0026-895X/59/020265.08$02.

OO/O Copyright (C by The American All rights

MOLECULAR

Society
in any form

for

Pharmacology

and

Experimental

Therapeutics

of

reproduction
PHARMACOLOGY,

reserved.

36:265-272

Multiple Opioid Receptors: Binding Site Signatures

AVRAM Department Received GOLDSTEIN and ASHA NAIDU

Ligand

Selectivity

Profiles

and

of Pharmacology,
February 1 0, 1989;

Stanford
Accepted

University, Stanford,
May 30, 1989

California

94305

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

SUMMARY
signatures have been derived, which uniquely describe each of Methods are described for studying ,, 5, and ic opioid binding of sites. The z, 5, and i binding sites have sites, each without interference from the others. A large array the three types of ligands has been characterized by ligand selectivity profiles, interesting common features and distinctive differences. graphic depictions of affinities and selectivities. Binding site

To one studies receptor mistakenly type sumed for

z is

characterize type or with subtype1 opioid assumed binding

from

multiple nearly receptors had that site

other

receptors, exclusively. (2) been a ligand label

For sites.

it illustrates showing that

example,

is The

necessary history this

insignificant to label primary sites of binding report point. After We

even

when use

substantially of specific

more

secondary

than binding

are present. here the

radioreceptor each The ligand constants set

heterogeneity of to

demonstrated site the defined use as used receptor great that goal. affinity the binding at
from the

would #{244} sites, as

assays (3), it was often LSP. selectivity for a one without significant a given
DADLE was

for opioid z, t5, and K sites to characterize LSP is the set of equilibrium dissociation ligand at the three types of sites for (7). a BSS constants a unique LSP,

by of LSP

for

interference

label

only as great effects

whereas for into was one obtained. and of the at that

affinity EKC, as as an type, but

almost activation,

#{244}. Likewise,

pharmacologic receptor sites even Selective membranes populations radioligands in several tive ferred site
This

previously came affinity (4-6) to be toward for

a radiolabel in

data can be rearranged to yield astative rank order of dissociation of this ligand the three sites studied here has which elicited the conceptual difference between being due K to ligand, and BSS, which characterizes K binding for great
K. as

each site, a quantiat that site. Each of signature. We characterizes site. stress a

which a binding

though alkylation with could were laboratories that great

its respect not

is almost

for enrich Materials.

unless contains made
(Research is the

attempt

to homogeneous type-selective

Experimental
Reagents
otherwise. NaCl, (mM): Organics, same as

Procedures
grade
50,
MgCl2,

Sufficiently progress Sufficiently constant

stated

required, the site ratio to so

by
a

has

been type at

means (primary) is

selecKRB the pre-

were Baker analytical contains (mM): Tris-HC1, 118; KC1, 4.8; CaCl2, 2.5; Cleveland, OH), 25; pH adjusted TB
KHB with 1.2 mM KH2PO4

or
pH
1.2;

equivalent
HEPES

7.4. KHB NaOH. mM choline 7.4. stated, of DYN 9. 3, The DAKLI

to

7.4
and

with 0.02

25 mM NaHCO

instead

of HEPES

and, with from DYN

in

addition,

11 mM

glucose also 37 to

and present. maintain unless

that

next-preferred

enough
was supported

(secondary) chloride; secondary sites bubbledis

Peptides
National Institute on and

mepyramine
constantly were

maleate
5% Peninsula CO2

(125M) n
in 02

was at

KRB
pH otherwise

was

Laboratories,

work

Grants

DA-1199

were
DYN

purified
A and

by reverse
B and

phase
various

HPLC and its

2,

in our

laboratory
and derivatives ref. see Figs.

as required.

Drug
1

Abuse We call

and BNS-84-16617 the z, #{244}, and

from
opioid who

the

National

Science

Foundation.

For

fragments code

introduced Like a, subscripts.

by Ahlquist and a2, opioid

(1), receptor

receptors so described subtypes (if

types after the terminology A, see i. the and fl-adrenergic receptors. they exist) would have numerical following

Ref.
2.

8,

and Dr.

for
are

DAKLI
listed by

ligands

derivatives, number;see also

SUF (Janssen);

3-5 and

Table

1, OMF,

Chi

Zhiqiang

(10);

ABBREVIATIONS: enkephalin; SUF, DAKLI, dynorphin

DADLE, [D-A1a2,D-Leu5]enkephalin; LSP, ligand selectivity profile; BSS, binding site signature; DAGO, [D-Ala2,MePhe4,Gly-ol] sufentanil citrate; OMF, ohmefentanyl hydrochloride, N-[1-(fl-hydroxy-3-phenethyI)-3-methyl-4-piperidyI]-N-phenylpropionamide; A analogue c Iigand; CTOP, cyclic D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr amide; BREM, bremazocine hydrochloride; EKC,

ethylketazocine methanesulfonate; j3-NTA, fl-naltrexamine; TB, Tris-HCI buffer; hydroxyethyl piperazine-N-2-ethanesuffonic acid; DYN A-(1 -13)-NH2, dynorphin
A; DYN B, dynorphin penicillamine5]enkephalin; B; HPLC, high SDL, specific

KHB, Krebs-HEPES buffer; KRB, Krebs Ringer buffer; HEPES, N-2. A-(1-13) amide; Me, methyl; Et, ethyl; Pr, propyl; DYN A, dynorphin
DPDPE, cyclic [D-penicillamine2,Dtartrate; DEX, dextrorphan tartrate; fi-

pressure liquid chromatography; DSLET, [D-Ser2,Leu5]enkephalin-Thr; displacement ligand; B,,-END, human fl-endorphin; LEV, levorphanol

NTA-I2BH,

diiodinated

Bolton-Hunter

reagent

coupled

to f3-naltrexamine;

fl-NTA-F,

fluorescein

coupled

to fl-naltrexamine;

DPLPE,

[D-penicillamine2,L265

penicillamine5]enkephalin.

