Conformationally restricted peptides MCQs With Answer

Introduction: Conformationally restricted peptides are engineered to limit backbone or side-chain flexibility, improving pharmacological properties important for M.Pharm students to understand. This blog presents targeted multiple-choice questions covering methods such as cyclization, hydrocarbon “stapling,” N‑methylation, incorporation of constrained residues (e.g., Aib, proline analogs), and impacts on helicity, target affinity, proteolytic resistance, and cell permeability. Questions also address synthetic strategies (solid-phase synthesis, ring-closing metathesis), analytical tools (CD, NMR, HPLC, MS), formulation implications (solubility, aggregation, excipients) and regulatory/stability considerations. These MCQs emphasize mechanistic understanding useful for drug design, formulation, and analytical development in peptide therapeutics.

Q1. What best defines a conformationally restricted peptide?

• Peptides engineered to have limited backbone or side‑chain flexibility
• Peptides synthesized only from D‑amino acids
• Peptides with increased free rotatable bonds for flexibility
• Peptides that are exclusively linear and unmodified

Correct Answer: Peptides engineered to have limited backbone or side‑chain flexibility

Q2. Which primary benefit is most commonly sought by introducing conformational constraints into a peptide?

• Improved target binding affinity and proteolytic stability
• Increased molecular weight without functional changes
• Enhancement of random coil content to improve solubility
• Reduction of hydrophobicity to eliminate membrane crossing

Correct Answer: Improved target binding affinity and proteolytic stability

Q3. Which of the following is a widely used chemical strategy to impose an α‑helix constraint in peptides?

• Hydrocarbon stapling via ring‑closing metathesis
• Glycosylation at N‑terminus
• PEGylation of lysine residues
• Terminal amidation only

Correct Answer: Hydrocarbon stapling via ring‑closing metathesis

Q4. Incorporation of alpha‑alpha disubstituted amino acids like Aib (α‑aminoisobutyric acid) primarily promotes which secondary structure?

• Promotion of 3₁₀‑ and α‑helical conformations
• Formation of extended β‑sheets exclusively
• Conversion to random coil states
• Induction of cis‑peptide bonds at multiple sites

Correct Answer: Promotion of 3₁₀‑ and α‑helical conformations

Q5. N‑methylation of backbone amide nitrogens in peptides mainly affects which property?

• Protease resistance and hydrogen‑bonding pattern
• Increased net negative charge of the peptide
• Directly increases molecular weight by >500 Da
• Removes the need for cyclization to stabilize structure

Correct Answer: Protease resistance and hydrogen‑bonding pattern

Q6. Which analytical technique is most informative for assessing secondary structure (helicity) of conformationally restricted peptides in solution?

• Circular dichroism (CD) spectroscopy
• Size‑exclusion chromatography (SEC) alone
• Gas chromatography (GC)
• Ion‑selective electrode measurement

Correct Answer: Circular dichroism (CD) spectroscopy

Q7. Lactam bridges used to constrain peptides are formed between which functional groups?

• Side‑chain carboxylate (Asp/Glu) and side‑chain amine (Lys) to form an amide (lactam)
• Alpha carbon to sulfur bond via thiolation
• Terminal hydroxyl groups forming esters only
• Peptide backbone carbonyl and aromatic ring forming imines

Correct Answer: Side‑chain carboxylate (Asp/Glu) and side‑chain amine (Lys) to form an amide (lactam)

Q8. Stapled peptides often show enhanced cell permeability. Which mechanism most plausibly explains this?

• Helical stabilization reduces polar surface area and presents a favorable amphipathic face for membrane transit
• Stapling increases total charge to promote endocytosis
• Crosslinking directly forms a pore through membranes
• Stapling causes irreversible aggregation that is taken up by phagocytosis

Correct Answer: Helical stabilization reduces polar surface area and presents a favorable amphipathic face for membrane transit

Q9. Which synthetic approach is commonly used on solid phase to install hydrocarbon staples?

• Incorporation of olefinic non‑natural amino acids followed by ring‑closing metathesis (RCM)
• Native chemical ligation at the N‑terminus only
• Direct enzymatic cyclization on resin using proteases
• Metal‑catalyzed azide‑alkyne cycloaddition in solution only

Correct Answer: Incorporation of olefinic non‑natural amino acids followed by ring‑closing metathesis (RCM)

Q10. Introducing disulfide bridges for constraint is limited by which factor in therapeutic peptides?

