Lecture 4.1 Medchem

• Kd and Bmax values are essential for quantifying the degree of drug binding based on its receptor affinity in response to changing drug concentrations.

Binding Curves

• Binding curves help determine if a drug delivers its benefit through direct interaction with its target.
• Binding curves are generated by plotting the binding response against the drug concentration to understand how a drug interacts with its target receptor.
• The shape and characteristics of the curve provide information about the affinity and specificity of the drug-receptor interaction.

Types of Binding

• Total Binding: Overall binding of the drug to the receptor, including both specific and non-specific binding.
• Non-Specific Binding: Drug binds to other proteins or components in the sample, not the intended receptor.
• Specific Binding: Drug binds to the target receptor only, obtained by subtracting non-specific binding from the total binding; it reflects the direct interaction of the drug with the intended target.

Generating a Binding Curve

• A sample containing the receptor is incubated with a radiolabeled drug.
• Receptor sample types include a purified protein, a tissue sample, or a cell sample that expresses the receptor target.
• The radiolabeled drug binds to the receptor, and unbound drug is washed off.
• The remaining target and bound drug, which has a readable label, are measured.
• Radioactivity amount is plotted against the drug concentration to generate the binding curve.
• Specific binding is measured using a parallel experiment where the target is saturated with an unlabeled version of the drug and then incubated the labelled drug, which binds non-specifically. Specific binding is then found by subtracting non-specific binding from total

Quantitative Measures from Binding Curves

• Bmax (Maximum Binding): The concentration at which maximal binding occurs, indicating that all available receptors are occupied.
  • It is an indirect measure of the number of receptor molecules in a tissue and can be affected by genetics, disease, age, sex, and long-term drug use.
• Kd (Dissociation Constant): Reflects the drug's affinity for the target; a lower value indicates a stronger binding affinity.

Specific Binding Characteristics

• The binding curve should have a normal slope, following the law of mass action.
• The drug concentration should achieve an 80% response within a 100-fold window.

Methods for Studying Drug-Target Interactions

• Radiolabeled Drug-Binding: Uses a drug labeled with a radioactive isotope measured by a scintillation counter, providing direct measurement of drug binding with the limitation to being resource-intensive and safety issues.
• Fluorescence-Based Methods: Employs fluorescence tags to label the drug or receptor, is more benign but susceptible to signal loss and interference.
• Biotin-Avidin Tagging: Labels the drug with biotin and uses avidin labeled with a fluorophore to form a stable complex; it has a strong interaction between biotin and avidin, but fluorescein signal is not very long-lasting.
• Homogeneous Time-Resolved: Uses fluorescence resonance energy transfer (FRET) to minimize noise and enhance signal, done with less material in a more native state, but requires specific labeling of both target and drug.
• Label-Free Methods: Do not require labeling, eliminating the need to label molecules but potentially requiring specialized equipment and expertise.
  • Surface Plasmon Resonance (SPR): Immobilizes the target on a sensor chip and measures changes in current as the drug binds. It allows the study of both association and dissociation and reach saturation, but requires target immobilization.
  • Isothermal Titration Calorimetry: Measures the emitted/absorbed heat during binding, providing thermodynamic parameters but can be challenging to optimize.
  • Nuclear Magnetic Resonance (NMR): Uses magnetic fields to study molecular structure and dynamics but requires high sample concentrations.
  • Mass Spectrometry: Measures the mass-to-charge ratio of ions to identify and quantify binding, but analysis can be complex.

Role of Bmax and Slope

• Bmax indicates the maximum number of binding sites, serving as an indirect measure of receptor molecules in a tissue.
• Slope of the binding curve reflects the specificity of drug-receptor interaction.

Hill Coefficient

• The Hill coefficient, derived from the Hill plot, provides insights into the cooperativity of ligand binding.
  • A coefficient of 1 indicates that one drug molecule binds to one receptor molecule, with no cooperativity.
  • A coefficient greater than 1 suggests positive cooperativity, with an increased receptor affinity for subsequent molecules, resulting in a steeper slope.
  • A coefficient less than 1 indicates negative cooperativity, where binding decreases the receptor affinity for subsequent molecules.

Allosteric Modulators and Hill Coefficient

• Positive Allosteric Modulators (PAMs): Increase receptor affinity for its ligand, resulting in a Hill coefficient greater than 1, and can also increase the Bmax value.
• Negative Allosteric Modulators (NAMs): Decrease receptor affinity for its ligand, potentially resulting in a Hill coefficient less than 1.

Competition-Binding Curves

• Used when labeled molecules are unavailable to determine if different molecules have the same mechanism of action.
• Involves allowing a labeled ligand (known drug) to bind to the receptor and then treating the complex with an unlabeled drug; of the unlabeled drug has a higher affinity, it displaces the labeled ligand.
• IC50 (Inhibitory Concentration 50%): Concentration of unlabeled drug required to inhibit 50% of the labeled ligand's binding; a lower IC50 signifies that a lower drug concentration is needed to inhibit 50% of the labeled ligand's binding.
• Ki (Inhibition Constant): The affinity of the competing drug for the receptor, calculated from the IC50 value using the Cheng-Prusoff equation.

Cheng-Prusoff Equation

• Formula: IC50 = Ki (1 + [Ligand]/Kd)
  • IC50: Inhibitory concentration.
  • Ki: Inhibition constant.
  • [Ligand]: Concentration of the labeled ligand.
  • Kd: Dissociation constant of the labeled ligand.

Structural Changes and Affinity

• Stereochemical Changes: Alterations in the drug molecule’s three-dimensional shape can change Kd and Ki values.
• Receptor Mutations: Modifications in the shape of the receptor or its binding pocket can also affect Kd and Ki values.
• Structure-Affinity Relationship: Building such a relationship informs decisions on the best analog of a given drug for achieving maximum benefit.

Applications of Competition Curves

• Identifying Novel Compounds: Confirms if a compound from a drug screen works at the intended receptor.
• Assessing Selectivity: Helps determine if a compound selectively binds to a specific receptor isoform over others.
• Comparing Compounds: Enables comparison of a compound's affinity and selectivity with competitor compounds.
• Guiding Medicinal Chemistry: Can guide medicinal chemistry efforts to improve therapeutic benefit and reduce harm.