266

Goldstein

and Naidu V. J. Hruby (11); 5, naltrexone 7, BREM (Sandoz) (12); 8,

hydrochloride (Endo);

(9); 4, CTOP, Dr. 6, LEV (Roche);

flh-END;

test 9,
buffer

tube:
alone;

buffer,

0.5

ml,

containing
suspension, 1.0

DYN

A-(1-13)-NH2;

membrane

SDL and blockers ml kept agitated

as required, by swirling;

or

10,

Winthrop); 12, for preparation;

hydrochloride

to DAKLI, below; 18,

tional ratory Institute by

10 l of methanol/0.1 M HCI (1:1, v/v) containing competing ligand or hydrochloride (Endo); 1 1, EKC (Sterlingmethanol/HC1 alone; and radioligand in buffer, 0.5 ml. Incubation (23, DAGO (13, 14); 13, biotinylated DAKLI, see Ref. 9 demonstrated, with each radioligand and competing ligand, 14, [N-Me-TyrJDYN A-(1-13)-NH2; 15, naloxone2 hr) was to be long enough for equilibration. Samples were kept on ice (less than (Endo); 16, diiodinated Bolton-Hunter reagent coupled filtration (S&S No. 32 glass fiber filters, 24 mm, presoaked see Ref. 9 for preparation; 17, fl-NTA-I2BH, see code 1520 mm) until in water that was saturated with t-amyl alcohol) in an DADLE; 19, morphine sulfate (Merck); 20, 3-NTA, Na- glass-distilled

oxymorphone

on

Drug coupling

Abuse;

derivatives procedures and to reverse DSLET, (-)-enantiomer 20 above;

were purified

prepared by prepared HPLC B. 29, P. [U63,640] DPLPE(Me)

in reverse

our

aluminum labo-

filter

block.

Alternatively,

No.

32

glass

fiber

strips

were

standard

prepared phase
Gaithersburg, with 4 ml fluoand allowed 24, (Westchem, dissolve

HPLC;
(Upjohn) rescein

21,
(15);

DYN
23,

A (8);
and

22,

spiradoline
coupled on 25, code

(+)-enantiomer
DAKLI, phase Dr.

(U63,639)
with (9); (Up-

fluorescein purified (16);

in the same way and used in a cell harvester (M24R; Brandel, MD). Tubes were washed onto the filters three times ofcold buffer. Filters were transferred to scintillation vials
to San stand Diego, SD or at CA) room for temperature at least 5 hr, in the 5 ml time of Cytoscint required to

isothiocyanate

[D-Abu2,D-t-Leu5]enkephalin

Roques (17);

26,
john)

DYN
(15);

A-(1-13);
28,

27,
3-NTA-F,

spiradoline
see

all
to

radioligands
1.5% binding
t

counted (16);

and attain for 100 mm.

constants with were

maximum
(Kd) the obtained were

cpm.
determined

Radioactivity
in program TB model, and (22).

was

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

morphiceptin (18); 31, DPDPE(Me) (16); 32, 33, DPDPE(Me)2 (16); 34, DPLPE (16); 35, DYN 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate (U50,488) (Upjohn) (19); 37,
38, (1-6); (16); DEX (Roche); 39, DPDPE, Dr. H. I. Mosberg

30,

DPLPE(Et) B36, (trarw)(8);

DYN (20); A-(1-9); 40,

(16); Radioligand KHB from

fits for the

dissociation isotherms
K

LIGAND with

Best but

and

systems

a one-site

[3H]DAGO
DYN elaborated A-

binding was fitted best bytwo-site a upon in the Discussion, we used

Kd
in Bmax in

the

model high
ligands.

(Fig. 1). As affinity estimate

Data are
2-fold

41, DPLPE(Pr) (16); 42, DPDPE(Et) 44, DYN A-(1-7); 45, [D-t-Leu2,D-t-Leu5jenkephalin (5a,7a,8fl)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-(4,5)dec-8yljbenzeneacetamide (U69,593) (Upjohn) (21); Membranes. A guinea pig was decapitated
some
-70

(16);

43,
(16);

DPDPE(Pr) of DAGO

in

computations
Table 1.Kd values

of K

for
differed

competing
less than

46,

D-

summarized buffers.

varied
KHB and

widely
TB

at
were

different
usually

47,

DYN and the

(about

A-(1-8). whole brain

3 g) and

therms

inasmuch (in
in frozen different

as Bmaa differences
experiments with

as large
the same

thein two times and among brains; isonot carried out concurrently and, as severalfold were also observed
buffer, we cannot attribute the

experiments,

less

cerebellum)

was

weighed

immediately
without

on
Tissumizer,

dry
loss

ice.
of

Brains
binding

could
capacity.

be
in
(37,

stored
The

for
tissue

several
was

months
homogenized

differences at
To After
in

observed
errors due

here
to
parallel

to the

buffers.
depletion (23), free
were

avoid

unrecognized

radioligand

(Tekmar centrifugation

Cincinnati, x g, 23,
incubated

OH) 10 mm),

30 the

ml pellet

of

KRB was

at

37.

concentrations
supernatants in of instead

were
from

measured

routinely
sets of

by
tubes,

determining
which

radioactivity
centrifuged

(12,000

resuspended

KRB to about 90 ml and tubes to destroy endogenous membranes were resuspended

portions above. with There each followed the made two

mm) in tightly 20 capped opioids. After another centrifugation, to about 180 ml, subdivided into
200 of ml, capped again, and (16,000 incubated centrifugation

filtered

(concentration the percentage three

was 2%)

after the incubation, and interpolating to the ICse at which specific binding is reduced by half). The of depletion by binding in the absence of a competing ligand
2%) (11 for [3H]DAGO experiments) (3 but experiments) 43% (SE, and 1%) 8% for corn(SE, [3H]

up

to

as 10

only for

6% (SE,

rounds

x g, 23,

mm) was

and resuspension to be used in

in the
binding

same

volume
assay).

