Since the end of the 1980s by molecular cloning and genome sequencing projects were identified genes coding unknown GPCRs

1
Target identification
Orphan GPCRs

• Since the end of the 1980s by molecular cloning
  and genome sequencing projects were identified
  genes coding unknown GPCRs
• These receptors are termed orphan since their
  corresponding ligand(s) remains to be identified
• - oGPCR could be new targets

375 GPCRs
2
We have found a target!!!
Congratulation you have been put in charge of a
drug discovery project on this target!!!!
..and now????
3
Target validation

• The target validation process is aimed at
  gathering convincing proofs that the target under
  study is a key player in the development and/or
  progression of a disease.

An appropriate target validation increases
success in drug discovery
4
Target validation flow-chart
Target validation
5
A New Paradigm for Drug Discovery-
Gene-to-Function -
Target validation
A case study SP1999 an orphan receptor
6
A case study SP1999
Target validation
(Zhang et al. 2001)
7
Target validation flow-chart
Target validation
8
How do we do an expression profile?
Target validation
9
Gene expression analysis
Target validation

• Methods
• Dot and Northern blot
• RT-PCR
• QRT-PCR
• In situ hybridization

10
Northern blot
Target validation
11
Target validation
A case study SP1999
Dot blot and Northern blot expression analysis
in human tissues
12
Polimerase chain reaction (PCR)
Target validation
The basis of PCR
13
Target validation
A case study SP1999
14
In situ hybridization
Target validation
15
A case study SP1999
Target validation
In situ hybridization
16
What next?
Target validation
17
Target validation
Strategies for deorphanizing a GPCRs
1. Orphan receptor strategy
2. Reverse pharmacology

• Exposure of cell line to random panel of
  potential receptor transmitters

18
Target validation
A case study SP1999
Size exclusion chromatography
19
Target validation
A case study SP1999
The endogenous ligand of SP1999 is a purine
ADP Thus the receptor belongs to the purinergic
family The deorphanized receptor has been
called P2Y12