Lead identification: combinatorial chemistry and HTS MCQs With Answer

Lead identification: combinatorial chemistry and HTS MCQs With Answer

This set of MCQs is designed for M.Pharm students studying principles of drug discovery, focusing on lead identification through combinatorial chemistry and high-throughput screening (HTS). Questions cover library design strategies, synthesis methods (split-and-pool, parallel synthesis), diversity and focused libraries, assay formats used in HTS (biochemical, cell-based, FP, FRET, AlphaScreen), quality metrics (Z’-factor, signal-to-background), hit triage and counterscreens, automation and plate formats, and integration with cheminformatics and fragment-based approaches. The aim is to deepen conceptual understanding and practical considerations encountered in modern lead-finding campaigns.

Q1. Which statement best defines combinatorial chemistry in the context of lead identification?

• Systematic synthesis and testing of single optimized compounds one at a time
• Parallel or mixture-based synthesis of large numbers of related compounds to create libraries for screening
• Computational docking of virtual compounds without any synthesis
• Fragment-based design focusing exclusively on sub-millimolar binders

Correct Answer: Parallel or mixture-based synthesis of large numbers of related compounds to create libraries for screening

Q2. What is the main advantage of split-and-pool synthesis for library generation?

• It produces single, fully characterized compounds without mixtures
• It allows exponential expansion of library size with minimal synthetic operations
• It guarantees uniform physicochemical properties across all compounds
• It eliminates the need for purification and analysis entirely

Correct Answer: It allows exponential expansion of library size with minimal synthetic operations

Q3. Which library design strategy is most appropriate when structural information about the target is available and specific interactions are desired?

• Diversity-oriented synthesis (DOS)
• Focused libraries designed around a known ligand or pharmacophore
• Random combinatorial libraries with maximal chemical diversity
• Fragment libraries composed solely of hydrophobic fragments

Correct Answer: Focused libraries designed around a known ligand or pharmacophore

Q4. Which assay format is most commonly used in biochemical HTS to measure enzymatic activity using fluorescence changes?

• Surface plasmon resonance (SPR)
• Fluorescence resonance energy transfer (FRET)
• Mass spectrometry-based readout
• Cell viability luminescence assay

Correct Answer: Fluorescence resonance energy transfer (FRET)

Q5. The Z’-factor in HTS is a measure of:

• Compound solubility in the assay buffer
• Assay quality and separation between positive and negative controls
• Throughput of the robotic liquid handler
• Number of false positives expected from a screen

Correct Answer: Assay quality and separation between positive and negative controls

Q6. Which of the following is a common cause of false positives in fluorescence-based HTS assays?

• Plate sealing during incubation
• Intrinsic compound fluorescence or quenching (optical interference)
• Using water instead of buffer
• Target protein stability over long-term storage

Correct Answer: Intrinsic compound fluorescence or quenching (optical interference)

Q7. What is the primary purpose of counterscreens during hit triage in an HTS campaign?

• To increase library size by adding analogs
• To confirm hit potency in orthogonal assay formats and eliminate artifacts
• To measure compound purity only
• To scale up synthesis of hits immediately

Correct Answer: To confirm hit potency in orthogonal assay formats and eliminate artifacts

Q8. Which plate format is most commonly used to balance throughput and reagent consumption in modern HTS?

• 6-well plates
• 96-well plates
• 384-well plates
• 1536-well plates exclusively

Correct Answer: 384-well plates

Q9. In combinatorial library analysis, ‘scaffold hopping’ refers to:

• Changing assay conditions between screens
• Replacing core molecular frameworks to explore new chemotypes while retaining key interactions
• Increasing the number of substituents on the same scaffold only
• Combining two active compounds into a single molecule without structural changes

Correct Answer: Replacing core molecular frameworks to explore new chemotypes while retaining key interactions

Q10. Which property set is typically targeted when designing ‘lead-like’ molecules compared to ‘drug-like’ molecules?

