Drug Discovery 10 Flashcards
what does high throughput sequencing require?
process requires sophisticated robotics and liquid handling devices capable to dispensing nanolitre volumes
multi-well plate formates are used - 384, 1536 and now 3456 plates are used
it is cheaper to do assays on smaller plates
what is high throughput sequencing ?
it is an automated process which allows large numbers of compounds to be screened to assess potential efficacy
how many compounds can be assayed per day ?
chemical libraries of 100000 compounds can be assayed per day
if the high throughput screening doesnt attempt to fully characterise each compound, what does it do ?
strategy is usually to test three different concentrations in order to ascertain whether the compound is a hit or a miss - a quick 3 point dose-response curve is carried out
any hits determined need to be re-tested more thoroughly to produce a full dose response curve
what are GPCRs?
they are 7 transmembrane receptors with an extracellular N-terminus and an intracellular C-terminus
how many different GPCRs are there in our genome?
about 800 - many are potential drug targets
what is the main difference in structure of the active and inactive state beta 2 adrenoreceptor ?
main difference is the movement of the TM6 and TM5 at the cytoplasmic side
what happens when a GPCR is activated ?
the intracellular G protein undergoes conformational changes causing the release of the GDP from the alpha subunit and this causes GTP to bind to the alpha subunit enabling it to be released from the receptor and beta-gamma dimer
the alpha subunit and beta-gamma subunit can now initiate down stream signalling pathways
the hydrolysis of the alpha subunit causes the reformation of the G protein heterodimer and stops signalling
what happen in the signalling pathways associated with Gi and Gs ?
Gs- activates adenylate cyclase which increases cAMP
Gi- inhibits adenylate cyclase which decreases cAMP
why is important to understand what happens when a specific G protein is activated ?
because then we can judge if the compound has activated or blocked the receptor by measuring the levels/activities of some molecules down stream
what signalling pathway is associated with Gq?
inositol pathway is activated
it increases phospholipase c which increases the levels of IP3 and DAG and this leads to the release of intracellular calcium
what factors are a measure of affinity ?
Kd, IC50/Ki, Kb/pA2
what are measures of efficacy ?
efficacy is a combination of both potency and intrinsic activity
what are measures of potency ?
Ec50
what are measures of intrinsic activity ?
Emax
what is affinity ?
it is the strength of binding
- it is the drug concentration which determines the percentage of sites occupied
- a drug with a greater affinity compared to another will occupy a greater number of receptors if both drugs are added at the same concentration
what is Kd?
it is the concentration of the drug that is required to occupy 50% of the receptors
what does a lower Kd mean ?
the lower the Kd is the higher the affinity of the drug
describe affinity :
it is a measure of how tightly a ligand binds to a receptor
it is independent of efficacy - both agonists and antagonists can have a high affinity
it is described by the equilibrium dissociation constant Kd
concentration is the unit measurement of Kd
what method is used to measure affinity ?
radioligand binding assays
what do radiolabelled ligands permit ?
it enables the determination of the concentration of drug required to occupy 50% of receptors
it is incubated with the target and then the free radioligand is washed away
this leaves the total binding = specific binding of radiolabelled ligand to receptor as well as non-specfic binding of the radiolabelled ligand to random sites
- the number of non-specific sites is infinite and not saturable therefore it cannot be competed off
then using a massive excess of unlabelled ligand to compete off the saturable specific binding what remains is the non-specific binding
this will enable the calculation of the specific binding
what is the relationship produced with non-specific binding ?
the more radioligand added the more non-specific binding you get - this is a linear relationship
how is the saturation curve for specific binding determined?
it is obtained by subtracting the non-specific binding from the total binding
what can be determined from the specific binding curve?
it is possible to determine the maximal binding (Bmax) and therefore it is possible to determine the Kd value
what are the issues with competition radiolabelled ligand assays ?
problem is it requires you to have lots of radiolabelled ligand
it is also not suitable for high throughput sequencing because it only gives the Kd for the radiolabelled ligand
describe the process of estimating Kd for saturation binding :
it is a direct measurement of affinity Kd
affinity of the radioligand only not the actual compound
requires saturating concentrations of radiolabelled ligandd
not suitable for HTS
what is another way for estimating the Kd?
it is to calculate an IC50 using competition radioligand binding assay
what happens in competition radioligand binding assays ?
label a fraction of the target population with a radioligand and then compete it off with an unlabelled ligand
when the unlabelled ligand occupies 50% of the receptors (i.e. at its Kd) it should have removed 50% of the radioligand
what is the IC50 ?
