Drug Target Validation Flashcards
21st October 2024 - flashcards contain notes from further reading as well
1
Q
Why is validating drug targets important?
A
- Ensures that drug discovery efforts focus on targets that play key roles in disease, maximising the return on investment
- Provides evidence that modulating a target can impact disease and confirms the association between the target and disease mechanism
2
Q
What are the key aspects of drug target validation?
A
- Druggability assessment (confirms sequence-related properties of proteins and determines 3D structure)
- Assayability assessment (evaluates the ability to develop assays for high-throughput screening)
- Genetic assessment (studies genetic sequences and patient data to predict therapy efficacy and potential side effects)
3
Q
Steps for Validating Drug Targets Using CRISPR/Cas9
A
- Design and synthesise sgRNAs targeting the drug target gene
- Transfect sgRNAs into cells or animals using methods like electroporation, lipofection, or viral transduction
- Knock out or knock down the drug target gene by creating double-stranded DNA breaks
- Analyse effects on disease-relevant phenotypes using assays like cell proliferation, apoptosis or animal disease models.
- Confirm target validity if knockout/knockdown results in the disease phenotype
4
Q
Post-Validation steps
A
- Validate biochemical assays
- Use high-throughput screening approaches
- Identify promising chemotypes during lead identification
5
Q
What is a drug target?
A
A drug target is a biological entity (protein or gene) interacting with a drug to produce clinical effects
6
Q
Challenges with the one-target concept
A
- Many drugs exhibit polypharmacology
- Proteins may form complexes or contain multiple binding pockets
- Target-based screening may not always lead to effective drugs
7
Q
Target-based screening
A
- Involves identifying new targets first and screening for molecules that bind to them
- Advanced tools like gene sequencing and bioinformatics have revolutionised this strategy
- Limitations: Often lacks clinical efficacy, high R&D costs with fewer drugs over time
8
Q
Phenotypic screening
A
- Drugs can be developed without prior target identification
- Advantages: targets are identified post-discovery, useful for discovering less obvious targets
9
Q
Fast follower strategy
A
- Focuses on improving existing drugs
- Advantages: lower risk, validated targets, known therapeutic profiles
- Challenges: limited innovation
10
Q
Good targets
A
- Receptors or proteins modulating cell functions
- G-protein-coupled receptors (GPCRs) are the most common drug targets
11
Q
Target deconvolution
A
- Identifying the target of an active compound discovered via phenotypic screening
- Can be done in parallel with drug developement
12
Q
Methods for target identification and validation
A
- Affinity chromatography
- Genetic methods
- Haploinsufficiency profiling in yeast
- Analysis of resistant mutants
- siRNA for target validation
- Yeast three-hybrid system
13
Q
Affinity chromatography
A
- Small molecules are immobilised to isolate binding proteins from a mixture
- Works with proteins in their natural state
- Complex setup, high affinity required, difficulty with membrane proteins
14
Q
Genetic methods
A
- Include siRNA, knockdowns, and gene editing to modulate protein expression
- Used for target discovery, deconvolution, and validation
15
Q
Haploinsufficiency Profiling in Yeast
A
- Deletes one copy of a gene to observe changes in drug sensitivity
- Useful for studying multi-target interactions
16
Q
Analysis of resistant mutants
A
Mutants resistant to a drug are analysed to identify target mutations
17
Q
siRNA for Target Validation
A
Silences genes to mimic drug effects, validating potential targets
18
Q
Yeast Three-Hybrid System
A
Modified yeast cells express proteins interacting with small molecules to identify targets
19
Q
RNA Interference (RNAi)
A
- Uses double-stranded RNA (dsRNA) to silence genes post-transcriptionally
- Dicer cleaves dsRNA into siRNA, which integrates into the RISC complex to degrade complementary mRNA
- Used in large-scale screens for cancer dependencies and identifying key regulatory genes
20
Q
Zinc-finger nucleases (ZFNs)
A
- Target specific DNA sequences using zinc-finger motifs fused with nucleases
- Used in introducing double-stranded breaks (DSBs) for gene knockout or insertion
- Used in studies related to renal injury, hypertension, cancer, and immune disorders
21
Q
TALENs (Transcription Activator-Like Effector Nucleases)
A
- Artificial restriction enzymes targeting DNA using TALE proteins from Xanthomonas bacteria
- Used in generating precise mutations in model organisms like zebrafish and pigs
22
Q
Historical Development of CRISPR/Cas9
A
- 2012: Discovery that Cas9 endonuclease can be guided by CRISPR RNA (crRNA) to mediate programmable DNA cleavage
- 2013: CRISPR/Cas9 was successfully used in eukaryotic cells, opening up new directions for biomedical research.
23
Q
CRISPR/Cas9 components
A
- Cas9 nuclease: binds to DNA and creates DSBs at specific locations, requires a short RNA sequence adjacent to the target
- gRNA: provides sequence specificity through Watson-Crick base pairing with the target DNA
24
Q
DNA cleavage in CRISPR/Cas9
A
- Cas9 contains two catalytic domains: HNH domain cuts the DNA strand complementary to the gRNA and RuvC domain cuts the non-complementary strand
- DSB can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR)
25
Q
NHEJ vs HDR
A
- Non-Homologous End Joining (NHEJ): Error-prone, leading to insertions or deletions (indels) that disrupt the gene.
- Homology-Directed Repair (HDR): High fidelity, requiring a repair template for precise modifications.
26
Q
Applications of CRISPR/Cas9
A
- Gene knockout (introducing indels via NHEJ to disrupt the reading frame)
- Precise genome editing (using HDR for target modifications)
- Non-coding regions (investigating regulatory elements and lncRNAs)
- Screening (high-throughput CRISPR screens to identify novel drug targets or understand disease mechanisms)
27
Q
Advantages of CRISPR/Cas9
A
- Flexibility: Can target nearly any DNA sequence with a PAM site.
- Ease of Use: The system is simpler compared to earlier technologies like ZFNs and TALENs.
- Scalability: Easily adaptable for genome-wide applications.
28
Q
Challenges and limitations of CRISPR/Cas9
A
- Off-target effects: imperfect gRNA-DNA pairing can lead to unintended edits. This could be improved by optimising gRNA design and using high-fidelity Cas9 variants
- Delivery: Effective delivery of CRISPR components (e.g., gRNA and Cas9) is critical and can be achieved using plasmids, mRNA, or ribonucleoproteins (RNPs)
- Complex genomic contexts: Polyploidy, copy number variations, and chromatin accessibility can influence editing efficiency
29
Q
Design and Optimization of CRISPR Experiments
A
- gRNA design: GC content optimal range is 40-60%, avoid regions prone to exon skipping or alternative splicing. CRISPOR and CHOPCHOP provide predictions for gRNA efficiency and specificity
- Cell model selection: Immortalised cell lines (e.g. HEK293) are commonly used but may present challenges like aneuploidy. Haploid embryonic stem cells offer higher targeting efficiency
- Confirmation of editing: Edited cells must be validated at the genomic, transcriptomic, and protein levels to ensure accuracy.
30
Q
Future directions of CRISPR/Cas9
A
- Integration with NGS for enhanced target validation
- Development of more precise and less invasive delivery methods.
- CRISPRa/i: Activating or inhibiting gene expression without altering the DNA sequence.
- Base Editing: Directly changing specific nucleotides without DSBs.
