Non-random screening methods MCQs With Answer

Non-random screening methods are targeted strategies used in pharmaceutical screening that prioritize compounds based on prior knowledge, biology, or chemistry to improve hit rates and efficiency. For B.Pharm students, understanding non-random approaches—such as focused libraries, structure-based and virtual screening, fragment-based discovery, and phenotypic screening—is essential for modern drug discovery and assay development. Key concepts include assay design, hit validation, counterscreens, Z’-factor, cheminformatics, ADMET filtering, and avoiding assay artifacts like PAINS. These methods reduce wasted resources, guide lead identification, and integrate computational and experimental workflows. This primer emphasizes practical principles and screening cascades relevant to pharmaceutics and medicinal chemistry. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. Which statement best describes non-random screening?

• Screening that tests every compound in a collection indiscriminately
• Targeted screening based on prior knowledge or bias
• Automated high-throughput random sampling
• Blind phenotypic screening without any hypothesis

Correct Answer: Targeted screening based on prior knowledge or bias

Q2. What is the primary advantage of non-random screening compared with unbiased high-throughput screening?

• It always identifies novel scaffolds with no prior information
• It reduces experimental throughput and increases cost
• It increases hit quality and relevance by leveraging prior knowledge
• It eliminates the need for secondary assays

Correct Answer: It increases hit quality and relevance by leveraging prior knowledge

Q3. Which of the following is an example of a non-random screening approach?

• Random combinatorial screening
• Focused library screening based on target biology
• Purely phenotypic high-throughput screening without selection criteria
• Screening using randomly synthesized fragments

Correct Answer: Focused library screening based on target biology

Q4. What is a major disadvantage of non-random (biased) screening?

• Higher probability of false positives than random screens
• Greater reagent consumption than full-deck HTS
• Risk of missing novel chemotypes outside the focused space
• Inability to use computational tools

Correct Answer: Risk of missing novel chemotypes outside the focused space

Q5. The Z’-factor in assay development is used to assess which attribute?

• The cost per data point in the assay
• The assay signal window and separation between positive and negative controls
• The number of compounds that can be screened per day
• The chemical diversity of the screening library

Correct Answer: The assay signal window and separation between positive and negative controls

Q6. What is the role of orthogonal assays in a non-random screening cascade?

• To increase throughput by duplicating the primary readout
• To confirm hits using an independent detection method
• To randomly select new library subsets
• To replace counterscreens entirely

Correct Answer: To confirm hits using an independent detection method

Q7. Why are counterscreens essential in non-random screening?

• They speed up primary screening by eliminating controls
• They help identify assay artifacts and nonspecific or interfering compounds
• They always increase the number of hits taken forward
• They are used to increase library diversity

Correct Answer: They help identify assay artifacts and nonspecific or interfering compounds

Q8. Focused libraries are typically designed how?

• By including as many unrelated scaffolds as possible
• By selecting compounds around known SAR, scaffolds, or target motifs
• By random synthesis of large numbers of compounds
• By using only natural products without modification

Correct Answer: By selecting compounds around known SAR, scaffolds, or target motifs

Q9. Virtual screening in a non-random approach most commonly employs which techniques?

• Random PCR amplification and sequencing
• Molecular docking and pharmacophore modeling
• High-content imaging without image analysis
• Purely wet-lab plate-based screening

Correct Answer: Molecular docking and pharmacophore modeling

Q10. Fragment-based non-random screening is characterized by what principle?

• Screening large macrocycles only
• Screening small, low-molecular-weight fragments and growing or linking them into leads
• Screening full drug-like molecules exclusively
• Screening only natural product extracts

Correct Answer: Screening small, low-molecular-weight fragments and growing or linking them into leads

Q11. Phenotypic non-random screening differs from target-based approaches because it:

• Requires detailed target structure before screening
• Identifies compounds based on cellular phenotype without a predefined molecular target
• Only uses purified enzymes in vitro
• Always relies on virtual libraries

Correct Answer: Identifies compounds based on cellular phenotype without a predefined molecular target

Q12. Structure-based non-random screening primarily requires which resource?

• High-resolution three-dimensional structure of the target
• Large-scale animal testing data
• Only ADMET predictions without structural input
• Randomized compound libraries

Correct Answer: High-resolution three-dimensional structure of the target

Q13. High-content screening (HCS) contributes to non-random strategies by providing:

• Single-value biochemical readouts only
• Multiparametric cellular phenotypic data for deeper hit characterization
• Only information on compound solubility
• Guaranteed absence of false positives

Correct Answer: Multiparametric cellular phenotypic data for deeper hit characterization

Q14. During hit-to-lead in a non-random workflow, priority optimization typically includes:

• Only reducing molecular weight regardless of activity
• Improving potency, selectivity, and ADMET properties
• Eliminating any secondary assay testing
• Focusing solely on synthetic route efficiency

Correct Answer: Improving potency, selectivity, and ADMET properties

Q15. Which class of compounds is notorious for producing frequent false positives in screening assays?

