Lecture 3 (target validation) Flashcards
Target validation is needed to demonstrate properties such as:
- confirmed role in the pathophysiology of disease
- Target expression is not evenly distributed throughout the body
- The target’s 3D-structure is available to assess druggability
- easily ‘assayable
- promising toxicity profile
- favorable intellectual property status
What does the ‘technical feasibility’ of a target include?
- druggability
- assayability
3, biomarker availability
What are the assessment blocks in the ‘critical path arrangement’ of target discovery?
- disease linkage
- target-related safety
- strategic issues
- technical feasibility
What is to be done for each of the assessment blocks after defining the critical path?
- Experimental approach
questions - Data quality questions
- experiments tasks
- if all milestones fulfilled –>
‘go’ decision
What is target validation?
A process that demonstrates:
- A molecular target is critically involved in a disease process
- Modulation of the target will lead to the desired therapeutic effect
A molecular target needs to be validated in vitro prior to the validation of its therapeutic concept in vivo and its clinical potential.
True or false.
True
What kind of tissues/models does validation study require?
- intact animals
- disease-related cell-based
models
What is the ‘ultimate target validation’?
human clinical trials
What parameters are evaluated in In vitro laboratory tests involving cells or tissues?
ionic concentration
enzyme activities
expression profiles
What is the importance of In vitro and In vivo studies?
In vitro:
to study the biochemical functions of the target upon
binding to potential ligands.
In vivo:
Animal model indicates how the target is involved in the disease.
evaluation of drug-target interactions in a living system
What is the role of In Silico methods in target validation?
- DNA sequence of the putative target is compared with those of known liganded receptors.
- study pathways of genes and products
–> reveal additional drug target
–> understand side effect profiles
Do animal models always predict the outcome in patients?
No
What are some techniques used in target validation?
- Gene knockouts, antisense DNA/RNA, RNA interference (RNAi) –> sense reversal
- proteomics
- Transgenic models, knockout/in, gain-of-function
- Chemical knockouts in chemical biology
What technique is used to carry out gene knockouts in target validation studies?
CRISPR-Cas9
What is the role of antisense DNA/RNA and RNA interference in target validation studies?
disrupting gene expression to reduce the amount of the
corresponding protein, and thus identify the physiological role of the target
How can the transcription of mRNA be modulated at the transcriptional level?
peptide nucleic acid (PNA), locked nucleic acid (LNA), zinc fingers, ribozymes
How can protein function be modulated in target validation experiments?
- through expression of dominant negative and wild-
type protein - using blocking/stimulating antibodies or aptamers.
What are dominant negative proteins?
dominant negative proteins –> mutant forms of a protein that have lost their normal function but retain the ability to interact with other wild-type proteins
How are dominant negative proteins used in target validation studies to modulate protein function?
By co-expressing wild-type proteins along with dominant negative proteins, the dominant negative proteins can outcompete the wild-type proteins for binding partners, leading to a reduction in the overall functional activity of the protein complex
Apart from target validation, what can mRNA modulation through siRNA demonstrate?
demonstrate the ‘value’ of a target by siRNA –>
siRNA allows one to mimic the effect of the drug via mRNA modulation resulting in the temporary suppression of the drug target.
What are the advantages of using siRNA in target validation?
- Can investigate the effect of inhibiting a potential target without having a drug
- Mimics the effect of a drug more closely than if one were to ‘knockout’ a gene
- Knowledge of protein structure not required
- inexpensive
What are the disadvantages of using siRNA in target validation?
- Down-regulating a gene is not the same as inhibiting a specific region of the drug target
- Down-regulating a gene can have an exaggerated effect compared to that seen with decreasing the concentration of drug target
- Residual protein synthesis may be present as it is not possible to achieve 100% down-regulation
- Off-target effect may need to be excluded
What are the major disadvantages of doing target validation at the genetic level?
- Many genes produce several different protein isoforms that often have subtly different functions.
- PTMs can result in protein variations.
What is the major advantage of using proteomics for target validation as opposed to doing it at the genetic level?
- Proteomics makes it easier to distinguish and target just one specific form of a protein.
- Avoid unwanted changes in the expression of other proteins
In target validation, proteomics approach can help to:
- determine in what tissues and cell components the putative target appears and in which development stage
- understand when the target gets expressed and degraded
–> expression patterns over multiple time points as opposed to normal vs disease state
-Verify the target protein’s specific role within the protein family
-Determine the effect when the putative target is inhibited.
–> use RNAi
What technique is used to determine in what tissues and cell components the putative target appears and in which development state?
Sequence analysis can identify
known location-specifying signal
peptides on the protein.
What kind of assays are performed to determine whether the target (protein) is membrane associated?
Protein-phospholipid interaction assays
Animal models for which diseases are extremely difficult to develop?
psychiatric illness
What assumption is made in validation studies using gene knockouts?
the assumption that knocking out the gene for the potential target has the same effect as administering a highly specific inhibitor of the target protein in vivo.
What are ‘inducible knockouts’?
Transgenic models where the transgene can be switched on or off at will. Can be used to study the function of such essential genes.
What is a ‘disease model’?
Transgenic animal models which present a phenotype that bears the hallmarks of a certain disease.
What is the disadvantage of using mice as opposed to zebrafish as disease models?
Working with mice is often time-consuming and expensive, the fish are more affordable and easier to keep
What are the advantages of using zebrafish as model animal?
- more affordable and easier to keep.
- Suitable for large-scale HTS screening
–> faster to raise than mammals
–> Transparent zebrafish embryos - Genes that cause disease in zebrafish
are similar to those in humans
e.g. angiogenesis, inflammation, insulin
regulation
What disease genes in zebrafish are similar to those in humans?
angiogenesis, inflammation, insulin regulation
What is the technique ‘chemical knockouts in validation studies?
chemical biology method uses compound collections to screen for phenotypes generated upon exposure to molecules.
–> A chemical knocks out the function of a gene and leads to a readable phenotype.
–> Relies on the availability of chemical libraries of many diverse compounds.
