Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 2971
Ta ble 2. Binding of Bivalent and Monovalent Ligands to δ and
κ Opioid Receptors Expressed in HEK Cells
are more consistent with in vivo data when compared
to singly expressed receptors.
Ki (nM)a
Ack n ow led gm en t. We thank Mary Lunzer for
capable technical assistance. This research was sup-
ported by Grants DA01533 and DA 015091 from the
National Institute on Drug Abuse.
compd
compd name
coexpressed δ-κb
mixed δ + κc
3
4
5
6
7
8
9
KDN-15
KDN-20
KDN-21
KDN-22
KDN-23
KN-21
315 ( 15
0.5 ( 0.1
0.3 ( 0.0
1.4 ( 0.3
4.8 ( 3.2
>1000
6.3 ( 2.9
58.8 ( 13.7
63 ( 6.3
1.1 ( 0.2
3.3 ( 2.4
54.8 ( 15.3
199 ( 90
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails including synthetic methods and physical data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
DN-21
>1000
a
Binding was conducted on intact HEK cells using [3H]di-
prenorphine as radioligand. b Coexpressed δ and κ opioid receptors
in HEK cells. c Mixed HEK cells containing singly expressed δ or
κ receptors.
Refer en ces
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distance for bridging. The very low binding affinity of
monovalent ligands, KN-21 (8) and DN-21 (9), for the
coexpressed cell lines is consistent with this conclusion.
These results support the i.t. studies that suggested that
KDN-21 bridges δ-κ opioid receptor heterodimers. We
were unable to adequately compare the antagonist
selectivity of i.t.-administered KDN-20 with that of
KDN-21 because the partial agonist activity associated
with the former precluded testing at equal doses.
Consequently, the correlation between the i.t. antago-
nist selectivity and binding for KDN-20 is not as robust
as for KDN-21.
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In conclusion, KDN-21 (5) is a member of a series of
bivalent ligands that contains δ and κ opioid antagonist
pharmacophores attached to variable-length spacers. In
contrast to other members of the series, i.t.-adminis-
tered KDN-21 exhibited selective δ1 and κ2 opioid
antagonist activity and was devoid of antinociceptive
activity. Considering the previously reported5 allosteric
property of spinal δ-κ heterodimeric opioid receptors
that possess the δ1 phenotype, we conclude that δ and
κ opioid receptor recognition sites in the heterodimer
are bound in the antagonist state by a single KDN-21
molecule. The substantially greater affinity of KDN-21
for coexpressed δ and κ receptors in cultured cells, when
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bridging of one KDN-21 molecule to δ and κ recognition
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dimeric opioid receptor by KDN-21, which contains a
21-atom spacer, suggests that bivalent ligands can
serve as molecular rulers to obtain insight on the
distance between recognition sites for the identifi-
cation of possible monomer interfaces involved in GPCR
dimerization. Moreover, the present study exemplifies
the power of the bivalent ligand approach for the
development of pharmacological tools to investigate
the association of different GPCRs in an oligomeric
array. Significantly, the different i.t. and icv antagonist
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G-protein-coupled receptors as an approach for targeting
different tissues (e.g., spinal cord vs brain). Finally, the
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and κ opioid receptors has broad implications in regard
to screening paradigms of GPCRs because the pharma-
cological properties and signal transduction pathways
may be altered by such association. In such cases,
screening of coexpressed receptors may give results that
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