Read Microsoft PowerPoint - Seminar 3 Ligand Binding text version

binding ­ get tight and cooperate or it'll turn nasty

Receptor-Ligand Binding

basic concepts the dose-response curve tissue drug concentration competitive antagonism non-competitive antagonism irreversible antagonism kinetics of drug action

Why do radioligand binding assays?

does my compound interact with a particular protein? what chemical features should a drug have to interact with the channel of interest? how potent is my drug? how is the protein of interest regulated? can my channel interact with more than one drug? how much of the protein is expressed?

The Ingredients of a Binding Assay

receptor source some basic pharmacology (i.e. activators, inhibitors) radiolabelled ligand (125I, 3H, 14C etc) with high specific activity method to separate bound and non-bound material binding assays do not provide details on the biological effects

[3H]-drug

protein membranes or cell fragments incubate filter (collect particulates)

the simplest experiment

keep the receptor amount constant vary the radioligand concentration

how do you analyse the numbers?

[R] + [L]

k1 k2

[RL]

old fashioned maybe

release radioactivity (scintillation fluid) measure dpm/cpm (scintillation counting)

the law of mass action states that

formation = k1[R]·[L] breakdown = k2[RL]

at equilibrium

formation = breakdown

[L] is known and [RL] is measured k2· [RL] = k1[R]·[L] substitute Kd = k2/k1 and [Rt] = [R] + [RL] Kd·[RL] = [L] ·([Rt] ­ [RL]) Kd·[RL] + [L] ·[RL] = [L]·[Rt] [RL]·(Kd + [L]) = [L]·[Rt] [L]·[Rt] [RL] = (Kd + [L])

Equilibrium Binding:

the "Langmuir Adsorption Isotherm"

Amount Bound

1.0

Rt = Bmax

0.5

Kd

0.0 0 50 100 150 200

[L]·[Rt] [RL] = (Kd + [L])

[ligand]

Bmax = binding capacity and reflects the receptor number Kd = dissociation constant & reflects the affinity

that's the theory ­ what about the reality?

material for binding will comprise real or artificial membranes

The reality of binding assays

Amount Bound

1.5 total 1.0 specific NSB

0.5

0.0

0

50

100

150

200

· drugs (the usual ligands) are normally quite hydrophobic · membranes are a sink for lipid soluble material · total binding is measured by filtration of membranes/liposomes through glass fibre filters · the non-specific binding component is made up of nontarget proteins, lipid intercalation and filter adsorption · non-specific binding is linear

[ligand]

· total binding is measured by filtration · non-specific binding also measured binding remaining in the presence of a competing ligand · specific binding is derived as Bs = Bt - Bnsb

the importance of [ligand]

the [ligand] drives formation of the receptor-ligand complex depletion of radioligand renders the simple Langmuir Isotherm invalid high receptor concentrations (e.g. recombinant systems) are frequently associated with depletion overcome with: less protein (downside is the low dpm) increase volume (downside is cost of ligand)

[ligand] problems: continued!

[ligand] refers to the free concentration ­ i.e. the amount added to tubes ligands are usually hydrophobic and under certain conditions the nsb may cause depletion of [ligand]free overcome by: alter experimental conditions (e.g. receptor) measure [ligand]free (ok for dialysis or centrifugation) use complex analysis techniques

not all ligands are available in labelled form

"Langmuir" analysis is not feasible

"competition" binding

conduct competition binding assays

· fix [receptor] and [radioligand] · vary the [competing drug] · measure binding

heterologous displacement assays

Amount Radioligand Bound

1.0

heterologous displacement assays

do not indicate whether the interaction is competitive or non-competitive

Cheng-Prusoff correction then? Ki = EC50/(1+[L]/Kd) i.e. EC50Ki ONLY if there is a true competitive interaction

what do these assays tell us?

IC50

0.5

0.0 -1 0 1 2 3

log10[competitor]

(i) if a compound interacts with the protein (ii) an idea of the compound potency

NOTE: · IC50 (or EC50) values are NOT constants · the IC50 depends absolutely on the reaction conditions (i.e. amount of receptor, [ligand] & volumes) DO NOT COMPARE POTENCIES!!!!!!!!!!

do not show cooperativity of interaction do not discriminate between reversible and irreversible processes do not reveal non-equilibrium conditions

what can we do to answer the following? do drugs interact competitively? what is the true potency of the compound? how many binding sites exist on the protein? do the sites interact (allosterically)?

this is difficult to analyse

answer:

heterologous binding curves

i.e. Langmuir analysis with varying amounts of competitor

Amount Bound

1.0

0.5

[competitor]

0.0 0 50 100 150 200

[ligand]

Amount Bound

1.0

Langmuir Isotherm

Y=

0.5

(Kd + [L])

Amount Bound

Y

max

· [L]

[antagonist]

1.0

now let's try those heterologous binding assays again

increased [antagonist] causes a shift in the dose response curve to the right

0.5

0.0

0

50

100

150

200

0.0

[ligand]

-3

-2

-1

0

1

2

3

4

5

log10[ligand]

Amount Bound

1.0

Dose-Response

Amount Bound

1.0

Km no Bmax

0.5

Y = Ymin +

0.0 -3 -2 -1 0 1 2 3 4 5

1 + 10(logEC50 -[L ])^ n

(Ymax - Ymin )

0.5

0.0

log10[ligand]

when n=1

-3

-2

-1

0

1

2

3

4

5

log10[ligand]

they can't both be competitive?

competitive

[antagonist]

Amount Bound

1.0

Log(dr-1)

Schild Analysis

-1 0 1 2 3 4 5

2

Schild Analysis

slope = 1 KB

-0.50 -0.25 0.00 0.25 0.50

1

0.5

0.0

-3

-2

log10[ligand]

derived, both experimentally and mathematically, a relationship between the shift in the doseresponse curves an [antagonist] log(dr-1) = log[B] ­ logKB

0

-1 -0.75

Log[antagonist]

log(dr-1) = log[B] ­ logKB

the analysis only holds where the slope = 1 equilibrium conditions must be met KB is the [antagonist] that produces a dose-ratio of 2 i.e. receptor population is equally bound by agonists & antagonists KB corresponds to the affinity of the antagonist

2

Log(dr-1)

1

dr = dose ratio

slope = 1 KB

-0.50 -0.25 0.00 0.25 0.50

0

-1 -0.75

Log[antagonist]

i.e.concentration of agonist required to provide similar binding to that observed in the absence of antagonist ([B])

Amount Bound

what about this one?

