GPCR Therapeutics

Question 1

What percentage of drug targets are GPCRs?

Answer 1

30%

Question 2

How many GPCRs in humans? How many olfactory?

Answer 2

> 800

~400 olfactory

Question 3

How are the non-olfactory GPCRs classified?

Answer 3

GRAFS system OR Family A, B, C

Question 4

What is the GRAFS system of classification for GPCRs?

Answer 4

Glutamate 
Rhodopsin
Adhesion
Frizzled
Secretin

Question 5

GPCRs have a proven history as drug targets. Give some examples.

Answer 5

Advair = Beta2-AR agonist for asthma
Plavix = P2Y12 antag for thrombosis
Zyprexa = 5-HT2C antag(?) for schizophrenia/ bipolar

Question 6

Which is the second best selling drug worldwide? What does it target?

Answer 6

Advair

Acts as a B2-AR agonist

Question 7

What is the basic principle of reverse pharmacology?

Answer 7

Using a ligand to ‘fish’ for a receptor

Question 8

Give an example of how a GPCR may be targeted by a therapeutic antibody

Answer 8

HIV-gp120 binds to CD4, causing a conformational change that exposes binding site for chemokine receptor (CCR5/CXCR4), allowing entry of the virus into the cell.
This is blocked by anti-GPCR antibody

Question 9

Outline how reverse pharmacology could be used to identify active compounds for a receptor.

Answer 9

1. Identify GPCR in silico using sequence/ hydropathy plot.
2. Examine tissue expression pattern &
3. Analyse family tree
4. Express in surrogate cell type (eg CHO)
5. Cellular assay with ligand bank (endogenous/ synthetic)
6. Identify active compounds

Question 10

Give some ways receptors could be identified as targets for drug development.

Answer 10

Bioinformatics
Expression profiling
Knowledge (literature/ experiments)

Question 11

What is a useful assay system in drug development for screening lots (>1million) of compounds?

Answer 11

High Throughput Screening (HTS)

Question 12

Give an example of a second messenger that could be used in HTS assays.

Answer 12

Calcium

Question 13

What does HTS stand for and why is it useful?

Answer 13

High throughput screening.

Useful for screening lots (>1million) of compounds to find a ‘hit’ for drug development

Question 14

How can Ca responses be used to determine whether a compound binds to a GPCR?

Answer 14

FLIPR

Monitor coupling by measuring calcium response. Uses Gq pathway.

Question 15

How do G proteins interact with GPCRs?

Answer 15

Via the C-terminal region of Galpha.

Question 16

How does Gq interact with its GPCR?

Answer 16

Through its Alpha subunit C-terminal residues- EYNLV

Question 17

How can Gq be modified so it still activated PLC, altering Ca levels, but does not couple to the Gq receptor?

Answer 17

Swap the EYNLV residues in C-term of alpha subunit for DCGLF (amino acids for Gi) to make a Gq(i) fusion protein

Question 18

How can cAMP be measured?

Answer 18

HTRF (homogeneous time resolved fluorescence)

Question 19

How do Gs and Gi affect cAMP levels?

Answer 19

Gs increase

Gi decreases

Question 20

Using HTRF, what will be seen as cAMP increases?

Answer 20

Decreased FRET

Question 21

What is FLIPR?

Answer 21

Fluorescence Imaging Plate Reader

A HTS that measures calcium responses

Question 22

Molecules identified as influencers of the activity of the target receptor require development to produce compounds with ‘drug-like’ properties. Give 4 examples of such properties.

Answer 22

High affinity
Selectivity
Good bioavailability
Orally active small molecules (tablet form)

Question 23

GPCR targets that naturally couple via Gq produce a ligand-dependent increase in intracellular calcium that can be measured using a calcium sensitive due. How can Gi/o-coupled receptor activation be ‘switched’ to induce and increase in intracellular calcium?

Answer 23

Through the use of chimeric G proteins (Gqi5 or Gqo5)

By engineering the cells to over-express a promiscuous G protein (G16/G15)