of KHB
The

or TB

(whichever
pellet

BREM
peting

[3H]DPDPE (17 experiments).

ligand:

A full
(24)

membrane

Cheng-Prusoff was

approximation

equation (23) was used to convert IC50 toK, for

instead
each

of the

dispersed with a Dounce B homogenizer, at 23, to make a homogeneous suspension (typically 150 ml) ofwhich each milliliter represents 20 mg K, = l1/[(2L/L0) + (L/Kd) 111 1CM) of original brain arid has about 0.2 mg of protein. the measured free radioligand concentrations are L0 in the Radioreceptor binding assay. Each radioligand was used at where the absence of a competing ligand and at the IC. L In over 100 experiments lowest practical concentration, to provide about 1500-3000 cpm of siteconducted over a period of many months (23), IC (and therefore K,) specific binding. SDL signifies a specific-displacement competing ligand,
was was at
tt

define
least

specific
because 10 times it

binding
produced the d K of

by difference.
a plateau the given in

Each
the radioligand.

SDL

concentration
curve Conditions

was

determined

with

a SD

of

0.1

decimal

log
whenever

units

(i.e.,

about

25%).

chosen

competition

Because or

it
unlabeled

is impractical
competing

to

measure

free

foraffinity

ligands,

concentrations observed an IC

of is less

high

the radioligand K,, the computed value K, may of be too large by the system were [3HJDAGO (48 Ci/mmol; NEN, Boston, MA)nM 0.8 than amount (23). in TB and 2.0 nM in KHB, with 300 nM DAGO as SDL; for the an 5indeterminate Radioligands and competing ligands were checked for purity system, [3HJDPDPE (28 Ci/mmol; Amersham, Chicago, IL)nM 1.0in reverse phase HPLC (Waters Associates C15 zBondaPak column, TB and 2.0 nM in KHB, with 100 nM DPDPE as SDL. Specific binding x 30 cm, 30-mm linear gradient of acetonitrile in 5 mM trifluoroacetic was 83 and 70% of total binding for [3H]DAGO and [3H]DPDPE, acid with gradients optimized for each ligand) and were purified respectively. HPLC as necessary. The integrity of the three radioligandsfter a the For labeling K sites, no sufficiently selective radioligand was avail-

by

3.9

by

able, mmol;
K

so a paired-tube NEN) is a high

whereas for
K

paradigm
affinity radioligand

was
with

devised.
modest

[3H]BREM
selectivity

sites,

U50,488 sites. of 500 Specific

(not
K

available was, in

as

radioligand) defined

selective difference
and

binding

therefore, pairs

2-hr with membranes was examined by the same method; no Ci! contact degradation was observed. Peptide competing ligands (e.g., dynorphin peptides), at low concentrations, are known to be degraded to a variable is highly and no suitable protective cocktails could be devised. Some as the extent,

(30

for

between

the
nM

binding
U50,488

of pM [3H]BREM 75
matched

in the
of and

absence
tubes at

such
not every

ligands
know

are
how

protected
general this

while

occupying
phenomenon is;

a binding
therefore,

site
some

(9)

but

we

do

presence

peptide

concentration of a competing aM DAGO and 50 nM DPDPE the paired-tube differences

without the blockers.
sites) was

ligand.
were

In some
present to

of these
block

experiments,
#{244} sites,

competing 5
but

ligands

may

have

higher

affinities

than

we estimate.

proved the
35%

to be residual
of total

virtually
binding

the
was

same
plotted

with

and
against

Results
than sites,
erroneous

When

[3H]BREM

log concentration to other than Components

of U50,488,
K

binding
binding.

at to

the

plateau

about

(i.e., If more secondary

mary sites,

than a few especially

K,

percent of if secondary
will be

radioligand sites for

is bound outnumber competing

to pri-

were

added

in the

following

order

a polypropylene

values

computed

Opioid TABLE Binding

For
C 0
0

Receptor

LSPs

and BSSs

267

1 isotherm
see wet

results
Experimental

in the #{244}, s, and

Procedures.

systems
independent experiments

details original

n denotes

performed
of

on different
weight

days and with different

of brain tissue. Data

brains.B,,,
have been

is expressed in terms rounded to two significant

figures.
U Q) 0. 0

abed radioligand

Buffer

Kd

SE

B,,.
ptnol/g

SE

flM
0

E
0.

DAGO High Low

Free 4.00 conc. (nM)

affinity affinity

DPDPE BREM

TB KHB TB KHB TB KHB TB KHB

of this

3 3 3 3 7 3 6 3
estimate,

0.14 0.24 1 .3 270. 2.0 1 .5 0.020 0.020

based on multiple

0.01 0.04 0.1 74. 0.1 0.1 0.002 0.003

points

1.6 0.82 2.0 12. 5.0 1 .1 3.9 2.8

near

0.2 0.10 0.2 5. 0.3 0.1 0.4 0.4

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

0
C

#{176}

Note

uncertainty
100 80 60

saturation.

3.00

0 0 .0 U 5) 0. U,

/
0 I

2.00

I)
0

40 20 0 100

E
0.

1 .00

0.00 0.0

-Ic
-

cfr
at)5,, %

[3H]DPDPE

5.0

10.0

15.0

2C .0

5,

80 60 40 20 0 100
.

ISYSTEM

Free

conc

(nM)

C,

z
0

Z\

z 0.015
C,

0
0

U. C.) Lu

5.

0
5) 5) U-

U)

0.010

0 0

_1;l,b,.
80 60
-

(3H]BREM 5\%#{149}
K

SYSTEM

0 0

0.005

0 0 0
0

40 20 !! -10 -9 -8 LOG -7 CONCENTRATION -6 (M) U5488H -5 -4 -3

0.000 0.0

Qn
1.0 2.0 30 4.0

Bound

(pmol/g)

Fig. 1. [3H]DAGO note wide range Bottom, Scatchard

binding. Top and middle, Saturation of concentrations tested and

transformation of all the data.