• Highly specific irreversible inhibitors
• Pan-assay interference compounds (PAINS)
• Strictly water-soluble peptides
• Monoclonal antibodies

Correct Answer: Pan-assay interference compounds (PAINS)

Q16. In pooled compound screening, deconvolution is used to:

• Mix more compounds into each pool without testing
• Identify the single active compound within a positive pool
• Increase background noise deliberately
• Skip secondary confirmation assays

Correct Answer: Identify the single active compound within a positive pool

Q17. When designing a biochemical screening assay for an enzyme, which parameter is critical to set appropriately?

• Substrate concentration relative to the enzyme Km
• The number of wells per plate irrespective of controls
• Only the color of the plate seal
• Ignoring enzyme stability

Correct Answer: Substrate concentration relative to the enzyme Km

Q18. Which statement about signal-to-background (S/B) ratio is correct?

• Lower S/B ratios are always preferable
• Higher S/B ratios generally increase assay robustness and detection of true signal
• S/B is irrelevant if you have many replicates
• S/B ratio should always be exactly 1

Correct Answer: Higher S/B ratios generally increase assay robustness and detection of true signal

Q19. The goal of maximizing library diversity in non-random screening is to:

• Reduce chemical space coverage intentionally
• Cover a broad portion of chemical space to increase the chance of finding varied actives
• Ensure every compound is a peptide
• Exclude drug-like properties

Correct Answer: Cover a broad portion of chemical space to increase the chance of finding varied actives

Q20. Applying ADMET filters early in a non-random screening pipeline helps to:

• Increase the number of late-stage failures
• Reduce the likelihood of advancing compounds with poor pharmacokinetics or toxicity
• Guarantee clinical success
• Replace the need for in vivo studies entirely

Correct Answer: Reduce the likelihood of advancing compounds with poor pharmacokinetics or toxicity

Q21. Scaffold hopping in medicinal chemistry refers to:

• Replacing the core scaffold to retain activity while improving properties
• Randomly adding bulky groups without rationale
• Only changing peripheral substituents but keeping the core identical
• Switching from small molecules to proteins exclusively

Correct Answer: Replacing the core scaffold to retain activity while improving properties

Q22. A key element of hit validation is:

• Reporting the initial hit without replication
• Resynthesizing or retesting fresh compound and generating dose–response data
• Advancing all primary hits directly to animal studies
• Skipping purity assessment of the hit

Correct Answer: Resynthesizing or retesting fresh compound and generating dose–response data

Q23. Why are positive and negative controls used in screening plates?

• Only to decorate the plate
• To normalize data, calculate assay metrics like Z’, and ensure assay performance
• To intentionally alter the hit rate
• They are obsolete in non-random screening

Correct Answer: To normalize data, calculate assay metrics like Z’, and ensure assay performance

Q24. Autofluorescent compounds interfere with fluorescence-based assays. Which approach helps mitigate this problem?

• Using higher compound concentrations to swamp the signal
• Implementing orthogonal readouts such as luminescence or absorbance counterscreens
• Eliminating negative controls from the plate
• Only screening colored compounds

Correct Answer: Implementing orthogonal readouts such as luminescence or absorbance counterscreens

Q25. A screening cascade in non-random methods is best described as:

• A single assay that answers all questions about a compound
• A sequential set of primary, secondary, and tertiary assays to triage and validate hits
• Randomly selecting compounds for clinical trials
• Only computational filtering without experimental follow-up

Correct Answer: A sequential set of primary, secondary, and tertiary assays to triage and validate hits

Q26. Assay miniaturization (e.g., 1536-well plates) is important because it:

• Increases reagent consumption and cost
• Reduces reagent use, lowers cost, and increases throughput
• Eliminates the need for quality controls
• Prevents the use of robotics

Correct Answer: Reduces reagent use, lowers cost, and increases throughput

Q27. How are machine learning and Bayesian methods used in non-random screening?

• To replace all wet-lab assays completely
• To prioritize compounds and predict actives using models trained on existing data
• Only for image storage without analysis
• To randomize screening plates

Correct Answer: To prioritize compounds and predict actives using models trained on existing data

Q28. What is the main aim of a primary non-random screen?

• To fully optimize ADMET and confirm clinical safety
• To identify initial hits that meet predefined criteria for further testing
• To perform full toxicology studies
• To finalize lead compounds without secondary assays

Correct Answer: To identify initial hits that meet predefined criteria for further testing

Q29. Counterscreens in non-random screening commonly help to eliminate which type of compounds?

• Highly selective inhibitors with single-target activity
• Promiscuous binders and frequent hitters that act nonspecifically
• Compounds with excellent ADME profiles
• Validated clinical drugs only

Correct Answer: Promiscuous binders and frequent hitters that act nonspecifically

Q30. Which practice is critical to ensure reliability and reproducibility in non-random screening data?

• Omitting technical replicates to save time
• Maintaining rigorous controls, documentation, and data validation workflows
• Publishing raw data without quality checks
• Using subjective selection without predefined criteria

Correct Answer: Maintaining rigorous controls, documentation, and data validation workflows

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