AL - Ligand Binding II Flashcards
What are the key properties of an ideal ligand for imaging and quantification? (5)
- High Affinity – Strong binding to receptor (K_D < 1 nM).
- Slow Dissociation Kinetics – Ensures stable binding and prolonged signal.
- Antagonists > Agonists – Antagonists provide more stable measurements.
- High Selectivity – Minimises non-specific binding for accuracy.
- Labeling & Stability – Must be compatible with labelling and remain stable under experimental conditions.
What are the differences between radioligands (6) and fluorescent ligands (6) ?
Radioligands –
- Very high sensitivity
- easy incorporation
- minimal biological interference
- simple detection
- low cost
- but require radioactive safety precautions.
Fluorescent ligands –
- High sensitivity
- harder to incorporate
- more biological interference
- complex detection
- high cost
- but allow real-time imaging and multiplexing.
Q: What biologically relevant isotopes are used in imaging and quantification? (5)
- Tritium (³H), Carbon-14 (¹⁴C), Phosphorous-32 (³²P), Sulfur-35 (³⁵S), Iodine-131 (¹³¹I)
- Used in labelling for imaging and quantification.
Q: What are key measures of radioactivity? (3)
- Becquerel (Bq) – Disintegrations per second.
- Curie (Ci) – 2.22 × 10¹² disintegrations per minute (d.p.m).
- Specific Activity (SA) – Radioactivity per unit amount (Ci/mmol).
What are the properties of commonly used isotopes? (3)
³H (Tritium) – β emitter, ~30 Ci/mmol, ~12-year half-life.
³²P (Phosphorus-32) – β emitter, ~9000 Ci/mmol, ~14-day half-life.
¹³¹I (Iodine-131) – γ emitter, ~2000 Ci/mmol, ~60-day half-life.
Q: How do we image (2D) radioactivity? (5)
- Cryosectioning – Tissue samples are sectioned and mounted on slides.
- Radioligand Binding – Tissue incubated with radiolabeled ligand.
- Washing & Fixation – Excess ligand washed off, tissue fixed.
- Phosphor Imaging Scan – Imaging plate captures radioactive emissions.
- Quantification – Regions of interest are analyzed for radioactivity levels.
Key Takeaway: Autoradiography maps receptor distributions and drug-binding sites.
Q: What are the key steps in PET imaging? (3)
- Radiotracer Production – Cyclotron generates positron-emitting radionuclides (e.g., ¹⁸F, ¹¹C).
- Administration & Detection – Radiotracer injected, positron emission leads to gamma photon detection.
- Image Generation – Signals reconstructed into a PET image.
Key Takeaway: PET enables non-invasive imaging of biological processes.
Q: What are the key features of immunohistochemistry (IHC) and cytochemistry? (3)
Antibody-Based Detection – Uses labeled antibodies targeting extracellular epitopes.
Labeling Strategies:
- Radiolabeling – Uses ¹³¹I via tyrosine oxidation.
- Fluorescent labeling – Antibodies conjugated with fluorophores.
- Enzyme labeling – HRP (Horseradish Peroxidase) with DAB reaction for colorimetric detection.
Methods:
- Indirect Immunohistochemistry – HRP-labeled secondary antibody, DAB reaction for visualization.
- Immunofluorescence – Fluorescent-tagged antibodies detected via light emission.
Key Takeaway: IHC & immunofluorescence localize proteins with high specificity.
Q: What is In Situ Hybridisation (ISH), and how is it used? (3)
Uses radio-labelled antisense oligonucleotides to target specific mRNA.
* Example: ³²P-labeled probes via polynucleotide kinase.
Advantages:
- Useful when no ligand is available for a receptor.
- Faster & simpler than fluorescence in situ hybridisation (FISH).
Application: Maps H₃ receptor isoform expression in rat brain.
Key Takeaway: ISH is crucial for mapping gene expression when ligands are unavailable.
Q: What are genetic approaches for receptor expression? (3)
Using Reporter Genes – Tags like fluorescent proteins track receptor expression in cells.
- Example: GAL4-UAS system enables controlled gene expression.
Advantages –
- No need for selective ligands or antibodies.
- Allows precise spatial and temporal control of gene expression.
Application –
- Ionotropic glutamate receptor expression studied in different brain regions and larval muscles.
