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Receptor Theory & Toxicant-Receptor Interactions. Richard B. Mailman. 1 . 2 . ligand. Ion. ligand. E. R. 1. R. R. a. a. b. g. b. g. E. 2. ligand. 3 . 4 . ligand. R. R. R. R. R. R. ATP. ATP. ADP. ADP. P. P. P. P. nucleus. E. Some examples of receptors.

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Receptor Theory & Toxicant-Receptor Interactions

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Receptor theory toxicant receptor interactions l.jpg

Receptor Theory & Toxicant-Receptor Interactions

Richard B. Mailman


Some examples of receptors l.jpg

1

2

ligand

Ion

ligand

E

R

1

R

R

a

a

b

g

b

g

E

2

ligand

3

4

ligand

R

R

R

R

R

R

ATP

ATP

ADP

ADP

P

P

P

P

nucleus

E

Some examples of receptors


What is a receptor l.jpg

What is a receptor?

  • To a neuroscientist

    • A protein that binds a neurotransmitter/modulator

  • To a cell biologist or biochemist

    • A protein that binds a small molecule

    • A protein that binds another protein

    • A nucleic acid that binds a protein

  • To a toxicologist

    • A macromolecule that binds a toxicant

  • Etc.


Definitions l.jpg

Definitions

  • Affinity:

    • the “tenacity” by which a ligand binds to its receptor

  • Intrinsic activity (= “efficacy”):

    • the relative maximal response caused by a drug in a tissue preparation. A full agonist causes a maximal effect equal to that of the endogenous ligand (or sometimes another reference compound if the endogenous ligand is not known); a partial agonist causes less than a maximal response.

    • Intrinsic efficacy (outmoded): the property of how a ligand causes biological responses via a single receptor (hence a property of a drug).

  • Potency:

    • how much of a ligand is needed to cause a measured change (usually functional).


Radioactivity principles l.jpg

Radioactivity Principles

  • Specific activity depends on half-life, and is totally independent of mode or energy of decay.

  • When decay occurs for all of the biologically important isotopes (14C; 3H; 32P; 35S; 125I; etc.), the decay event changes the chemical identity of the decaying atom, and in the process, destroys the molecule on which the atom resided.

    • e.g., 3H He

    • Do NOT adjust the specific activity of your radiochemical based on decay – for every decay, there is a loss of the parent molecule.


Drug receptor interactions l.jpg

Drug-Receptor Interactions

Lgand-Receptor

Complex

Ligand + Receptor

Response(s)


Bimolecular interactions foundation of most studies l.jpg

Bimolecular Interactions: Foundation of Most Studies

Ligand-ReceptorComplex

Ligand + Receptor

Response(s)

At equilibrium:

Rearrange that equation to define the equilibrium dissociation constant KD.


Saturation equations l.jpg

Saturation Equations

Michealis-Menten form

Scatchard form


Linear semilog l.jpg

1

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

-2

-1

0

1

2

Linear & Semilog

Linear Plot

Bound

20

40

60

80

100

0

Free

Semi-Log Plot

Bound

log [Free]


Saturation equations10 l.jpg

Saturation Equations

Michealis-Menten form

Scatchard form


Saturation radioreceptor assays l.jpg

Saturation Radioreceptor Assays

receptor

preparation

radiolabeled

drug

TissuePreparation

drug-receptorcomplex

BetaCounter

Filtration

unbound labeled drug + unbound test drug


Characterizing drug receptor interactions saturation curves l.jpg

800

600

400

200

0

0

2

4

6

8

10

12

14

16

18

Characterizing Drug-Receptor Interactions:Saturation curves

Total Binding

Specific Binding! (calculated)

Amount Bound

Non-Specific

Radioligand Added (cpm x 1000)


Saturation equations13 l.jpg

Saturation Equations

Michealis-Menten form

Scatchard form


Scatchard plot l.jpg

Scatchard plot

-1/KD

B/F

(Specific Binding/ Free Radioligand)

Bmax

B(Specific Binding)


Competition radioreceptor assays l.jpg

Competition Radioreceptor Assays

receptor

preparation

radiolabeled

drug

test

drug

TissuePreparation

drug-receptorcomplex

BetaCounter

Filtration

unbound labeled drug + unbound test drug


Competition curve l.jpg

100

90

80

70

60

50

40

30

20

10

0

0.01

0.1

1.0

10

100

Competition Curve

Top

Total Binding (dpm *10, e.g.)