• Higher molecular weight and lipophilicity than drug-like compounds
• Smaller size and fewer rotatable bonds to facilitate optimization (e.g., lower MW, lower clogP)
• Maximum polar surface area above 200 Ų
• Minimum number of hydrogen bond donors equal to zero

Correct Answer: Smaller size and fewer rotatable bonds to facilitate optimization (e.g., lower MW, lower clogP)

Q11. Fragment-based lead discovery differs from typical combinatorial screening because fragments are:

• Always nonpolar and insoluble
• Small, low-affinity compounds that can be optimized by growing or linking
• High molecular weight scaffolds screened in mixtures
• Identical to drug-like molecules and therefore not useful for optimization

Correct Answer: Small, low-affinity compounds that can be optimized by growing or linking

Q12. Which biophysical technique provides real-time kinetic binding data and is often used to validate hits from HTS?

• High-content imaging
• Surface plasmon resonance (SPR)
• Luciferase reporter assay
• pKa titration

Correct Answer: Surface plasmon resonance (SPR)

Q13. In assay development for HTS, a high signal-to-background ratio is important because it:

• Ensures shorter assay run times only
• Improves the ability to detect true modulators above assay noise
• Means the assay will never produce false negatives
• Allows use of any plate reader without optimization

Correct Answer: Improves the ability to detect true modulators above assay noise

Q14. PAINS (Pan-Assay Interference Compounds) are problematic because they:

• Always make ideal lead candidates due to high potency
• Frequently give false positives across many unrelated assays due to artifact mechanisms
• Are exclusively metal-containing chelators with no other effects
• Are only problematic in cell-based assays, not biochemical assays

Correct Answer: Frequently give false positives across many unrelated assays due to artifact mechanisms

Q15. Which readout is label-free and measures target stabilization by ligand binding in thermal shift assays?

• Fluorescence polarization (FP)
• Differential scanning fluorimetry (DSF) / thermal shift
• Time-resolved FRET (TR-FRET)
• AlphaScreen luminescence

Correct Answer: Differential scanning fluorimetry (DSF) / thermal shift

Q16. What role does cheminformatics play in combinatorial chemistry and HTS?

• Only used for storing raw plate images
• Helps design libraries, prioritize compounds, analyze SAR, and filter undesirable chemotypes
• Replaces all biological assays with computational predictions exclusively
• Is irrelevant when large physical libraries are available

Correct Answer: Helps design libraries, prioritize compounds, analyze SAR, and filter undesirable chemotypes

Q17. Which optimization objective is typically prioritized immediately after identifying HTS hits for progression to leads?

• Maximizing molecular weight to increase affinity
• Improving potency, selectivity, and basic ADME properties (solubility, metabolic stability)
• Eliminating all polar functionality regardless of activity
• Converting hits into irreversible covalent binders as first step

Correct Answer: Improving potency, selectivity, and basic ADME properties (solubility, metabolic stability)

Q18. AlphaLISA and HTRF are examples of assay technologies primarily used because they:

• Require radioactive labels
• Are homogenous, no-wash assays suitable for miniaturized HTS with low background
• Only work with membrane proteins in native cells
• Are incompatible with automated liquid handling

Correct Answer: Are homogenous, no-wash assays suitable for miniaturized HTS with low background

Q19. In HTS automation, acoustic droplet ejection (ADE) is valued because it:

• Uses high temperatures to improve solubility
• Enables contactless, low-volume compound transfer reducing DMSO and tip usage
• Replaces the need for any quality control checks
• Requires manual pipetting for each plate

Correct Answer: Enables contactless, low-volume compound transfer reducing DMSO and tip usage

Q20. When integrating virtual screening with physical HTS, a common strategy is to:

• Exclude all virtual hits from physical screening to avoid redundancy
• Prioritize virtual screening hits for focused physical screening and enrichment of actives
• Use virtual screening only after lead optimization is complete
• Rely solely on virtual screening for final lead nomination without experimental confirmation

Correct Answer: Prioritize virtual screening hits for focused physical screening and enrichment of actives

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