tox142 - receptor theory - 2006

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Receptor Theory

&

Toxicant-Receptor Interactions

Richard B. Mailman



Some examples of receptors

1

E2

R E1

ligand

F K

FK

E E

2 Ion

R R

ligand

ligand

nucleus

R

R

3 ligand

E

R R

4

R R

ATP

ADP

P

ATP

ADP

PP

P



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

Aff inity:

± the ³tenacity´ by which a ligand binds to its receptor

Intrinsic activity (= ³efficacy´):

± 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 eff icacy (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

S pecific activity depends on half-life, and is totally independent of

mode or energy of decay.

W

hen 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

Ligand + Receptor Lgand-Receptor

Complex

Response(s)



Bimolecular Interactions:

Foundation of Most Studies

Ligand + Receptor Ligand-Receptork on

[Ligand] [Receptor] k [Ligand Receptor] k on off !

Rearrange that equation to define the equilibrium dissociation constant K D.

[Ligand] [Receptor]

[Ligand Receptor]

k

k K

off

on

D

! !

At equilibrium:

Ligand + Receptor Ligand-Receptor

ComplexResponse(s)



Saturation Equations

D D K

B B

K F

B

B

max

D

max

1

F+K

FB

!

!

Fractional occupancy [Ligand][Ligand] K D

!



Linear & Semilog

0 20 40 60 80 100Free

Linear Plot

0

0.2

0.4

0.6

0.8

1

B o u n d

-2 -1 0 1 2log [Free]

Semi-Log Plot

0

0.2

0.4

0.6

0.8

1

B o u n d



Radioreceptor Assay (Simple)

unbound labeled drug +

unbound test drug

drug-receptor

complex

radiolabeled drugreceptor preparation test drug

FiltrationBeta

Counter

Tissue

Preparation



Characterizing Drug-Receptor Interactions:

Saturation curves

0 2 4 6 8 10 12 14 16 18

RadioligandAdded (cpm x 1000)

S pecific Binding! (calculated)

Non-Specif ic

Total Binding

800

600

400

200

0



Scatchard plot

S pecificBinding

S p e c

i f i c B i n d i n g / F r e e R a d i o l i g a n d

Bmax

-1/KD



Competition Curve

log [ligand] (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100

IC50

Top

Bottom

Specif ic

Binding

NSB



Calculations from Basic Theory (I)

log [competing ligand] (M)

S p e c i f i c B

i n d i n g ( % )

10-9 10-8 10-7 10-6 10-5 10-4 10-3

0

25

50

75

100

90%

10%

81 Fold



Calculations from Basic Theory (II)

log [competing ligand] (M)

S p e c i f i c B

i n d i n g ( % )

10-9 10-8 10-7 10-6 10-5 10-4 10-3

0

25

50

75

100

91%

9%

100-fold

Commit this to memory!!!!!



Competition Curves

Log [ligand] (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100 1000

B

A



Concentration (nM)

0

10

20

30

40

50

60

70

80

90

100

0.10.01 1.0 10 100 1000

A DCB



Functional effects & antagonists

Log Agonist Concentration (M)

0

0.2

0.4

0.6

0.8

1.0

-10-11 -9 -8 -7 -6

Control

(agonist with no

antagonist)

+ Increasing

concentrations

of antagonist B

Raw Data



S pare receptors and ³full agonists´

ED1

E1

F K

E2

ER

E1

F K

cAMP stimulation????

????

D1 D1



Full & Partial Agonists

Concentration (nM)

0

20

40

60

80

100

Full agonist

Partial agonist

1 10 100 1000 10000 100000

tox142 - receptor theory - 2006

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