Receptors / Dose Response Flashcards
Ligands
Molecules bound by receptors
Agonist
Molecule that binds receptor —> produces biological response
Antagonist
Molecule that binds a receptor but does not induce a biological response (endogenous mediators blocked)
Characteristics of a receptor
Saturability
Specificity
Reversibility
Bifunctional role
Classes of receptors
Ion channel receptors
G-protein coupled receptors
Tyrosine kinase receptors
Transcription factor receptors
Examples of ion channel receptors
Neurotransmitter receptors (e.g., acetylcholine, GABA, Aspartate, glycine)
Characteristic of ion channel receptors
Very fast (millisecond)
Examples of G-protein coupled receptors
Neurotransmitters, peptide hormones, biogenic amines
40-50% of pharmaceutical targets are GPCRs
Characteristics of GPCRs
G-protein - transducing agent that couples active receptor to cellular response; binds GTP then uncouples from activated receptor
Fast 100s of ms to second
Receptor pathways
Receptor to activate and/or inhibit pathway
Receptor can act on / activate multiple pathways
Pathways of GPCRs
GPCRs can link to more than one G-protein
GPCRs may initiate cell signaling not traditionally associated with G-proteins
Examples of tyrosine kinase receptors
Receptor for insulin, growth factors
Characteristics of tyrosine kinase receptor
Slower action - order of minutes
Transcription factor receptors
Receptors for steroids, thyroid hormone, vit D, retinoids
Intracellular receptor —> agonists must be hydrophobic to pass through the membrane and bind
Very slow - hours to days
Rate of receptor classes
Ion channel > GPCR > tyrosine kinase > transcription factor
Receptor subtypes
May have differential distribution —> can exploit for tissue specificity
Receptor affinity
ratio of off rate of a ligand binding a receptor to on rate (dissociation constant, Kd)
High affinity
Very slow off rate
Lower affinity
Faster off rate
No affinity
No on rate
Saturation isotherm
Directly measures binding of a radio labeled ligand to receptor
Competition binding assay
Compares ability of nonradioactive ligands to compete with radioligand to bind to a receptor
Measuring total binding
- combine tissue + radiolabeled drug
- Incubate
- Wash off unbound drug
- Measure radioactivity
Total binding curve
Binding experiment done with the same amount of tissue in each tube but with INCREASING conc of radioligand
Aka saturation isotherm
Measuring non-specific binding
- Combine tissue + radiolabeled drug + excess of non-radioactive competing ligand
- Incubate
- Wash off unbound drug
- Measure radioactivity
Non-specific binding curve
Binding experiment the same as for the total binding curve, but in the presence of high does of competing non-radioactivity ligand
Used to correct the total curve to get the specific binding curve
Plateau of the binding curve represents
Saturability
A lower Kd corresponds to a _____ receptor affinity
Higher
Kd
Concentration of ligand where half maximal binding is obtained
Competition binding analysis
Measure of % specific radioligand binding relative to increasing concentration of competitor
IC50 in competition binding analysis
Concentration of competitor which inhibits binding by 50%
Lower IC50 in competition binding analysis means …
The competitor has a higher receptor affinity
Dose response curves
Linear plot - hemiparabolic with plateau at saturation; proportional to receptors occupied
Semi log plot - used to be able to display a wider range of doses; sigmoid shape
Dose-response curve of antagonist / non-binder
Flat line on semi log curve
Dose response curves of agonists
Sigmoid shape
EC50 - concentration with half-maximal response
Lower EC50 —> more potent agonist
Dose-response curve for partial agonist
Fails to reach full response (i.e. doesn’t reach saturation point of full agonist)
Intrinsic efficacy
Ability of ligands to activate receptors
Full agonists - max response; intrinsic efficacy = 1
Partial agonist - less than maximal; 0 < intrinsic efficacy < 1
Antagonist - no response, intrinsic efficacy = 0
Molecular basis of intrinsic efficacy
Full agonist: full activation of receptor (i.e. binding + full interaction with activation domain)
Partial agonist: partial activation of receptor (i.e. binding + partial interaction with activation domain)
Antagonist: no response (i.e. binds receptor but not activation domain)
Potency of a ligand is determined by …
Fractional occupancy (binding)
+
Intrinsic efficacy (ability to activate)
Effect of competitive antagonist on dose response curve
Pretreatment with competitive antagonist directly blocks binding, but with high enough agonist concentration will be displaced
Curve shifts to the right (need higher conc to reach saturation)
Effect of non competitive antagonist on dose-response curve
Non-competitive antagonist doesn’t block binding of agonist, but prevents subsequent activity upon binding
With antagonist pre-treatment, will need a higher conc to illicit activity, but also will not reach maximum
Dose response curve shifts right and downward
Inverse agonist
Binds and induces a biological response in the opposite direction of an agonist
Other factors impacting the biological response
Spare receptors - shift does response curve left
(Greater response despite equivalent concentration of agonist - % receptors bound is the same, but the overall # bound is higher)
Large threshold for response - shift curve right
Receptor desensitization
Receptor can still bind agonist, but cell signaling is disrupted
Receptor downregulation
Receptor pulled off cell surface
Drug tolerance develops in response to
Receptor desensitization
Receptor downregulation
Elongates/flattens out the slope of the dose response curve (down/right shift)
Drug vacation
When receptor desensitization / downregulation occur; switch to a medication that acts by a DIFFERENT pathway to allow original pathway to resensitize
Receptor supersensitivity
Response following denervation —> nerve dies —> upregulation of receptors that make tissue supersensitive to any neurotransmitter
