receptors be separated from the reaction mixture in a bioassay?

Answer 46

They have a high MW

Question 47

What happens after the target and bioassay have been defined?

Answer 47

Screening of the compounds

Question 48

What are the two main types of compounds we can screen?

Answer 48

Pure compound libraries

Natural product sources

Question 49

What are the two types of screening for pure compound libraries?

Answer 49

Diversity screening where we test a vast number of compounds

Focused screening where we test a more limited number of compounds which we think are more likely to be active (this gives us more information. We are able to find the pharmacophore using this type of screening).

Question 50

What is the aim if screening?

Answer 50

To find a compound that shows the desired pharmaceutical activity (hit)

This hit then progresses to a lead, and ultimately to a new pharmaceutical product

Question 51

What is the underlying idea behind the mass random screening approach ?

Answer 51

A large number of compounds will contain a wide range of different structural types of molecules.

Question 52

What is a limitation of diversity screening?

Answer 52

It will only target about 1 million compounds which is a very small proportion when compared to 10^40

Question 53

What will large pharmaceutical companies often do?

Answer 53

Purchase new compounds from smaller companies or academic researchers, especially those with interesting or new chemical structures.

Once they have these compounds they will screen them through all their bioassays to find a hit

Question 54

What is the “me too” philosophy that focused screening is driven by?

Answer 54

Where someone else has discovered that a certain class of compound has a certain biological property so if 100,000 structurally related compounds are tested then we would almost be certain of a few hits

Question 55

What is the clinical example of a drug discovered using diversity screening?

Answer 55

Linezolid which is a new oxazolidinone class of antibacterial agent.

DuP721 was a synthetic modification of the agent, achieved using focused screening