TB is shown;

data from several

Scatchard in equal

experiments
plot is no numbers

are
proof and

Fig. 2. Homologous and and K systems. The buffer details of the assays. Open binding isotherms; independent experiments different x-axis scales. lines are log-logit slopes A typical experiment in summarized in Table 1. interaction. of site differing

heterologous competition curves in , the was TB. See Experimental Procedures for and filled symbols on each graph represent carried out at least 1 month apart. Straight of unity, corresponding to simple mass-law

ligands. A linear two sites present

homogeneity; binding by some small amount, by 5-fold followed in by reduction of the Kd yield a straight line with linear regression coefficient >0.99. higher concentrations. Absence Nor is a smooth, symmetrical, self-competition curve with logerologous competition curves logit slope of unity any better proof; self-competition is very a radioligand. insensitive to site heterogeneity. The most conclusive way to prove single homogeneous site population ligands
sites yield Fig. 2 shows typical smooth

to produce a distinct plateau, remaining bound radioligand

of such early plateaus in het-

at of

indicates
competition

adequate
curves

selectivity
obtained in

that is for
with

a radioligand to demonstrate
appropriate

labels TB a at that Procedures.

the

three Similar have

sites results

labeled were for the of

as

described

in in KHB

Experimental (not shown). the selfcurves

obtained

that appreciable site, even an

are
smooth

highly fraction though that the

selective
curves

nonpreferred is bound curve is secondary

competition

(secondary) Here we slopes. competition at smooth, a secondwith a greater ligands for

depicted, curve and the affinities own

each radioligand, both heterologous competition which sites of than such are for curves, orders the we of labeled found

If an ary

of

a radioligand self-competition

magnitude primary IC50

their

preferred

competing produce

ligand irregular

is curve.

selective At low

for the concentrations

site site. will it will reduce From

log-logit

transformations

268
values as site. ciation

Goldstein by linear

and

Naidu analysis, is described each secondary increments and by site we the to in computed values I( ratio that at of the the for S or K. OMF is a novel fentanyl dissosuperior to [3H]DAGO as a ti-selective affinity and selectivity, as compared primary (7) and site derivative (10); radioligand with morphine, it could and its might be high well

regression Selectivity at concentration

described. constant Because

a competition

are geometric, linear difference Figs. tivities the of and topmost for than LSP that known that was contained effect of order the three U50,488, LSP for a primary 3-5 of show which

selectivity is best on a logarithmic LSPs are uganda 36) 3A, more is Figs. for evident affinity (DAGO, can than be 4, and 33 at

displayed in a LSP scale of K values. the z site. 12; by of added respectively; site. salts The and well affinities Code The DPDPE, inspection each magnitude numbers a glance.

ligands, for the Code assessed SB, 2 orders secondary without

make it a superior analgesic agent. The higher z selectivity of curve morphiceptin than of morphine shows that selectivity and as a affinity can vary independently. fib-END is clearly selective for IL comparison of LEV and DEX shows that selec- over t5 and K. The for the (-)over the (+)-enanapplies sites. (unpublished has amino reagent interesting (in much to fi3-NTA) to The orders of three types to of of and traces naltrexone keto to without magnitude, of binding of LEV

ofdecreasing key Code in site the determined salts salts

the stereoselective preference reflect tiomer, to the extent of several selectivity the same degree to all Code about 39; with of results the data). affinity Substitution greater effects (Fig. in DEX on 3B). are the not due

6-position of

next-preferred in TB at on K physiologic

Conversion iodinated

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

concentrations.

causes a dramatic loss KHB in K affinity. Coupling

6 affinity Bolton-Hunter

change

NTA (fi-NTA-12 BH) causes a selective loss K affinity. of Finally, coupling with fluorescein (fl-NTA-F) causes differenaverage 2-fold increase but is sometimes negligible or absent tial changes of affinity that result in a t5-selective ligand. and is occasionally very large. Especially interesting is the large In Fig. 4, we show LSPs for three #{244} ligands; data for many shift with CTOP, in view of the fact that this compound is a others, published elsewhere (16), are not repeated here. The receptor antagonist (28); it would be interesting to study the LSP of DADLE shows very clearly that this compound, aleffect of sodium alone on CTOP in this regard, to test whether though technically #{244} selective, barely distinguishes #{246} from . the sodium shift necessarily distinguishes generically between DSLET is a modest improvement, but it is the cyclic structure agonists and antagonists (25, 26). Also interesting is the effect DPDPE of that evidently stabilizes an optimal conformation of salts on DYN A-(1-9), changing its selectivity Kfrom s. to for #{244} sites. Fig. 3 shows LSPs for several pt-selective ligands. The rela- Fig. 5A is consistent with our previous findings (29, 30) on tively poor selectivity of morphine for sites is evident here, how the K selectivity of DYN A depends primarily on residues

(25-27)

is seen

here

as

about

an

as that, due

compared in to this increased

with series,

DAGO, higher affinity

i,at

SUF, selectivity without

ti
)

and much

OMF. appears change

It to

is

noteworthy 11 and primarily fragments in affinity ported be

13. (29),

DYN are

A-(1-9) actually

is ic-selective selective.

only DYN

in TB, and smaller B, as previously re-

isK selective.

12.

DAGO

1-

2.

SUF

))-

1.

OMF

4.

CTOP
t

K&

1 0.

Oxymorphone

19.

MorphIne

_____________ S
K

1 5.

Naloxone
L-K-5

30.

Morphiceptln
It

I)

K5
K

5.

Naltrexone

IL-K-S 1LK-S

9.

h.END

I-

20.

t-NTA
I
K-

S
-S

6.

LEV

(-)

IL

K-S

17.

t-NTA-l2

BH

38.

DEX

(+)

ILKS

28.

