receptor theory toxicant receptor interactions
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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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Presentation Transcript
some examples of receptors

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
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
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
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
Drug-Receptor Interactions

Lgand-Receptor

Complex

Ligand + Receptor

Response(s)

bimolecular interactions foundation of most studies
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
Saturation Equations

Michealis-Menten form

Scatchard form

linear semilog

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
Saturation Equations

Michealis-Menten form

Scatchard form

saturation radioreceptor assays
Saturation Radioreceptor Assays

receptor

preparation

radiolabeled

drug

TissuePreparation

drug-receptorcomplex

BetaCounter

Filtration

unbound labeled drug + unbound test drug

characterizing drug receptor interactions saturation curves

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
Saturation Equations

Michealis-Menten form

Scatchard form

scatchard plot
Scatchard plot

-1/KD

B/F

(Specific Binding/ Free Radioligand)

Bmax

B(Specific Binding)

competition radioreceptor assays
Competition Radioreceptor Assays

receptor

preparation

radiolabeled

drug

test

drug

TissuePreparation

drug-receptorcomplex

BetaCounter

Filtration

unbound labeled drug + unbound test drug

competition curve

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

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

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

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

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

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

E1

E1

a

a

b

b

g

g

R

E2

Spare receptors and “full agonists”

D1

D1

D1

cAMP stimulation

????

????

full partial agonists
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

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

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

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

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

SideEffect 1

  • TherapeuticEffect 1
  • SideEffect 2
Activation of three pathways via a single receptor:“Functionally selective” compound
lessons of functional selectivity
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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