Schild plot is non-linear

1.0

another possible scenario for heterologous binding assays

[antagonist]

-3 -2 -1 0 1 2 3 4 5

0.5

0.0

log10[ligand]

Amount Bound

1.0

increased [competitor] causes a shift in the dose response curve to the right

0.5

the dose-response curves shift to the right shifts are shifted with ever decreasing "quanta" may be explained as an allosteric effect

i.e. antagonist binds to a distinct site

no Km

0.0 -4 -3 -2 -1 0 1 2 3 4

Bmax

log10[ligand]

as the allosteric sites become occupied the apparent shift in Kd is reduced

non-competitive

· the reduced Bmax suggests that the number of available receptors is decreased · however, those that are available display unaltered affinity · this effect is an insurmountable antagonism and thus clearly non-competitive non-

non-competitive (allosteric) interaction

Maximal Binding Capacity

1.0

Kinetics of Drug Action

[R] + [L]

k1 k2

[RL]

0.5

K = allosteric quotient

K

-1 0 1 2 3

0.0

log10[antagonist]

kinetics examines the rate of change in the amount of receptor-drug complex as a function of time the amount of receptor-ligand complex at any time (prior to equilibrium) will vary according to the respective rates of association and dissociation

the relationship between Bmax and the [antagonist] provides an indication of the allosteric potency of the compound

not the true affinity constant for the antagonist

i.e.

d[RL] dt

= k [R] · [L] - k [RL]

1 2

Dissociation kinetics

Fraction Maximal Binding

1.0

Fraction Maximal Binding

Bt -k ·t =e Bmax -k ·t Bt = Bmax · e

2 2

Dissociation kinetics

if the antagonist (B) is truly competitive then the formation of the RB can only occur once the RL complex has dissociated

1.0 no inhibitor competitive inhibitor 0.5 allosteric inhibitor

0.5

or Bt Ln Bmax = k · t

2

0.0

0

50

100

150

0.0

0

50

100

150

time (min)

time (min)

[RL]

[R] + [L] [M] [B]

· allow binding equilibrium to be reached between R & L · "block" the association reaction either by: excess unlabelled ligand large dilution (i.e. [L] & kon) · measure the amount bound over time · estimate kon by non-linear regression

the koff will NOT be altered by a competitive antagonist

[MRL]

[RB]

change in koff could however occur in the presence of an allosteric antagonist (M)

[L] [MR]

Fraction Maximal Binding

Association kinetics Bt = Bmax·(1-e-kobs·t)

1.0

Fraction Maximal Binding

Irreversible Antagonism

0 50 100 150

1.0

0.5

0.5

time

0.0

time (min)

0.0

-2

-1

0

1

2

3

log10[ligand]

however, the curve above is actually the sum of association and dissociation reactions

· effects continue to display temporal characteristics · no equilibrium is reached · KD cannot be determined since there is no koff · propensity/accessibility to labelling (e.g. NEM modification) is described by the rate constant

kobs = kon.[L] + koff

· plot kobs versus [L] and the slope corresponds to the association rate constant · alternatively substitute the experimentally derived value for kon to calculate the off rate

Multiple Agonist Binding Sites (non-interacting)

[R] + [L]

k1 k-1

Multiple Non-interacting Sites

the independent dissociation constants are:

[RL]

S2 S1

Kd1 =

[R] · [L] [RL]

Kd 2 =

[R] · [L] [RL2 ]

[RL] + [L]

k2 k-2

[RL2]

· an initial equilibrium may be attained with association and dissociation rate constants k1 and k-1 respectively · the [RL] complex may then bind a further ligand with rate constants k2/k-2

re-arranging & substituting leads to the Adair equation describing the fraction binding as a function of ligand concentration

F.B = Kd2 · [L] + 2[L]2 2 Kd1 · Kd2 + Kd2 · [L] + [L]2

Multiple Non-interacting Sites

kd2/kd1 ratio

1.0

Multiple Agonist Binding Sites (interacting)

[L] [R] [M] [RL] [M] [MR] [MRL] [L]

fraction bound

S1

S2

0.5

0.0 10 -1

10 0

10 1

10 2

10 3

10 4

10 5

10 6

[L]

· protein has >1 binding sites initially of equal affinity · usually, but not always, a quaternary protein · binding to one site may allosterically influence binding to the other(s) i.e. alter Kd · this situation will also occur in allosteric inhibition

the separation of curves only becomes apparent at high differences between the two binding sites careful and meticulous analysis is required

co-operative binding

increasing slope factor

fraction bound

flashy arrays may get you a trophy and all the glory.........................

1.0

n=0.5

0.5

n=2

0.0 -15 -12 -9 -6

log[ligand]

..................but here in the trenches, we know a thing or two about binding together for a common cause.

Y = Ymin +

(Ymax - Ymin ) 1 + 10(logEC50 -[L ])^ n

Information

Microsoft PowerPoint - Seminar 3 Ligand Binding

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