-NTA-F
5IL-K

5IL

A
Fig. 3. Ligand
values

t
-ii

I -

I
-

-7 LOGK1

-6

-5

-4

-11

-10

-9

-8 LOGKI

-7

-6

-5

-4

ligands. For each ligand, the logarithms of the dissociation constants, were computed K,, from log of a log-logit transformation of the competition curves. Greek letters, and K, are positioned , o, at log (K) for each binding site, with highest affinities at the Assays left. were carried out in TB without mineral salts (upper of pair, solid line)and in KHB containing salts at physiologic concentrations (lower ofpair, broken line). Data pointsare means of the values fromat least two or three independent competition experiments. The SD of such independent estimates of IC in our hands was 0.1 log units. In each experiment, the competition curve was generated by concentration doublings of the competing ligand, with triplicate incubation tubes at each concentration. selectMty Ugand is represented here by the horizontal distancebetween the preferred (highest affinity) site and the next-preferred site. See Experimental Procedures for details of the assay systems. This legend applies also to Figs. 4 and 5. Numerical correspond codes to the order of affinities for sites; these same code numbers areused to represent the ligands in Table 2.
(IC50) by

selectivity profiles linear regression

of hz-selective

analysis

Oploid
39. DPDPE
S 5IL (K)

Receptor coefficient

LSPs

and BSSs

269
For Thus, than
K

(Fig. against 0.05), the

L

6A), and,

the

correlation 6B), the

K

is 0.47 < (p 0.01). statistic it is 0.54 than from

i

IL)K)

t5 (Fig.

corresponding (Fig. 6C),

is more

0.19 > (p

18.

DADLE

SIL 5IL

K K

for L against

(p 0.01). < and

25.

DSLET

SIL

K K

S signature and K differ

differs fromK more from each other.

SIL

I
-11

Discussion
-5 -4

-10

-9

-8

-7

-6

LOG K1

The For additional ciation #{246}according radioligands

graphic

LSP

offers

the

following IC

advantages. of visual

(a)

Dissovary an The same affinexperIC (23).

Fig. 4. Ligand selectivity profiles of #{244}-selective ligands. selective ligands, see Ref. 1 6. See also Fig. 3 legend.

constants are to affinities used. and scale the The decimal error from which (b) readily means same

compared, not and concentrations The that, horizontal constants, horizontal units) 25%) in for essential interpretable as distance regardless

values, which the particular makes impact. for represents of the that roughly absolute is to derived the the the (c) comparisons

information

instantaneous Fig. and affinity U63,640 affinity, selective, without SB U69,S93 but and highly major presents and poorer U63,639 selective, by change rank uniquely the BSS includes the of LSP BREM selectivity
K

of

the and
K for

highly EKC, sites. The

K-selective which have enantiomers

US0,488 logarithmic higher selectivity, of ratio ities. high (about imental values (d) 0.1

is appropriate

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

of dissociation smallest

are extremely ligand becomes of or a #{244} affinity.

interesting-, here, a a high affinity, highly loss of

K

distance in constants selectivity in affinity the determining are

discernible

log

corresponds

IL ligand

virtue in

10,000-fold affinities that type the

affinity of of by

(approximately dissociation

(e)

The quantitative at a binding site Table study ligands mental There sites, site ers analogues few and ofthe of the orders Fig. When of
K

order of defines

a set of site

ligands comparing In a BSS. two the possible

ligands that differ causes, difference in we ligands that affinity by eye.

for a given site, for the preferred site, confirmasufficient orders

2 presents here. Each

patterns of the set -log(K,) of by order numerical

three values codes as among

site and sites under for 47 instantly in the Experi- Concerning tion that three selectivity,

difference distinguished LSPs, certain namely,

next-preferred quantitative requirement

are

that are designated Procedures. is a consensus rank by high distinguish DPLPE their
t5

have provided meet the there be for at

of 2 or for 3

of affinities have The are DPDPE very of poor the

least

of

disrupted but very that

a few affmity the (Code more

ligands for S 34) which than

that another. sites and have 2 orders

low affinity consensus cyclic (Code 39)

i

for magnitude one difference break- the to next-preferred enkephalin ohmefentanyl and DPLPE a for suitable the but, top unlike noted are at
K

in affinity site. DAGO and for latter it is now CTOP

the preferred is acceptable is better US0,488 yet. and

site

relative sites but

is better, acceptable sites; U50,488, in low that the

DPDPE U69,453 selective, radioliuncertainty is derived

and

S sites. is not available Procedures,

are

derivatives, catapult

affinities to at the affinity at pairs log(K,)

significantly as

more a 3H-labeled there estimate is

but

magnitude

BSS. Similarly, list at both i and

U50,488 S sites

and U69,593 are but have greater plots for #{244} plotted is affinities against

bottom gand. by 3 As

Experimental Kd. Our

of magnitude 6 presents each log(K,)

at sites. K correlation value at

about the of sites. from very i at centration

[3H]DAGO

high

affinity began with binding

concentrations. We yielded measurable

the lowest (9 pM),

conwith con-

21.

DYNA

KIL-5 K--IL--S

26.

DYN A-(1-13)

8.

DYN A-(1-13)-NH

KIL-S K--IL---5

14.

[N-Me-TyrJDYN (1-13)-NH2

A-

KIL-5

K---IL--5

36.

u50,488
K

IL

IL

S -S

37.

DYN A-(1-9)

KIL-5
IL KS

7.

BREM

KIL

47.

DYNA-(1_)

ILKS
(LK-5

11.

EKC

KIL5 K

Fig. 5. Ugand selectivity profiles ofKselective ligands. For additional K-selective ligands, see Ref. 9. See also Fig.
IL5

legend.
K K IL5 IL

44.

DYN

A-(1-7)
IL

46.

U69593

40.

DYNA-(1-.a)

IL

..... K

27.

u63,640

(-)

K)I

IL-

_S_

ILS-K

35.

DYNB

KIL-5 K--IL5

22.

u63,639

(+)

I IL-

K5 -

KS

) -10

-11

-9

-8

-7

-6-5-4

LOG K1

-11

-10

-9

-8 LOG

-7 K1

-6

-5 -4

TABLE Binding

centration

doublings

spanning

a very

large

range

(to

19

nM).

