5th Dec - Novel Drugs, Novel Uses for drugs and novel drug targets

Question 1

Why do we need novel drugs?

Answer 1

Most drugs don’t work in all patients
We need to enhance the specificity to reduce side effects
We need to enhance efficacy
There are still many untreatable diseases

Question 2

What is pharmacological efficacy?

Answer 2

The ability of a ligand to evoke a response

Question 3

What is clinical efficacy?

Answer 3

The ability of a drug to treat the disease

Question 4

What percentage of current drug targets are enzymes?

Answer 4

41%

Question 5

What percentage of current drug targets are GPCRs?

Answer 5

30%

Question 6

What is the globally top selling drug and what does it target?

Answer 6

Adalimumab which is an antibody that binds TNFalpha treating rheumatoid arthritis

Question 7

Roughly how many GPCRs are present in humans?

Answer 7

800

Question 8

What are the clues to an orphan GPCRs ligand?

Answer 8

Tissue distribution
Structure
Screen with specific ligands

Question 9

How is a GPCR deorphanised?

Answer 9

Using reverse pharmacology

1. In silico GPCR identification
2. Tissue Expression pattern and family tree analysis
3. Expression in surrogate cell type
4. Screen with synthetic ligand bank or biological extracts
5. Cellular Assay
6. Identify positive compounds

Question 10

What is a ligand bank?

Answer 10

A bank of between 1-2 million synthetic compounds used by big Pharma to manipulate the receptor

Question 11

What are possible sources of putative ligands for screening?

Answer 11

Tissue Extracts
Human haemofiltrate
Known and predicted ligand libraries
Synthetic ligands

Question 12

Outline the general process of drug development

Answer 12

1. Target identification via bioinformatics/expression profiling and validation by KO and OE to alter the cause of the disease
2. Assay generation
3. Lead finding
4. Lead optimisation
5. Development
   5i. Non-clinical safety tests
   5ii. Phase 1
   5iii. Phase 2
   5iv. Phase 3
   5v. Phase 4

Question 13

What is the approximate duration for drug development?

Answer 13

6-12 years

Question 14

What is the probability of success of drug development?

Answer 14

<0.01%

Question 15

What are the different assay systems used to screen drugs?

Answer 15

Monitoring coupling by [Ca2+i] response
cAMP assay
Assays of compound receptor interaction
Binding assays

Question 16

Outline how calcium can be used to monitor coupling

Answer 16

Use Gq receptors, promiscuous G proteins or chimeric g proteins to elicit a calcium response. Then monitor the calcium response.

This can be done in a high throughput screen by using the fluorimetric imaging plate reader (FLIPR). Which can screen about 1536 compounds in seconds. Normally use a control which doesn’t express the receptor of interest which should show no response

Question 17

How can a cAMP assay be used to find a successful ligand?

Answer 17

Both Gas and Gai directly affect cAMP levels.
In this process the G protein is fused to the receptor to make it more efficient at inhibiting/stimulating cAMP
cAMP levels are then monitored using Homogeneous time resolved fluorescence (HTRF) which uses FRET.
Anti-cAMP antibody has fluorophore which should cause cAMP’s fluorophore to fluoresce when bound to cAMP.

Then when new cAMP is produced, which is not labelled, it will displace the labelled cAMP –> loss of FRET as cAMP accumulates

Question 18

How can you use beta arrestin to detect active receptors?

Answer 18

Fuse a small fragment of beta galactosidase to the GPCR tail. The larger fragment of beta galactosidase is fused to beta arrestin.
When the GPCR is activated and phosphorylated beta arrestin will bind.
Beta galactosidase is now active –> chemiluminescent signal

Question 19

What is the advantage of the beta-galactosidase assay for receptor activation?

Answer 19

You do not need to know what the receptor couples to downstream

Question 20

What are the advantages for binding assays in drug screening?

Answer 20

It doesn’t matter if the ligand is an agonist or antagonist
Independent of efficacy
Can detect allosteric modulators

Question 21

Give an example of a drug which is a partial agonist, and because of this is beneficial

Answer 21

Buperomorphine is a partial agonist of the mu opiod receptor. It is used to treat heroine addiction as it out-competes heroine thus heroine will not have an effect but it does not elicit the response that heroine does

Question 22

What would be the benefit of using a biased ligand at the mu opiod receptor to target the G-protein pathway and not the Beta arrestin pathway?

Answer 22

Would increase analgesic efficacy
Reduce GI dysfunction
Reduce respiratory suppression

Question 23

What is TRV130?

Answer 23

A novel MOR G-protein biased ligand which has robust g protein signalling with similar efficacy to morphine but has far less beta arrestin recruitment

Question 24

How was TRV130, the mu opiod receptor g protein biased ligand, discovered?

Answer 24

Analgesia studies in mice
Used human embryonic kidney cells to OE b-arrestin 2 and then performed the path hunter assay
Measured cAMP changes using HTRF
Internalisation assay

Question 25

What is chronic heart failure?

Answer 25

When heart output does not match demands

Question 26

What diseases attribute to chronic heart failure?

Answer 26

Coronary artery disease
hypertension
idiopathic dilated cardiomyopathy
alcoholic cardiomyopathy

Question 27

What are the symptoms of chronic heart failure?

Answer 27

Fatigue
Dyspnoea (breathlessness)
Raised jugular venous pressure
Pulmonary and peripheral oedema
Left ventricular sytosolic dysfunction

Question 28

What are the current treatments for chronic heart failure?

Answer 28

Diuretics - remove oedema
ACE inhibitors - cause vasodialtion
Beta blockers

Question 29

Why could beta blockers used in the treatment of chronic heart failure?

Answer 29

Contrary to the hypothesis, in 1975 it was discovered that beta blockers actually reduced mortality in chronic heart failure as they have a protective effect when started on a low dose which slowly increases.

Question 30

What is the mechanism behind beta blockers effect on chronic heart failure?

Answer 30

1. Beta blocker e.g. carvedilol binds to beta adrenoceptor
2. Increased sympathetic drive normally reduces the number of beta adrenoceptors in chronic heart failure, beta blockers counteract this by acting as inverse agonists

OR

2. Ligand is biased i.e. certain beta blockers direct signalling down certain pathways. Believed that carevedilol can stimulate beta arrestin signalling whilst maintaining inverse efficacy for g protein pathway