- Specific subtypes (e.g., GluR, Nmdar1, KaiR1D) visualized in Drosophila.
Key Takeaway: Genetic tools offer an alternative for receptor expression studies when ligands or antibodies are unavailable.
Q: What are quantitative (radio)ligand binding assays used for? (3)
Purpose – In vitro characterization of receptor function (wild-type vs. mutant).
Screening Receptor Profile –
- Ligand affinity (K_D) – Measures binding strength.
- Number of binding sites (B_max) – Expressed per tissue/protein amount.
- Binding heterogeneity – Identifies multiple sites or cooperativity.
Competition Binding Assays –
- Unlabelled molecules compete with radioligand to infer K_D.
Key Takeaway: Binding assays quantify ligand-receptor interactions, crucial for drug discovery.
Q: What are the key principles of receptor-ligand binding assays?
1) Ligand Affinity (K_D) – Measures binding strength.
2) Number of Binding Sites (B_max) – Expressed per tissue/protein amount.
3) Binding Heterogeneity – Identifies multiple sites or cooperativity.
Competition Binding Assay:
- Unlabeled molecules compete with radioligand.
- Measures IC₅₀ (concentration needed to inhibit 50% of binding).
Q: What does the dissociation constant (K_D) represent? (3)
- K_D = ligand concentration at which half of receptors are occupied.
- Lower K_D → Higher affinity (stronger binding).
- Graph Interpretation – Saturation curve shows binding reaching B_max.
Q: How is measuring tissue responses different from ligand binding assays? (3)
Measuring Tissue Responses –
- Drug is added to tissue (e.g., organ bath).
- Physiological response (e.g., contraction) is measured.
- % response is plotted against drug concentration.
Occupancy Theory & EC₅₀ –
- If response is proportional to receptor occupancy, then EC₅₀ ≈ K_D.
- However, spare receptors or drug access issues can shift EC₅₀ from K_D.
Key Difference –
- K_D measures ligand binding affinity.
- EC₅₀ measures functional tissue response to a drug.
- EC₅₀ is NOT always equal to K_D but is useful for comparing agonist potency.
Key Takeaway: Occupancy theory states that response (E) is proportional to receptor occupancy but can be modulated by spare receptors.
Q: What are the limitations of dose-response measurements? (2)
Indirect measurement – Response does not directly reflect receptor binding (applies poorly to antagonists).
Not valid in many systems –
- Partial agonists – Activate receptors but do not produce a full response.
- Spare receptors – Full response can occur with only a fraction of receptors occupied.
- Ligand diffusion & degradation – Can alter apparent potency.
Key Takeaway: Dose-response curves do not always reflect true receptor occupancy, making K_D a more reliable measure of binding affinity.
Q: What are the key steps in designing ligand binding assays? (5)
1) Choose an Appropriate Ligand –
- Can be radioactive or fluorescent, similar to autoradiography considerations.
2) Select the Receptor Source –
- Options include membrane preparations, purified proteins, or whole cells.
3) Separate Bound vs. Unbound Ligand –
- Achieved using filtration, centrifugation, or precipitation methods.
4) Differentiate Specific vs. Non-Specific Binding –
- Use excess unlabeled ligand to determine non-specific binding.
5) Estimate Ligand Binding –
- Measure bound ligand at different concentrations to calculate K_D (binding affinity) and B_max (maximum binding capacity).
Q: What are the possible sources of receptors for ligand binding assays? (6)
- Solubilised receptor – Extracted and purified receptor proteins.
- Homogenised membranes – e.g., synaptosomal preparations.
- Freshly isolated or cultured cells – Naturally expressing the receptor.
- Cells transfected with cloned receptor – Used for studying specific receptor subtypes.
- Tissue homogenates or fresh slices – Typically 5-30 µm thick, used for ex vivo studies.
- Animal models (sometimes human) – For in vivo receptor studies.
Q: How is radioactivity measured in solution?
- Scintillation Counting – A technique used to measure radioactive decay by detecting emitted photons from a radiolabeled sample.
Q: What are techniques for separating bound and free ligand? (5)
- Filtration – Membrane filtration traps bound ligand-receptor complexes, while free ligand passes through.
- Centrifugation – Pellets bound receptor-ligand complexes, leaving free ligand in the supernatant.