Site Signatures Most data in the literature (e.g. Refs. 14 and 31) cover only the The same ligands depicted in Figs. 3-5 and others from Refs. 9 and1 6 are upper range and consequently reflect only the low affinity site, arranged here vertically, by numerical code,according to -log(K) in TB at the three 1 nM. For competition curves, we used types of binding sites, with highest affinity at the top. Numerical codes and other with Kd approximately information about the ligands in Experimental Procedures. Parentheses indicate 0.8 nM [3H]DAGO, 5.7 times the ofthe K,, high affinity site but that the true value of K was greater, -log(K,) numerically smaller, than could be only 0.62 times the Kd of the low affinity site. For the relative measured. For structures and binding data of derivatives of the cyclic enkephalin site densities given in Table 1, computation shows that 64% of analogues DPDPE and DPLPE, see Ref. 16. For structures and binding data for the DYN A analogue DAKLI series, see Ref. 9. the label is in high affinity sites, the properties of which should
-K)
,

)5

chiefly
K,

govern based in Figs. same be affinity

the on 3-5 2 all amount. that state in our guanyl

competition the would entries the high rather membrane nucleotides steps. suggested
however,

results. affinity be under affinity than

Kd,

If conversions

of IC50 at to the

to

11.1 11.0 10.9 10.8 10.7 10.6 10.5 10.4 10.3 10.2 10.1 10.0 9.9 9.8 9.7

were

low

all

-log(K,)
0.97 column log

values units would site subtype because the that but extensive it

sites and 2 3,8 13, 14 7, 16 11 21 3 4,5,6,7 8 9, 10, 11 12, 13 14 15, 16 34 18 25 39 27 26 by It high the

moved the

right down a that washit

in Table may

move represents and

[3H]DAGO a distinct preparation (32) during data,

our

predominates ofregulatory ing to

rigorously

of removal it corresponds cannot

the site(s)

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

and

preincubation (33) by the set is

(5,

The by

possibility our
under

is

not

be

excluded;

conditions

labeled 23 by atives
assays

[3H]DAGO profiles of of
29,

show(s) in Figs. and in

showing

classical z behavior, 3-5. otherwise

that selectivity K

as evidenced DYN of seen is The in DYN critical A denyA, role selective fragments
are

9.6 9.5
9.4

all The

truncated interest
30),

modified results

9.3
9.2 9.1

confirming A-(1-13)-NH2 beyond

pharmacologic

DYN 5, 36 46 20 on lysine-il ligand,

such

A-(1-13), C terminal

and truncation

DYN with
which

progressively of a novel

lost

9.0 8.9 8.8

8.7 8.6 8.5 8.4 8.3

17, 18 19

lysine-li. effectiveness (34),

have also

of

20
21,22 23,24 25, 26, 27

7,9
3 14 8, 29 11

is consistent [D-Pr&#{176}]DYN
as DYN A-(1-8),

the

A-(1-11) that

but
been

shorter
proposed,

not

suitable. enkephalin-Arg6, is tially sites, absolute with 1,2,17

affinities

It

is

interesting is 6) and degree LSPs

the

fragment

1-6,

i.e.,

[Leul

selective,
DEX of are However, (Code
greatly

whereas
(Code virtually a quite and
different

[Leu]enkephalin
38) demonstrate applies the same (+)
i5, but

itself
that essenfor is 22); of of the among receptors in defining in biologic a bioassays for (3). amino terminal aromatic to it each strong is N that the

8.2
8.1 8.0 7.9 7.8
7.7

16
5, 13, 23 1,2,28

6, 15
35

selective. LEV (Code the i.e., U63,640

are

same the affinities.

(-)

stereoselectivity

at

all except pattern

three seen

6, 21, 28,29 30 31 32 41 15,42 10 31,32 33 17,26

24

the

different the times

7.6
7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7

27)
K

U63,639 and at than 10,000 and as qua

(Code affinity

not

the 9 10, 37 19 28

(-)-enantiomer BSSs shed light binding naloxone indeed, as be 3B, and opioid (in Another by Finally, in
that surprising
K

at

is more the

(+)-enantiomer. on and such opioid. naloxone greater peptides at a certain common two all hydrophobic the their
there

similarities The naltrexone is sine the antagonists, of binding a preference for over z that the from rings opioid a-amino multiple

differences opioid

19, 20, 43 33,34 35,36 37 38, 39 40, 41 42,43

the assays;

opioid

sites. blockade With reflective shows preference require usually distance

all

recognize receptor

antagonists non for potencies affinities. of about bioassays have group or a display ligands.
quantitative rank

6.6
6.5 6.4 6.3

12 47 45

should in Fig. the over All group tyrosine) ring. produced

directly

Accordingly, 10-fold a basic of other T angle very Thus,

order

6.2
6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 44,45 46 47 40 27, 4 47 46 22 36 35, 37, 44

44
12 22 4

#{244}, in as a ligand a phenolic is at with for

receptors

requirement receptors

conformation

a certain

18 38 (25),40 45

other.
not

stereoselectivity of ligand affinities

interactions
is a consensus
t5,

5.2
5.1 5.0 4.9

across
that

the j.s, been

and

binding
distinguish

sites.
each

It is the
site. No

consensus

breakers

uniquely

4.8
4.7 4.6
(38), (30)

(39)
24, 30, 33 29, 31, 32, (34), 41, 42, 43 270

opioid

receptor

has

yet

cloned,

so

the

molecular splicing, of novel

basis or postopioid

of

the differences, different genes, differential translational modification, is unknown. Claims have been advanced for the existence

Opioid 1 .0

Receptor

LSPs

and BSSs

271

B
100T
)

C
Cb
0
,

0.0

,c
-)

0
0

9.0

t
-

90+

.u
0

8.0 7.0 6.C

n-___
Q]
t
)

,
::t
0C
O
fl
-0g

..

5. 0 4.0

0 0

C)

5.0k
4
.

