Topic 2: Target identification and validation

Question 1

successful targets

Answer 1

• Can be inhibited or activated
• Can be assayed
• Chemically tractable
• Rationale for disease link/ suitable models to test theory
• Ability to separate required effects from undesirable ones
• Ability to demonstrate efficacy in clinical setting
• Presence of a market

Question 2

Disease genes, are they good?

Answer 2

• More than 1000 genes are mutated in disease
• Very few are successfully targeted
• The intersect of druggable genes and disease modifying genes is approximately 600-1500 targets

Question 3

Issues with KO mice

Answer 3

• Correlation between mouse and human
• Relevance of KO to developing a small molecule drug
• Compensation by other genes for developing a KO
• Relevance of KO throughout development to adult behavior (day 0)
• Embryonic lethality

Question 4

Questions in target identification

Answer 4

1. Where is it expressed
2. Does its level change in disease
3. What does it do? What happened if you block/ enhance its activity

Question 5

Answering Where is it expressed

Answer 5

• Look for mRNA using (a) microarray or (b) RNA sequencing

Limitations: Not all of the mRNA is expressed. Immunohistochemistry provides a higher fidelity method

Question 6

Answering does its level change in disease and significance of this

Answer 6

• Conduct a microarray. Combine RTPCR products of experimental and control tissue labelled in different colors and see which predominates
• Can also do RTPCR

What does a change in regulation actually mean ?

• It could be the cause of the disease i.e. an important target
• It could be a protective effect

Question 7

Pharmacological approaches to question 3

Answer 7

• Use allosteric modulators
• i.e. it Strontium is an inhibitor of bone-resorbing osteoclasts in osteoporosis. It was hypothesized that it mediates its activity via the calcium-sensing receptor thereby if cinacalcet a calcium sensing receptor PAM would replicate or potentiate the effects of strontium. It did not :(

Question 8

animal approaches to question 3

Answer 8

i.e. double stranded RNA used to silence genes in worms to observe the phenotypic effects. These genes have homologous targets in man. Very high throughput

Question 9

question 3: transgenic approaches

Answer 9

Full KO, Conditional KO, and Inducible KO

Question 10

Human genetics:

Answer 10

• HIV virus required cell surface receptor to invade host (CD4 and chemokine receptors CXCR4 or CCR5)
• 4-12% of Europeans have CCR5-32 allele resulting in a non-functional receptor which renders them immune to R5-tropic HIV infection
• CCR5 antagonists have been developed for treatment of infection i.e. maraviroc

Question 11

Animals (pros and cons)

Answer 11

• Zebra fish
• Mouse
• Rats (Mammalian, easy to breed/handle, many disease models, //strain variability, low throughput)
• Fruit Flies ( replicate easy, complex nervous system, /50% of disease genes homologous to man, not mammalian, handling storage, low throughput)
• Worms (multicellular, easy to grow, similar signaling systems to man,// no immune system, not mammalian, 50% of genes unknown function)

Question 12

Human genetics

Answer 12

• HIV virus required cell surface receptor to invade host (CD4 and chemokine receptors CXCR4 or CCR5)
• 4-12% of Europeans have CCR5-32 allele resulting in a non-functional receptor which renders them immune to R5-tropic HIV infection
• CCR5 antagonists have been developed for treatment of infection i.e. maraviroc