EXAM 2 - Target Identification and Validation

Question 1

Answer 1

C) A target is not validated until it has been shown to be critically important to a particular disease state and that modulating the target results in a therapeutic effect.

Question 2

Explain what target identification is.

Answer 2

Identification of a biomolecule(s) or signal transduction pathway that plays a causative role in disease development or progression.

Question 3

What does a signal transduction pathway do?

Answer 3

Extraceullular stimulus initiates a cascade of cellular events ultimately leading to a specialized cellular response.
* stimulus –> cellular response –> overall result

Question 4

Explain why you must consider downstream events when you try to identify a drug target.

Answer 4

The target location is going to have downstream effects.
* upstream inhibition will cause downstream inhibition
* targeting drugs more upstream = less selective

Question 5

What is the primary target consideration?

Answer 5

Selectivity
(Is the protein/receptor selective for the disease state?)

Are there targets that are unique to certain pathogens?
* antibacterial = cell wall (not in humans)
* antiviral - HIV = reverse transcriptase (not in humans)

Determining selectivity for human disease
* target only present in disease state
* target specific receptor/enzyme subtype
* control dose to have pos. effect on disease state and neg. effect on normal cells

Question 6

Explain what a side effect is.

Answer 6

A biologial effect induced by a drug that is secondary to the intended effect of the drug.

Question 7

Describe the difference between on-target and off-target side effects

Answer 7

On-target - results from modulating desired target
* difficult to work around bc side effect occurs when the drug binds to the target
* do pos outweigh neg?

Off-target - results from modulating another cellular target
* occurs when drug binds to other proteins that are not the target
* can possibly be avoided with further drug design

Question 8

Does selectivity always matter?

Answer 8

No. Sometimes selectivity doesn’t matter.
* even though it isn’t very selective (has multiple targets), it may still be a good, effective drug.

Question 9

Name the 4 ‘historic’ drug target classes. Why?

Answer 9

1. enzymes
2. GCPRs
3. ion channels
4. ‘other’ receptors

• They naturally have binding sites that bind to small endogenous molecules –> easy to make a drug to bind to those receptors

Question 10

What method would you use to find out how a target works?

Answer 10

Affinity purification - biotin/avidin

Question 11

What method would you use to determine what drug targets are involved with a certain disease state of interest?

Answer 11

• genomics
• proteomics

Question 12

What are the steps of affinity purification?

Answer 12

1. Lead compound is “biotinylated” - covalently linked to biotin
2. Ligand of interest is attached to immobilized avidin
3. Column is filled with avidin-biotin-lead compound
4. Cell lysate is added to the column (lysed cells associated with the target disease)
5. Non-binding proteins are eluted from the column (proteins that are not wanted/dont bind are washed out of the column)
6. Super concentrated free ligand is washed through, eluting bound protein (competes with protein-compound in the column)
7. Protein is analyzed for structure and function.
   * you can identify what proteins can be bound to the targeet compound

Question 13

Explain the genomic approach to determine drug target.

Answer 13

Identify novel disease targets at the level of gene expression by comparing normal vs diseased tissues.
* you can see what genes are only present in diseased tissues and target them.

Question 14

How do you analyze a DNA microarray? What can it tell you?

Answer 14

The color of the spots have different significations:
* black - no cDNA bound in either system
* yellow - gene expression present in both disease and normal
* green - present in normal cells + not present in disease cells
* red - present in disease cells + not present in normal cells

(This can tell you what is a possible target bc it is only present in diseased cells)

Question 15

What is the proteomic approach to target identification? What does it tell you?

Answer 15

Looks at the expression of proteins across normal and disease levels
* Is there a clear difference at the protein level between the normal and disease state that can be exploited for therapeutic gain?

Question 16

What is the difference between target identification and target validation?

Answer 16

Target identification - identifying that a biomolecule/pathway has an active role in disease development (not sure if targeting it will change the progression of disease).

Target validation - determining that the target is critically involved in a disease and targeting it will have a therapeutic effect.

Question 17

Does association with a disease = validation?

Answer 17

No. Association does not equal validation
* doesn’t mean it is a suitable intervention strategy

Question 18

What are the different levels of target validation?

Answer 18

1. lowest - changes in cell culture
2. second - changes in single animal model
3. third - changes in multiple animal models
4. highest - reversal of disease symptoms in clinical setting

Question 19

What is the end goal of methods like RNAi and CRISPR?

Answer 19

They are DNA-modifying strategies
Their goal is to key to validate targets (does the expression of a specific protein have an effect on the disease state?)
* turn off gene expression for a particular protein –> prevents gene expression –> what happens to the disease state?
* without the protein expressed (inhibited), does it reduce disease state?

Question 20

How does a proteasome work?

Answer 20

Proteasome is a protein complex that degrades unwanted/damaged protein tagged by ubiquitin for degradation.

Question 21

Explain Trim-Away method for protein targeting.

Answer 21

Specific antibody binds the protein of interest and TRIM21 (ubiquitin ligase)
* after the target protein and TRIM21 are in close proximity, the target is degraded by proteasome

Question 22

Explain the PROTACs method for protein targeting.

Answer 22

Duo-sided small molecule that recruits E33 ubliquitin ligase and target protein.
* one on each side of the molecule

Because of close proximity, the target molecule gets ubiquinated –> sent to proteasome –> degraded