C!
4.D

-__-_----_ . . ..:

-.-----t

4
I

4.0

5.0

6.0
-H04

70
K

8.0 ot
mo
St5

9.0

fl

fl,

1 1.0

-o

Ct

CeOG

st

r,

k.cppo

Ctes

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

Fig. 6. Correlation highest affinities B, K versus tS; C,

plots for 47 ligands at pairs of sites. Each represents -109(K) point at a site on the y-axis and another are to the right and top of each plot. Least squares regression lines are shown; see text for correlation IL versus K.

site on the x-axis. Here coefficients. A, 5 versus

receptor conclusive ligand BSS selective

subtypes. proof, to binding, in be demonstrated. for

Until our

cloning opinion, Some (35) (36) sites,

rest

and

sequence site

analysis unequivocally

provide 5. Chavkin,
ligand by 6. James,

demonstrate

a novel

C., I. F. James, and A. Goldstein. Dynorphin is a specific endogenous of the s opioid receptor. Science ( Wash. D. C.) 15:413-415 2 (1982). I. F., and A. Goldstein. Site-directed alkylation of multiple opioid
I. Binding A. Binding Biology selectivity. 25:337-342 (1984). profiles for ligands of multiple receptors. Trends Pharmacol. Sd. 8:456-459 (1987). and chemistry of the dynorphin peptides, The selectivity Mol. Pharmacol. receptor in

requires that a uniquely such evidence for e sites and for has such A sites been z as(33)
on

that With

novel receptors. 7.areGoldstein,

types:
Goldstein, Peptides

$-endorphin epoxymorphinans other putative

arguments

that

are

selective 8.

focus

on opioid
A.

for certain respect to

(37-40), the

adduced. or K subtypes
poorly selective

primarily

(J. Meienhofer and S. Udenfriend, eds.), Vol. 6. Academic Press, New York, 95-145 (1984). 9. Goldstein, A., J. J. Nestor, Jr., A. Naidu, and S. R. Newman. A multipurpose radi- ligand with high affinity and selectivity for dynorphin(kappa opioid) binding
(1988).

oligands approach due to

sites. We

used with selective blockers leaves open the possibility of radioligand binding at unknown
believe that site characterization

of known sites. That sites. Proc. NatL Acad. Sci. USA 85:7375-7379 and Q. Chi. Ohmefentanyl: Z. heterogeneous labeling10. Xu, H., J. Chen, receptor. Sci. Sin. 28:504-511 (198.5). and poorly blocked Pelton, 11. J. T., W. Kazmierski, K. Gulya,
should be based on

a new

agonist

for

si-opiate
Hruby.

ligands ized. The topography is obvious.

that theoretical

are

highly value

selective of gaining

for

the

sites

being

character12.

Design logues

and with

synthesis high potency

(1986).

of

conformationally and specificity

H. I. Yamamura, and V. J. constrained somatostatin anafor opioid receptors. Med. J. Chem.

better

understanding

of

29:2370-2375 Romer, D., the W. Benson,

activity Gillan,

H. H. Buscher, E. Finner, W.
class and

R. C. Hill, Milkowski,

R. Maurer, and P. W.

T. J. Petcher, H. Thies. Unexpected

(1982). mu-, the deltaof 77:461J. Rance,

Zeugner, opioid

of related There are

receptor types, also practical

as through LSP and values. Autoradiography or for a very as

BSS,
13.

in a known M. G. C.,

of drug. Life Sci. 31:1217-1220 H. W. Kosterlitz. Spectrum

and

positron emission tomography izing specific receptor types degree high these of selectivity densities purposes, of for secondary it would the seem

scanning techniques place a premium on desired sites site, inasmuch may essential

kappa-binding local- 468 (1982).

14.

sites
B. K., A. C.

in homogenates
Lane, J. A. H.

of rat
Lord,

brain. Br.
B. A.

J. Pharmacol.
Morgan, M.

high

Hands,

and at

C.

localized

F. C. Smith. Analogues IL-opiate receptors. Eur.

P. F.,

of 9-LPH61.,

possessing

selective

agonist activity

otherwise mislead. to determine LSP the selectivity therapeutic for the target. To

15. Vonvoigtlander, For

forof spiradoline,
(1988).

J. Pharrnacol. 70:531-540 and R. A. Lewis. Analgesic a potent kappa opioid. J. PharmacoL

(1981). and mechanistic evaluation Exp. Ther. 246:259-262

each new candidate of a new drug should type useful tors, binding or subtype that new drugs site-selective selectivity,

ligand. Furthermore, be related to its will be its

value Mosberg, 16. )5 opioid receptor

17. develop Roques, Maigret,

H. I., J. R. Omnaas, receptor binding. Mol.

B. P., and J. mu and

and
PharmacoL

A. Goldstein. 31:599-602

Structural
(1987). J.

requirements
C. Meunier, properties

for
B. of highly

therapeutic

that distinguish binding assays such as described

between closely and quantitative here, will be

related recep- specific measures of33:321-331 essential.

18. Chang, analogs: Pharmacol.

M. C. Fournie-Zaluski, G. Gacel, L. Morgat. Rational design delta opioid peptides. Ado. Wei, A. Killian, relationships and and

M. David, and biological

Biochem.
J. K. Chang. morphine-like

Psychopharmcol.
Potent activities. morphiceptin

(1982). K. J., E. T. structure-activity Exp.

J. a selective
Exp. J. J. Galligan,

Ther. 227:403-408

19. Acknowledgments We
mental

Vonvoigtlander,
and structurally Ther. 224:7-12 Mosberg, H. and
specificity

P.
(1983).

F.,

(1983). R. A. Lahti, and

non-mu (kappa) V. J. Hruby, opioid K.

J.

H.

Ludens.
J.

U-50,488:
Pharmacol.

novel I., Burks. R. Hurst,

agonist. Gee, H.

thank

the generous

donors

of

ligands,

whose

names

are

listed

in

Experi- 20.

I. Yamamura,

Procedures.

