Target selection and validation

Question 1

In rational drug discovery a key molecular target that is important in a disease process has to be identified. How can this target be affected by a potential drug ?

Answer 1

1. Block binding of an endogenous ligand
2. Inhibit activity of the target
3. Alter expression of the target
4. Alter interactions of the target with other molecules in the cell

Question 2

What are the six steps of drug discovery and development ?

Answer 2

1. Disease selection
2. Target identification and validation
3. Lead compound identification
4. Lead compound optimization
5. Pre-clinical trial
6. Clinical trials

Question 3

What are the two main criteria on which the disease selection is based in academia ?

Answer 3

1. Personal interests
2. Available grant funding

Question 4

What are the three main criteria on which the disease selection is based in pharmaceutical industry ?

Answer 4

1. Disease prevalence / likely revenue
2. Resources
3. Competition

Question 5

Name the four main reasons for failures in drug development and the proportions (%) of these reasons.

Answer 5

1. Target-related failures (35%)
2. Metabolism failures (10%)
3. Pharmacokinetic failures (10%)
4. Toxicity failures (10%)

Question 6

What is the proportion of successful drug developments (of all drug development programs) ?

Answer 6

25%

Question 7

What are the two main reasons for drugs to fail in the clinical trials ?

Answer 7

1. The drugs do not work
2. The drugs are not safe

Question 8

Name two examples of pharmacokinetic failures in drug development.

Answer 8

• The drug is to long or not long enough stable in the body
• The drug can not be transported to the target

Question 9

Which kind of molecules are targets normally ?

Answer 9

Proteins, whose modulation might inhibit or reverse disease progression.

Question 10

Name four examples of target protein modulation.

Answer 10

1. Direct inhibition or activation in the active site
2. Allosteric activation or inhibition
3. Disruption of critical protein-protein interactions
4. Gene silencing

Question 11

A good target…

Answer 11

• is efficient and safe
• meets clinical and commercial needs
• has specific sites to match another molecule
• may change its structure upon molecule binding
• induces a physiological response by structural change
• induces a physiological response that have therapeutic effects on pathological conditions

Question 12

What are the three main types of drug target proteins ?

Answer 12

1. Enzymes
2. Receptors
3. Monoclonal antibodies

Question 13

What is the molecular approach in drug design ?

Answer 13

The identification of intracellular targets like regularory, structural and metabolic proteins

Question 14

What is the systems approach in drug design ?

Answer 14

When a phenotype is detected at organismal level and can be used as the read-out-system.

Question 15

Which kind of data are collected in a clinical sample ?

Answer 15

Genomics, Proteomics and Genetic Associations

Question 16

In cell models forward genetics are used. What does it mean ?

Answer 16

Based on a known phenotype, an associated gene is searched for. This often involves inducing random mutations and then analysing the changes in the phenotype.

Question 17

In animal models reverse genetics are used. What does it mean ?

Answer 17

A selective gene is manipulated by specific, known mutations and the phenotype is analysed afterwards.

Question 18

Which kind of data are collected in a clinical research (in patients) ?

Answer 18

Data of disease-relevant phenotypes

Question 19

Name four reasons for the differences between the transcriptome and the proteome.

Answer 19

1. mRNA processing
2. mRNA transport out of the nucleus
3. Differing mRNA degrading rates
4. Translational regulations

Question 20

For the identification of disease proteins the protein production has to be investigated. Which methods are usually used therefore ?

Answer 20

1. Gel elecrophoreses
   - IEF for the separation by charge
   - SDS-PAGE for the seperation by size
2. HPLC
   - Separation by e.g. affinity or size
3. Mass spectrometry
   - Protein sequencing by mass

Question 21

How does 2-Dimensional Difference Gel Elecrophorese (2D-DIGE) work ?

Answer 21

• Protein extract 1 contains the healthy sample and is labelled with flour 1
• Protein extract 2 contains the disease-associated sample and is labelled with flour 2
• The protein extracts are mixed and run through the PAGE where the proteins are co-separated
• The resulting gel shows spots of protein extract 1 under the excitation at wavelength 1 and it shows spots of protein extract 2 under the excitation at wavelength 2
• The two images are overlayed and the similarities and differences are analysed

Question 22

With which dysfunction is the grl mutation associated in zebrafish and how can it be treated ?

Answer 22

The mutation is associated with a disrupted blood flow, which can be treated with GS4012

Question 23

What are chemical genomics ?

Answer 23

It is a scientific approach that uses small molecules to study the functions of genes and proteins across a genome.

Question 24

Name three advantages of chemical genomics.

Answer 24

1. Confirms the “druggability” of an unknown target
2. Provides the lead compounds for drug development
3. Compounds that are too toxic are easily rejected

Question 25

What is ENU ?

Answer 25

- N-ethyl-N-nitrosourea - highly efficient chemical mutagen - it mutates DNA in sperm-producing cells - it is used to perform forward genetics

Question 26

What are the advantages of ENU mutagenesis compared to knockout mutagenesis?

Answer 26

- The induced mutations are more "disease-like" - There are no problems associated with knockout-lethality

Question 27

Name one disadnantage of ENU mutagenesis.

Answer 27

The target can not be identified directly and a genome sequencing is required.

Question 28

Name the three steps of target validation.

Answer 28

1. Defining the role of the target in the disease 2. Identifying the patient populations susceptible to interventions using this target 3. Performing experiments in a cellular system to confirm that regulation of the target have the expected effect

Question 29

What is in drug development the next step after the target discovery ?

Answer 29

Screening - Identification of hits - Requires a good read-out-system

Question 30

What means "Screening" in drug development ?

Answer 30

The filtration of active molecules against a target from a huge library

Question 31

Name two types of screening methods in drug development.

Answer 31

1. Assays (receptor, enzyme, cell or tissue -assays) 2. Robot-assisted high-throughput screening (HTS)

Question 32

Name two types of assays used for drug screening.

Answer 32

- Biophysical assays (NMR, SPR) - Fluorescence, luminescence, radioactive and absorbance -based technologies