Specific Binding

IC50

Bottom

NSB

log [ligand] (nM)


Calculations from basic theory i l.jpg

100

75

50

25

0

10-9

10-8

10-7

10-6

10-5

10-4

10-3

Calculations from Basic Theory (I)

90%

Specific Binding (%)

10%

81 Fold

log [competing ligand] (M)


Calculations from basic theory ii l.jpg

100

75

50

25

0

10-9

10-8

10-7

10-6

10-5

10-4

10-3

Calculations from Basic Theory (II)

Commit this to memory!!!!!

91%

Specific Binding (%)

9%

100-fold

log [competing ligand] (M)


Competition curves l.jpg

100

90

80

70

60

50

40

30

20

10

0

0.01

0.1

1.0

10

100

1000

Competition Curves

A

Specific Binding (%)

B

Log [ligand] (nM)


Slide20 l.jpg

100

90

80

70

60

50

40

30

20

10

0

0.01

0.1

1.0

10

100

1000

Specific Binding (%)

A

B

C

D

Concentration (nM)


Functional effects antagonists l.jpg

1.0

0.8

0.6

0.4

0.2

0

-11

-10

-9

-8

-7

-6

Functional effects & antagonists

+ Increasingconcentrationsof antagonist B

Raw Data

Control(agonist with no antagonist)

Response (Fraction of maximal)

Log Agonist Concentration (M)


Spare receptors and full agonists l.jpg

E1

E1

a

a

b

b

g

g

R

E2

Spare receptors and “full agonists”

D1

D1

D1

cAMP stimulation

????

????


Full partial agonists l.jpg

Full & Partial Agonists

100

Full agonist

80

cAMP synthesis

60

(% stimulation relative to dopamine)

Partial agonist

40

20

0

1

10

100

1000

10000

100000

Concentration (nM)


Slide24 l.jpg

bg

a

Ligand #1

Typical Agonist

Ligand #2

Functionally Selective Agonist

A

B

Normal Agonist

F.S. Drug

bg

Functional

Complex

#1

D2R

a

G-protein

C

D

Functional

Complex

#2

No activation


Ligand action on three pathways via a single receptor traditional view of full agonist l.jpg

  • SideEffect 1

  • TherapeuticEffect 1

  • SideEffect 2

Ligand action on three pathways via a single receptor: Traditional view of “full” agonist


Ligand action on three pathways via a single receptor traditional view of partial agonist l.jpg

  • SideEffect 1

  • TherapeuticEffect 1

  • SideEffect 2

Ligand action on three pathways via a single receptor: Traditional view of “partial” agonist


Ligand action on three pathways via a single receptor traditional view of antagonist l.jpg

  • SideEffect 1

  • TherapeuticEffect 1

  • SideEffect 2

Ligand action on three pathways via a single receptor: Traditional view of antagonist


Activation of three pathways via a single receptor functionally selective compound l.jpg

  • SideEffect 1

  • TherapeuticEffect 1

  • SideEffect 2

Activation of three pathways via a single receptor:“Functionally selective” compound


Lessons of functional selectivity l.jpg

Lessons of functional selectivity

  • Increases complexity in understanding mechanisms of toxicity.

  • BUT ….provides opportunities to dissociate toxicity from therapeutic effects mediated via a single receptor.

  • Universal to almost all targets for small molecules.


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