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A. Mayasundari et al. / Bioorg. Med. Chem. Lett. 18 (2008) 942–945
Table 1. IC50 (lM) of the binding of NHERF1 PDZ domains to their
cognate ligands measured by AlphaScreen assay
NHERF1 PDZ2 domain (Table 1). As expected, the
PDZ2 domain showed less significant preference for
compounds with the carboxylic acid at the indole-2-po-
sition (2, 4, 6).
Compound
GST-PDZ1: b2ARa
GST-PDZ2: CFTRb
1
2
3
4
5
6
1540
777
NAc
820
280
15
160
330
NAc
490
380
170
In summary, we have created the first non-peptide
small-molecule antagonist of NHERF1 PDZ domain
interactions by incorporating an extra carboxylic acid
on the 2-position of the indole carboxylate scaffolds that
we had utilized to design antagonists for MAGI3
PDZ216,17 and Dishevelled PDZ19 domains. Among
the three indoles, the indole-3-carbinol scaffold offered
the most potent antagonism. These results suggest that
incorporation of functional groups to mimic native li-
gands for each PDZ domain is a promising strategy to
discover new antagonists of these domains with in-
creased potency and selectivity. This method also offers
new small-molecule tools to investigate the pharmaco-
logical function of these PDZ domains. Pharmacologi-
cal studies using these compounds are under way and
will be reported in due course.
a Biotin-SQGRNCSTNDSLL.
b Biotin-KEETEEEVQDTRL.
c NA, No competition activity was observed up to 300 lM.
indole-2-carbinol and -2-amide scaffolds may be due to
the position of the hydroxyl group that mimics the
S(À2) not being enough appropriately positioned (1–
2), or replacement of the hydroxyl group with an amide
(3–4). Actually, distance of hydrogen bonds observed in
between His27/ Arg40/ His72 of the PDZ1 and carbox-
˚
ylate/ hydroxyl groups of 2 (3.19, 3.32, 3.52 A, respec-
tively; Fig. 2B) is slightly longer than that of the
13
˚
NDSLL peptide (2.71, 2.86, 2.52 A, respectively ). On
the other hand, indole-3-carbinol (5–6) showed not only
the greatest potency but also the greatest comparative
improvement (Fig. 4). This may be due to the proper
orientation of the 3-carbinol group to make better
replacement of the hydroxyl group of S(À2), that forms
essential interaction of the NDSLL peptide to the PDZ1
domain, in irreversible manner.17
Acknowledgments
We thank Randy A. Hall (Emory University) for the
generous gift of NHERF1 plasmid; St. Jude Protein
Production Facility for preparation of GST-NHERF1
PDZ domain proteins; St. Jude Hartwell Center for pep-
tide synthesis; Michele Connelly and Chandanamali
Punchihewa for excellent technical assistance; and Shar-
on Naron for editing of the manuscript. Financial sup-
port was provided by the American Lebanese Syrian
Associated Charities (ALSAC).
The NHERF1 PDZ2 binds to CFTR10 and b-catenin,3
which possesses the D-T-X-L motif, but also binds to
ligands that have amino acid residues other than
D(-3), such as parathyroid hormone receptor (PTH1R,
-E-T-V-M)21 and Yes-associated protein (YAP)-65
(-L-T-W-L).22 Therefore, we anticipated that the bind-
ing preference of the PDZ2 domain would be less spe-
cific to ligand possessing the carboxylic acid corre-
sponding to D(-3). We used a similar AlphaScreen
protocol to assay competition against the interaction
of a biotinylated peptide derived from the carboxy-ter-
minal sequence of CFTR and GST-fused human
Supplementary data
DOCK modeling method, chemical syntheses proce-
dure, analysis data, protein preparation, and protocols
for AlphaScreen biochemical assays associated with this
article can be found in the online version. Supplemen-
tary data associated with this article can be found, in
References and notes
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Figure 4. The effects of the compounds’ carboxylic acid moiety of the
indole-3-carbinol compound on the potency of their biochemical
antagonism. The GST-fused NHERF1 PDZ1 domain and biotin-
SQGRNCSTNDSLL peptide (b2AR carboxy-terminal sequence) were
allowed to equilibrate with the test compounds and were then
incubated for 30 and 45 min with anti-GST and streptavidin beads,
respectively, to titrate inhibition of PDZ1 domain ligand binding as
measured by AlphaScreen.