PHAR232 - Measurement in pharmacology -wk 5 wk 5 Flashcards
What are the 3 core approaches of a BINDING ESSAY?
- What is the target?
- target characteristics
- drug-target interaction
What is a BIOASSAY?
The physiological outcome of drug-target interactions
- Fixed number of receptors on a cell
- DIFFERENT CELLS WILL EXPRESS VARIOUS RECEPTORS AT DIFFERENT LEVELS (pending on what to achieve)
- ## ‘on’ and ‘off’ rates = affinity
Dissociation constant for equilibrium (as a whole is)
Is ‘association’ meaning ON OR OFF?= at receptor complexes?
ON
Does disassociation of receptor complexes mean ON or OFF?
OFF
At equilibrium rates of association and dissociation are equal? Y/N
yes
K+1 [A][R] = K-1 [AR]
What would Kd be in reference to?
Dissociation constant
What does Ntotal refer to?
The fixed number of receptors a cell has
What is (A) in reference to when reviewing drug-receptor complexes?
Exposure to the drug = (A)
What is the suffix for a set proportion of receptors occupied by an agonist?
(NA)
What would be the equation for deciphering the number of vacant receptors?
Ntotal - NA
What are the most IMPORTANT 2 factors that the PROPORTION OF receptors OCCUPIED depend on?
- AFFINITY of the drug for receptors (KD)
- CONCENTRATION (or dose) of he drug added [A]
What is KD in relation to the property of the drug and receptor?
AFFINITY CONSTANT
aka the DISSOCIATION constant
What is the definition of KD?
The concentration of drug REQUIRED TO OCCUPY 50% of the receptors at equilibrium
Why doesn’t AFFINITY reflect receptor EFFICACY?
Affinity is associated to BINDING not to the exerted EFFECTS of the ligand
Affinity is NOT associated with EFFICACY
This is how to assess 50% affinity
- Look at the graph of when it hits 0.5 = 50%
(MUST KNOW) what does a SMALL KD value (dissociation constant) affinity index of a drug mean?
that is has HIGH AFFINITY
(means that if only a small amount of drug is needed to occupy 50% of the cells receptors… the fit of binding must be very good!)
(MUST KNOW) what does a HIGH KD value (dissociation constant) affinity index of a drug mean?
POOR AFFINITY
Takes a lot of the drug dose to occupy 50% of the target receptors (done by force of high drug dose)
What does the suffix Bmax represent?
Maximum Number of receptors
B= binding
Used for tracking - (aging, drug tolerance, etc etc)
Tracking change over time
KD = the ease at which the drug binds to the receptor
That is all.
Choose the right tissue
- Are co-factors required?
- Usually for agonist binding
How would you help to discriminate signal from noise in an assay to separate specific interactions (ligand-receptor) from non-specific interactions (ligand-non-receptor)?
- Increased radioligand = ability to interact non-specificially with other proteins or lipids become easier
- Accomplished using specific non-radioactive (cold) ligand to define NON-SPECIFIC BINDING
> Cold ligand should occupy all receptors
» THEREFORE, ANY BOUND RADIOLIGAND IS ASSOCIATED WITH SITES OTHER THAN RECEPTOR OF INTEREST
What is a cold ligand’s major positive impact in an assay?
- Cold ligands associate with all receptors
- Allowing bound radioligans to bind with OTHER receptors of interest
Is a cold ligand radioactive?
no
Radioligands that use 3H require what for measurement?
scintillant
- amplifier of low level beta radiation 3H-ligands emit
What does a scintillant
- Low level radiation excites molecules of scintillant
- Scintillant generates photons of light
- Scintillation COUNTER MEASURES as INDEX amount of radioligand present
UNDERSTAND THIS MECHANISM AND CHARTING
What are 3 things that complicate binding assays analysis data?
- Evidence of cooperativitiy: binding of one molecule of ligand makes second binding event easier
- Receptor complexes: hetero& homodimers of GPCRs
- Multiple affinity states of receptors
What is the definition of bioassays?
Estimation of concentration of potency of substance by meansurement of BIOLOGICAL RESPONSE THAT IT PRODUCES
Roles of a bioassay
What can you measure in bioassays?
Can experiments require both negative and positive controls?
yes
What is a negative control?
A compound you know WON’T work in your system
What is a positive control?
Anyy compound that WILL WORK in your system
- Often = current standard of treatment
What are common bioassays for analgesic potency?
- radiant heat tail-flick
- Immersion tail-flick
- Hot plate test
What are another 3 common bioassays for analgesic potency?
- Tail pinch test
- Writhing test
- Thermal paw withdrawal reflex
What are another 2 common bioassays for analgesic potency?
- Formalin test
- Cold pressor test
Therapeutic index =
max non-toxic dose/min effective dose
