Drug Binding

• HILL-LANGMUIR equation = the central equation governing the interaction of a drug with its target
• KD = the equilibrium dissociation constant for a drug. It is the measure of affinity and has units of concentration. KD can also be defined as the concentration of drug that produces 50% of maximum binding
• BMAX = the maximum binding of a drug in a sample of tissue. It is a measure of the number of binding sites and is usually expressed as an amount
• AFFINITY = the tightness of binding of a drug to its target. It is normally measured using KD (As KD goes down, affinity goes up)
• SELECTIVITY = the relative affinity of a drug for one receptor type compared to another (usually expressed as the ratio of the two KD values)
• MICHAELIS-MENTON equation = a version of the hill-langmuir equation that describes how the rate of an enzyme-catalysed reaction varies with substrate concentration
• KM = the michaelis constant. It's one of the parameters in the michaelis-menton equation and is a measure of an enzyme's affinity for its substrate
• VMAX = the maximal velocity of an enzyme-catalysed reaction. It is the product of the enzyme concentration and the catalytic constant for the enzyme
• THE LAW OF MASS ACTION

  • formulated by Cato Guldberg and Peter Waage in 1864
  • states that the rate of reaction is directly proportional to the concentrations of the reactants
• THE HILL-LANGMUIR EQUATION

  • saturable binding-finite number of copies of a protein present. Once sites are occupied, there can be no more binding to target molecules
  • KD is irreversibly related to affinity (KD = 1/ Keq)

  $B =$$Bmax*$$[D]/[D] +$$KD$

  • BMAX = maximum binding capacity
  • B = amount of receptor with drug bound to it (same unit as Bmax)
  • KD = equilibrium dissociation constant
• PLOT OF B VS [D] YIELDS A RECTANGULAR HYPERBOLA

  
• RADIOLIGAND BINDING ASSAYS

  • Radiolabelled drugs are termed radio ligands (3H- tritium, 14C or 125I)
  • Used to detect binding to quantify how much of a drug has bound in a tissue sample

  1. Incubate the radioligand and tissue until equilibrium is reached
  2. seperate bound from free ligand: filtration and centrifugation
  3. measure the amount of radioactivity in the sample

  *[3H]: liquid scintillation

  *[125I]: gamma counter
• NON-SPECIFIC + SPECIFIC RADIOLIGAND BINDING ASSAYS

  • some radioligand will bind to non-receptor sites in the assay to give non-specific binding (NSB)

  1. NSB represents a very large, unsaturable pool of LOW AFFINITY binding sites

  • there's radio ligand in solution around the membranes to represent the control/ total binding reaction
  • receptor is mixed with radio ligand and allowed to come in equilibrium
  • the binding to receptor sites is specific binding and binding to membrane sites is non-specific binding

  $specific binding =$$total binding -NSB$
• SATURATION ASSAYS

  • Used to establish the affinity (KD) and receptor density (Bmax) for a radioligand in a tissue
  • Limitations include:

  1. requires a radioactive version of every drug being investigated
  2. time-consuming and costly to produce a radioligand
  3. produces lots of radioactive waste- expensive to dispose of

  • As affinity increases; KD decreases
• LINKS WITH ENZYME KINETICS