Target identification and validation strategies MCQs With Answer

Introduction:
Target identification and validation are fundamental steps in the drug discovery pipeline, linking disease biology to therapeutic opportunities. For M.Pharm students, understanding strategies used to discover, prioritize, and validate biological targets helps bridge basic science and translational research. This set of MCQs covers genetic, biochemical, cellular and in vivo approaches — including genomics, proteomics, RNAi/CRISPR technologies, phenotypic screening, structural biology, and target engagement assays. Questions emphasize practical considerations such as druggability, selectivity, on-/off-target effects, and model selection to ensure candidates have biological relevance and translational potential. Use these items to deepen comprehension and prepare for advanced coursework and research.

Q1. Which of the following best describes “target identification” in drug discovery?

• Evaluating a compound’s safety profile in animal models
• Discovering a biological molecule whose modulation may alter disease outcome
• Optimizing lead compound pharmacokinetics
• Performing clinical trials to assess efficacy

Correct Answer: Discovering a biological molecule whose modulation may alter disease outcome

Q2. What is the primary goal of target validation?

• To determine the toxicity of a lead compound
• To confirm that modulation of a candidate target produces a therapeutic effect in disease-relevant systems
• To identify biomarkers for patient stratification
• To solve the crystal structure of the target protein

Correct Answer: To confirm that modulation of a candidate target produces a therapeutic effect in disease-relevant systems

Q3. Which genomic approach directly reduces gene expression to assess target function?

• X-ray crystallography
• RNA interference (RNAi)
• Surface plasmon resonance (SPR)
• Mass spectrometry proteomics

Correct Answer: RNA interference (RNAi)

Q4. Compared with RNAi, CRISPR-Cas9 offers which main advantage for target validation?

• Only transient suppression of gene expression
• Permanent and specific gene knockout at the DNA level
• Enhanced off-target effects making phenotype detection easier
• Universal protein-level degradation without DNA change

Correct Answer: Permanent and specific gene knockout at the DNA level

Q5. Which technique measures direct binding affinity between a purified protein and a small molecule in real time?

• Cell viability assay
• Surface plasmon resonance (SPR)
• RNA-Seq differential expression
• shRNA knockdown

Correct Answer: Surface plasmon resonance (SPR)

Q6. What does “druggability” of a target primarily refer to?

• The ability to culture the target in vitro
• The likelihood that a target can bind a small molecule with high affinity and selectivity
• The expression level of the target mRNA in all tissues
• The target’s evolutionary conservation across species

Correct Answer: The likelihood that a target can bind a small molecule with high affinity and selectivity

Q7. Phenotypic screening differs from target-based screening primarily because:

• Phenotypic screens monitor organism or cell-level outcomes without predefined targets
• Phenotypic screens always use purified proteins
• Target-based screens cannot discover novel mechanisms
• Phenotypic screening requires crystal structures

Correct Answer: Phenotypic screens monitor organism or cell-level outcomes without predefined targets

Q8. Which assay is commonly used to demonstrate target engagement inside intact cells?

• Cellular thermal shift assay (CETSA)
• Western blot of purified protein
• In vitro enzymatic assay using recombinant enzyme
• RNA-Seq for gene expression

Correct Answer: Cellular thermal shift assay (CETSA)

Q9. Chemical genetics contributes to target validation by:

• Using small molecules to perturb biological pathways and infer target function
• Sequencing the genome to find mutations
• Measuring protein abundance by mass spectrometry only
• Generating transgenic animals exclusively

Correct Answer: Using small molecules to perturb biological pathways and infer target function

Q10. Which method is best suited to identify protein-protein interactions relevant to a target?

• Co-immunoprecipitation followed by mass spectrometry
• High-content phenotypic screening of cells
• qPCR measurement of mRNA levels
• In vitro kinase activity assays

Correct Answer: Co-immunoprecipitation followed by mass spectrometry

Q11. Why are orthogonal validation approaches recommended in target validation?

• To reduce costs by using only one type of experiment
• To confirm findings with independent methods that have distinct biases and artifacts
• To increase the number of variables in a single assay
• Because regulatory agencies require at least five different assays

Correct Answer: To confirm findings with independent methods that have distinct biases and artifacts

Q12. A conditional knockout mouse is most useful for:

• Knocking out a gene ubiquitously from conception to death without spatial control
• Avoiding embryonic lethality by deleting a gene in a tissue- or time-specific manner
• Rapid transient suppression of gene expression in cell lines
• Measuring protein binding kinetics in vitro

Correct Answer: Avoiding embryonic lethality by deleting a gene in a tissue- or time-specific manner

Q13. Which of the following is a limitation of RNAi for target validation?

• RNAi causes permanent genomic deletion
• Off-target knockdown and incomplete suppression of gene expression
• RNAi provides direct measurement of ligand binding affinities
• RNAi cannot be delivered to cultured cells

Correct Answer: Off-target knockdown and incomplete suppression of gene expression

Q14. Biomarkers in target validation are primarily used to:

• Replace in vivo efficacy studies entirely
• Provide measurable indicators of target modulation and downstream biological effects
• Guarantee clinical efficacy in all patient populations
• Determine crystal structures of target-ligand complexes

Correct Answer: Provide measurable indicators of target modulation and downstream biological effects

Q15. Which structural biology technique yields atomic-level detail useful for rational drug design?

• Cryo-electron microscopy (cryo-EM) and X-ray crystallography
• Flow cytometry
• Reverse transcription PCR
• ELISA for cytokine measurement

Correct Answer: Cryo-electron microscopy (cryo-EM) and X-ray crystallography

Q16. What is a primary advantage of using patient-derived xenograft (PDX) models for validation?

• They are genetically identical to cell lines and thus highly reproducible
• They preserve patient tumor heterogeneity and can better predict clinical response
• They eliminate the need for pharmacokinetic studies
• They guarantee success in human clinical trials

Correct Answer: They preserve patient tumor heterogeneity and can better predict clinical response

Q17. Which assay can quantify thermodynamic binding parameters (ΔG, ΔH, ΔS) between ligand and protein?

• Isothermal titration calorimetry (ITC)
• High-throughput cell imaging
• RNA-Seq
• Gel electrophoresis

Correct Answer: Isothermal titration calorimetry (ITC)

Q18. On-target toxicity during validation suggests what about a candidate target?

• The target is irrelevant to the disease
• Target modulation produces adverse effects in normal physiology, requiring therapeutic window assessment
• No further safety studies are needed
• The compound has poor solubility only

Correct Answer: Target modulation produces adverse effects in normal physiology, requiring therapeutic window assessment

Q19. Which bioinformatic resource is most useful for prioritizing targets based on human genetic evidence?

• Genome-wide association study (GWAS) databases
• Mass spectrometry peak lists
• Chromatography retention time libraries
• ELISA kit catalogs

Correct Answer: Genome-wide association study (GWAS) databases

Q20. A successful translational target validation strategy should include:

• Only in vitro enzyme assays with recombinant protein
• An integrated approach combining genetic, pharmacological, biochemical, and in vivo efficacy and safety data
• Exclusive reliance on high-throughput screening hits
• Validation in a single cell line without replication

Correct Answer: An integrated approach combining genetic, pharmacological, biochemical, and in vivo efficacy and safety data

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