T.
(1983).

F.

toward

Bis-penicillamine enkephalins delta opioid receptors. Proc. NatL

possess Acad.

highly improved Sci. USA 80:5871-

5874

References 1. Ahlquist,
153:586-600

21. R. P.
(1948).

study used

of

the

adrenotropic study Hughes, of opioid and H.

receptors.

J. Am. PhysioL
Rev.

2. 3.

Leslie,
39:197-249

F. M. Methods
(1987).

for the J.

receptors. PharmacoL W. Kosterlitz.

(Lond.)

22.

Lahti, R. A., M. M. Mickelson, J. M. McCall, and Vonvoigtlander. P. F. [3H] U-69593: a highly selective ligand for the opioid receptor. s Eur. J. PharmacoL 109:281-284 (1985). Munson, P. J., and D. Rodbard. LIGAND: a versatile computerized approach for characterization of ligand-binding systems.AnaL Biochem. 107:220-239
(1980).

Lord,

J. A., A. A. Waterfield,

4.

opioid peptides: multiple agonists and receptors.Nature 499 (1977). James, I. F., C. Chavkin, and A. Goldstein. Preparation containing a single type of opioid receptor highly Proc. NO.tL Acad. Sci. USA 79:7570-7574 (1982).

Endogenous 23. 267:495membranes dynorphin. 24.

Goldstein,
petition macoL Cheng, constant

A.,

and

R.

W.

Barrett.

Ligand
associated W. H.

dissociation
with Relationship ligand

constants
depletion. MoL between

from
Pharthe

com-

selective

of brain for

binding 31:603-609 Y. C.,

assays: errors (1987). and Prusoff,

inhibition

(K,)

and

the

concentration

of inhibitor

which

causes

50

per

cent

272
inhibition
(1973).

Goldstein

and Naidu
reaction. Biochem. S H. Snyder.
binding in PharmacoL

(ILu) of an enzymatic
Pasternak, by receptor

22:3099-3102 and
D.

guinea pig 33:423-431

cortical,
(1988).

NG1O8-15,

and

7315c

cell

membranes. MoL

Pharmo.coL and pharmaco-

25. Pert, C. B., G.

discriminated 1361 (1973).

and

Opiate

agonists
( Wash.

antagonists

33.
34.

brain. Science

C.) 82:13591

Pasternak, G. W. Multiple mu logical evidence for multiplicity.

Gairin, J. E., C. Gouarderes,

opiate

receptors:

biochemical
35:361-364 P. Alvinerie, selective

J. M. Hiller, J. Groth, and I. Edelman. Further properties ofProl0jDynorphin-(1-11) is a stereospecific opiate binding sites in rat brain: on the nature of the sodium opioid receptors. Eur. J. Pharmo.coL 106:457-458 (1984). effect. J. PharmacoL Exp. Ther. 192:531-537 (1975). 35. Toogood, C. I. A., K. G. McFarthing, E. C. Hulme, and D. G. Smyth. Use 27. Paterson, S. J., L. E. Robson, and H. Kosterlitz. W. Control by cations of 25I-Tyr7 fl-endorphin for the study offl-endorphin binding sites in rat cortex. opioid binding in guinea pig brain membranes. Proc. NatL Aced. Sci. USA Neuroendocrinology 43:629-634 (1986). 36. Grevel, J., V. Yu, and W. Sadee. Characterization ofa labile naloxone binding 83:6216-6220 (1986). 28. Shook, J. E., J. T. Pelton, P. K. Lemcke, F. Porreca, V. J. Hruby, and T. F. site (lambda site) in rat brain. J. Neurochem. 44:1647-1656 (1985). Burks. Mu opioid antagonist properties of a cyclic somatostatin octapeptide 37. Attali, B., C. Gouarderes, H. Mazarguil, Y. Audigier, and J. Cros. Evidence in viva: identification ofmu receptor-related functions. J. PharmacoL Exp. for multiple kappa binding sites by use of opioid peptides in the guinea-pig Ther. 242:1-7 (1987). lumbo-sacral spinal cord. Neuropeptides 3:53-64 (1982). 29. James, I. F., W. Fischli, and A. Goldstein. Opioid receptor selectivity 38.of Nock, B., A. Rajpara, L. H. OConnor, and T. J. Cicero. Autoradiography of
dynorphin gene products. J. PharmacoL Exp. Ther. 228:88-93
(1984).

26.

Simon,

E. J.,

Biochem. Pharmo.coL H. Mazarguil, highly potent and

(1986). and J. Cmx. [Dligand kappa for

of

30. Chavkin,

C.,

and

A.

Goldstein.

Specific

31.

dynorphin: structure-activity relationships. 78:6543-6547 (1981). Borea, P. A., G. M. Bertelli, and G. binding of1, 5 and sc agonists to the opiate J. PharmacoL 146:247-252 (1988).

receptor Proc. NatL

Temperature

for the Acad.

opioid peptide Sci. USA 39.

dependence

Gilli.

receptors

in guinea-pig

brain. Eur.

[3HJU-69593 binding sites in rat brain: evidence x opioid for receptor subtypes. Eur. J. PharmacoL 154:27-34 (1988). Zukin, R. S., M. Eghbali, D. Olive, E. M. Unterwald, and A. Tempel. Characterization and visualization of rat and guinea pig brainopioid K evidence forK1 and K2 opioid receptors. Proc. NatL Acad. Sci. USA of thereceptors: 85:4061-4065 (1988). 40. Clark, C. R, B. Birchmore, N. A. Sharif, J. C. Hunter, R and Hill, G. J.

Downloaded from molpharm.aspetjournals.org at MISSOURI UNIVERSITY COLUMBIA on June 17, 2011

32.

Werling, L. L., states of opioid

P. S. Puttfarcken, receptors: regulation

and B. M. of binding

Cox. Multiple agonist-affinity by guanyl nucleotides in

Hughes.
PharmacoL

PD117302:
93:618-626

selective
(1988).

agonist

for

the

e-opioid

receptor. Br.

J.