Target Validation Flashcards
(9 cards)
1
Q
Target validation
A
- Where is it expressed?
- Does level change in disease?
- What does it do?
2
Q
Where is it expressed?
A
RNAseq:
- bulk to measure throughout tissue
- or can measure within a single cell
- measure across a specific tissue and observe how levels of expression change
Immunohistology:
- staining to visualise
- can provide evidence that the target is expressed in the desired tissue and cell type
3
Q
Do levels change during disease?
A
- quantitative RT-PCR or RNAseq
- determine mRNA in normal vs disease tissue
- identify genes that are up or down-regulated in disease state
4
Q
What does it do?
A
- model systems
- manipulate novel targets to understand role
5
Q
e.g. cathepsin k - discovery
A
- ostoeporosis = chronic decrease in bone mass and weakened bones
- due to imbalance of osteoclasts vs osteoblasts
- cDNA prepped from osteoclasts and sequenced
- found cathepsin k
6
Q
e.g. cathepsin k - localisation
A
- localised cathepsin k and found mRNA in osteoclasts but not osteoblasts
- no other cathepsin expression in osteoclasts
- immunohistology showed expression at bone and osteoclast interface
7
Q
e.g. cathepsin k - genetic studies
A
- mutations in cathepsin k = osteochondodysplasia
- increased bone mass and fragility when mutated
- 3 mutations are loss of function
- mutant means osteoclasts do not degrade protein matrix
- confirmed cathepsin k as protease involved in bone degradation
8
Q
e.g. cathepsin k - gene to function
A
- antisense = inhibits osteoclastic bone pit formation
- KO mice = osteoporosis and produced osteoclasts with decreased ability to resorb bone
- proteolytic cleavage activates cell release of cathepsin k to degrade bone
9
Q
e.g. cathepsin k - search for inhibitors
A
- cathepsin k cloned and assays developed
- use crystal structures
- identified selective inhibitors with potent anti-resorptive activity in cell and animal models
- odanacatib = efficacy in phase III but discontinued due to cardiovascular risk