it is the concentration of unlabelled ligand required to displace 50% of a preset amount of labelled ligand
it is essentially equivalent to Kd
it is sometimes converted to Ki
what is Ki ?
is the inhibition constant for a drug - it is the concentration of competing ligand in a competition assay whihc would occupy 50% of the receptors if no radioligand was present
what is the difference between the IC50 and Ki?
the IC50 value for a compound may vary between experiments depending on the radioligand concentration whereas the Ki is an absolute value
what equation is used to calculate the Ki from the IC50 ?
cheng-prusoff equation
what can be determined from competition binding assay graphs ?
different unlabelled ligands can be compared for their affinities
lower affinity ligands will be further to the right because they require higher concentrations to remove the same amount of radioligand
describe competition binding :
- indirect measurement of affinity (IC50) but can be converted to equilibrium dissociation constant
- affinity of unlabelled ligands
- low concentrations of radiolabelled ligands
- much more useful for HTS than saturation binding
occupancy is a measure of ……… and activation is a measure of ………
affinity
intrinsic activity
what does a drug with a greater affinity mean ?
it means the drug is less likely to dissociate from its binding site
what are the 2 factors required for a compound to be an agonist ?
it must have affinity and intrinsic activity
other than the properties of a drug there are properties of the system which are important, what are they ?
number of receptors present in the system
the ability to convert a drug induced stimulus into a response
what are the 2 advantages of plotting a concentration response curve to the log base 10 ?
- it prevents clumping of data points when the concentration range is large
- it causes the graph to become S-shaped with a well defined linear central portion
what 2 basic properties of an agonist can be determined from a concentration response curve ?
potency and intrinsic activity
what is used as the measure of potency and intrinsic activity and when do both these properties define ?
potency= EC50
intrinsic activity = Emax
they define the efficacy of the ligand
define potency :
it is a measure of the concentration of drug needed to elicit a given effect - difficult to determine a maximum concentration because the graph is curved at the top so instead the concentration that gives 50% of the maximum response is used- this is the EC50
how can you compare potencies of drugs ?
using the concentration response curve and comparing the EC50s of the drugs
drugs further to the left are more potent because they require a smaller concentration to produce their EC50 values
what is intrinsic activity ?
a full agonist elicits a maximum respoonse= 1
whereas partial agonists are a fraction of the full agonist response
it is the ability of a drug-receptor complex to evoke a response
what is the intrinsic activity of an anatagonist ?
0 because it is not able to produce a response and therefore it has no intrinsic activity
what do full agonists and partial agonists do to GPCRs ?
they shift them into their active state
whereas partial agonists only move some of the receptors into the active state
what do inverse agonists do ?
they look like antagonists but they push receptors into the inactive state to reduce the activity of the receptors
they are only detected in your system if you system has a high basal activity because they cause this activity to decrease
what are inverse agonists useful for ?
may be useful in therapy in cases where disease is caused by over activity of the receptor
may also be useful in stabilising the inactive conformation of GPCRs used in X ray crystallographic studies
what happens between agonists and antagonists if the competitive antagonist is surmountable ?
then increasing the concentration of agonist will overcome the block- sometimes known as reversible competitive antagonism
define pA2:
it is the concentration of antagonist that requires a 2 fold increase in agonist concentration to regain original response level
how can you estimate the affinity of an antagonist ?
use antagonist/agonist experiments
what is the Kb/pA2 equivalent to ?
the Kd
what is a schild plot and what can be determined from it ?
it is a pharmacological method of receptor classification
it is constructed using the dose-effect curve for an agonist determined in the presence of various concentrations of a competitive antagonist
the pA2 is determined which is an estimate of affinity of the antagonist for its receptor
what is a schild plot sometimes referred to as?
a pA2 analysis
what happens once the actual experiments for a schild plot have been completed ?
series of dose ratios are calculated
e.g. the ratio of the dose of agonist to produce a specific effect in the presence of the antagonist - determined for several doses of antagonist
if the regression is linear with a slope of -1 then it indicates that the antagonism is competitive and by definition the agonist and antagonist act at the same receptor site. the x intercept is the estimate for the pA2
what is another name for pA2?
it is the equilibrium dissociation constant for the antagonist
what is required for the correct use of the schild plot ?
it requires the antagonist to be competitive
why is schild analysis not too good for HTS?
because it requires you to go through many different concentrations of antagonist to obtain an affinity value