- Sedimentation (Plated Cells) – Ligand binds to cells on a surface, unbound ligand is removed, and bound ligand is solubilized for measurement.
- Equilibrium Dialysis – Ligand diffuses across a membrane; bound ligand remains trapped, ensuring accurate K_D determination.
- Size Exclusion Chromatography (SEC) – Separates molecules by size; bound ligand elutes first, but may shift equilibrium if too slow.
Q: What are the types of ligand binding, and how is non-specific binding measured? (3)
Specific Binding (Saturable) –
- Binds to receptor sites.
- Follows a saturation curve, reaching B_max.
- Measured as Total Binding - Non-Specific Binding.
Non-Specific Binding (Not Saturable) –
- Occurs on non-receptor sites (e.g., membranes, proteins).
- Increases linearly with ligand concentration.
Measuring Non-Specific Binding –
- Use excess “cold” ligand (unlabeled competitor) to block specific sites.
- Measure remaining radioligand binding to determine non-specific binding.
Key Takeaway: Subtracting non-specific binding ensures accurate K_D and B_max determination.
Q: What are competition binding assays, and how do they work? (4)
1) Principle –
- An unlabeled competitor ligand competes with a radiolabeled ligand of known affinity for receptor binding.
- Measures binding affinity (K_D) and IC₅₀ (half-maximal inhibitory concentration).
2) Types of Competition –
- Homologous competition – Cold (unlabeled) version of the same ligand competes for binding.
- Heterologous competition – A different ligand or analogue competes for binding.
3) IC₅₀ & K_D Determination –
- IC₅₀ = Concentration of cold ligand that displaces 50% of specific binding.
- Cheng-Prusoff equation converts IC₅₀ to K_D of the competitor ligand.
4) Limitations –
- May not detect allosteric modulators (ligands binding at a different receptor site).
Q: What are techniques for measuring receptor-ligand binding? (6)
- NMR – Analyzes structure and dynamics of ligand binding.
- Fluorescence-based assays – Includes quenching, anisotropy, FRET, and BRET for interaction studies.
- Microscale Thermophoresis (MST) – Measures binding affinities in solution.
- Isothermal Titration Calorimetry (ITC) – Determines thermodynamic parameters of binding.
- Surface Plasmon Resonance (SPR) – Provides real-time, label-free kinetic analysis.
- Differential Calorimetry – Assesses thermal stability changes upon ligand binding.
Q: What is Bioluminescence Resonance Energy Transfer (BRET) and how does it work? (3)
Fluorescence-based technique for studying protein-protein interactions.
Mechanism:
- Luciferase catalyzes a reaction that emits light.
- If an acceptor fluorophore (EYFP) is within <10 nm of the donor (RLuc), energy transfer occurs, leading to fluorescence emission.
- No electromagnetic energy input is required.
Key Features:
- Donor: Renilla luciferase (RLuc) emits at 480 nm.
- Acceptor: Enhanced Yellow Fluorescent Protein (EYFP) emits at 530 nm if energy transfer occurs.
- Application: Used for detecting protein interactions in live cells.
Q: How is BRET used for screening selective A₃R agonists? (3)
Adenosine A₃ receptor (A₃R) ligand binding is studied using Bioluminescence Resonance Energy Transfer (BRET).
Experimental Setup:
- A₃R fused to NanoLuc (nLuc) and mixed with a fluorescent ligand (AVO39).
- BRET signal increases when AVO39 binds to A₃R (Panel B).
- Competitive Binding Assay (Panel C): Candidate ligands displace AVO39, reducing the BRET ratio.
Interpretation:
- Higher BRET ratio → Stronger ligand-receptor interaction.
- Greater displacement of AVO39 → Stronger binding affinity of the competing ligand.
Q: What are the advantages (4) and disadvantages (3) of receptor binding assays vs. dose-response assays?
Advantages:
- Direct measurement of ligand-receptor interactions.
- Quantitative assessment (e.g., Kₐ, Kᵢ, IC₅₀ values).
- Independent of pharmacokinetics, allowing focus on binding properties.
- High-throughput compatible, enabling rapid screening of multiple ligands.
Disadvantages:
- Cannot differentiate between agonists, partial agonists, and antagonists.
- Binding may be affected by non-physiological conditions (in situ effects).
- Long incubation times may cause receptor desensitization.