• Reductive environments in vivo can reduce disulfides and destabilize the constraint
• Disulfides universally increase membrane permeability, which is undesirable
• Disulfide formation always eliminates target binding
• Disulfides are not compatible with solid‑phase peptide synthesis

Correct Answer: Reductive environments in vivo can reduce disulfides and destabilize the constraint

Q11. Which effect on binding thermodynamics is often observed when a peptide is conformationally preorganized into the bioactive conformation?

• Reduced entropic penalty on binding leading to improved apparent affinity
• Increased entropic cost and therefore lower affinity
• Lack of enthalpic contribution from hydrogen bonds
• Complete abolishment of van der Waals interactions

Correct Answer: Reduced entropic penalty on binding leading to improved apparent affinity

Q12. For formulation scientists, a potential risk of strong conformational restriction is:

• Increased propensity to aggregate due to exposed hydrophobic patches
• Guaranteed improvement in aqueous solubility
• Complete elimination of immunogenic potential
• Universal compatibility with all excipients

Correct Answer: Increased propensity to aggregate due to exposed hydrophobic patches

Q13. Which modification would you choose to increase proteolytic stability without significantly altering backbone geometry?

• N‑methylation of specific backbone amides
• Adding extra glycine residues at termini
• Replacing all residues with charged amino acids
• Cleaving the peptide into two fragments

Correct Answer: N‑methylation of specific backbone amides

Q14. Which biophysical method provides residue‑level conformational information useful for validating a constrained peptide structure?

• Nuclear magnetic resonance (NMR) spectroscopy
• Ultraviolet visible (UV‑Vis) absorption without labeling
• Dynamic light scattering (DLS) for single‑residue detail
• Paper chromatography for secondary structure mapping

Correct Answer: Nuclear magnetic resonance (NMR) spectroscopy

Q15. In peptidomimetic design, replacing a peptide bond with a reduced amide or thioether linkage primarily aims to:

• Enhance metabolic stability by removing scissile amide bonds
• Increase backbone hydrogen bonding for α‑helix formation
• Promote enzymatic cleavage to decrease half‑life
• Make the peptide more susceptible to oxidation

Correct Answer: Enhance metabolic stability by removing scissile amide bonds

Q16. Which parameter is most critical to monitor during stability studies of a constrained peptide formulation?

• Retention of the constrained conformation and aggregation state over time
• Color change only, irrespective of chromatographic profile
• Total organic solvent content exclusively
• Melting point of the lyophilized cake without other assays

Correct Answer: Retention of the constrained conformation and aggregation state over time

Q17. Which excipient strategy can help mitigate aggregation of hydrophobic constrained peptides in solution?

• Addition of surfactants (e.g., polysorbate) and/or solubilizing co‑solvents at controlled levels
• Exclusion of all buffers to maintain neutral pH only
• Inclusion of proteases to cleave aggregated species
• Heating the solution to extreme temperatures during storage

Correct Answer: Addition of surfactants (e.g., polysorbate) and/or solubilizing co‑solvents at controlled levels

Q18. Which in vitro assay is most appropriate to evaluate increased target engagement due to conformational constraint?

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for binding kinetics and thermodynamics
• Gram staining to assess binding to bacteria
• Standard urine dipstick test for peptide concentration
• pH titration only without binding readout

Correct Answer: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) for binding kinetics and thermodynamics

Q19. A potential regulatory concern specific to non‑natural amino acids used to enforce constraints is:

• Unknown immunogenicity and metabolite profiles requiring additional safety data
• Automatic approval due to novelty
• Elimination of the need for toxicology studies
• Guaranteed oral bioavailability without testing

Correct Answer: Unknown immunogenicity and metabolite profiles requiring additional safety data

Q20. Which design trade‑off is often encountered when maximizing constraint to increase affinity?

• Higher affinity may come at the cost of reduced solubility or increased aggregation tendency
• Maximum constraint always yields optimal PK without downsides
• Constraint reduces manufacturing cost proportionally
• Increased constraint eliminates the need for formulation development

Correct Answer: Higher affinity may come at the cost of reduced solubility or increased aggregation tendency

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