medchem exam 2 quantitative pharmacology

slide 9, class 11, april 2nd, all done

what properties to know to compare target ligands (1:2)

affinity for target (how strong does it bind), functional activity (potency ie at what concentration does it have an effect, efficacy ie how strong is the effect?)

How to quantify drug target interaction

Add drug concentration and then watch it increased and then plateau over time

Law of mass action

Velocity of a chemical reaction is proportional to the product of concentrations (or mass) of reactants

what is Kd

ligand affinity, how good ligand is interacting → dissociation constant (at equilibrium); shows at what [A] value you reach saturation

Kd unit

nanomolar

what do diff Kd numbers mean

• The lower the Kd - the better it sits on the receptor, the less you need of it

• The higher the Kd - the more you need for it to sit on the receptor (it is worse at it)

Can 100% ever be bound? why/why not?

interactions are reversible, usually can never bind 100%

why is it important to wait till equilibrium when doing these experiments

• If not at equilibrium - incubate for shorter than it takes to each equilibrium

  • Highest one will be at equilibrium but lower values will not be

  • Underestimate affinity of ligand binding to receptor

  • if you test at equilibrium- much more likely to be accurate data!

what is the total receptor number

Bmax (or Rtotal)

association rate formula

= k₊₁ x [R] x [A]

Dissociation rate formula

= k_-1 x [AR]

association and dissociation at equilibrium

they are equal - ie k₊₁ x [R] x [A] = k_-1 x [AR]

what is the equilibrium dissociation constant equal to?

Affinity

K_d formula

k_-1/k₊₁
= [A] x [R]/ [AR}

Bmax formula

[R] + [AR]

rearranged Bmax formula

[R] = Bmax - [AR]

Kd formula using Bmax

[AR] rearanged formula

benefit of the [AR] rearranged formula?

relation between something you can measure [AR], something you know [A] and 2 parameters that you like to determine

K_D[A meaning

[A] that occupies 50% of all receptors

what is it when [A] > > > > K_D

[AR] = Bmax

what is it when [A] = K_D

[AR] = ½ Bmax

fractional receptor occupancy (fAR)

= [A]/ K_D + [A]

what are diff curves here

different compounds with different K_D values

what is this?

fractional receptor occupancy but on a ¹⁰Log scale so they are S-curves instead

why is it helpful to use log scales here

S curves are easy to do curve fitting

when is f = 0.5 on log scales

At the inflection point

pKd = 9 (bc thats the one thats the smallest number since its 1 × 10^-9§

how to make your ligand visible so you can measure things w/ it

but something radioactive on it which is detectable, can then measure how much of it is bound to the receptor as the rest is washed away (this method was used more in the past though)

displacement (competition) binding descr

indirect measure of affinity of unlabeled ligands, displace fixed [radioligand] from receptor by increasing concentrations of unlabeled ligand; eg with B present not all targets are accessible to A* the labelled ligand

when would you get a graph like this

when doing competition of A* with B in increasing []s

way to compare ligands when doing competition binding

IC50 - inhibitory [] at 50%, measure of the potency of B to displace A*; BUT not the affinity of B (that is Kd(B))

what is the IC50 of B not equal to the affinity of B

IC40 value in binding experiments depends on experimental conditions and is not just a property of compound B; if you change the [A*] and eg make it higher, it is more difficult for unlabeled ligand B to displace → higher [B] are needed to fully occupy the target so the IC50 value shifts to the right

Ki value meaning

actual affinity

cheng-prusoff equation

examples of cheng prusoff equation calc

what is the cheng-prusoff equation used to find

equation to derive an affinity parameter for a ligand that is independent of experimental condition, convert IC50 to Ki values

what is this a summary of?

direct binding - from A* + R <=> A*R

what is this a summary of

reversible competition binding, convert IC50 into Ki value

what can agonist binding to receptor lead to?

cellular response

what is this

dosis-response curve

possible eg’s of a response that can happen after agonist binding (4) (that can be measured)

contraction muscle, frequency heart (cell), level of 2nd messengers, gene expression

agonist dosis response - potency descr

potency of agonist [A] to produce a given response

EC50 formula

[A] giving half a maximal response, -log EC50 also used

partial agonists descr

reduced maximal response at 100% occupancy, a partial agonist needs to occupy all receptors to produce its (low) maximal response

alpha formula

max response (partial agonist) / max response (full agonist)

diff alphas for each line (top to bottom)

alpha = 1
alpha = 0.9
alpha = 0.5
alpha = 0.1

conformational sensors descr

can report on TM6 movement of GCPRs upon activation; can use bioluminescent emitter (BRET) when brought over emits red cause energy transfer to fluorophore

what do diff H3R agonist induce

different conformational states of the H3R sensor, although they all bind to the sensor

what are A and B here

A - agonist
B - antagonist

competitive antagonism descr

agonist dose response in presence of an antagonist - competition for common binding site, shift EC50 of agonist A, Emax still obtained if [A] is high enough and competes w/ B

how can you see competitive antagonism on a graph w/ diff conditions

increasing [antagonist] shift against DRC rightwards
parallel shifts if similar increase in [antagonist B] is tested
if graph increases in [B] in log-steps and half-log steps are shown

steps how to quantify strength of a blockade

1. determine EC50,A ([B] = 0) and EC50,A’ (with B present) for all tested [B]

   1. determine dose ratio DR = EC50,A’/EC50,A for schild analysis

label red point

EC50,A’

what is affinity of antagonist [B'] expressed as?

Kd,b or pKd,b

what relationship do log([B]) and log(DR-1) have and result; what should the slop plot be for competitive antagonism?

linear relationship, if [B] increases 10-fold, DR also increases 10 fold; for competitive antagonist - slope = 1

what happens w/ insurmountable/non-competitive antagonism?

irreversible binding of B to receptor, less receptor available for A - reduced response by agonist A, agonist DRC in presence of irreversible antagonist, covalent bond formed, messenger blocked from binding site