Arylamide derivatives as peptidomimetic inhibitors of calmodulin

Article abstract of DOI:10.1021/ol052478j

Many peptides bind to calmodulin (CaM) in a helical conformation. Here we describe a group of synthetic inhibitors of CaM based on an arylamide scaffold that is intended to mimic smMLCK, a CaM-binding helical peptide. Compound 1 showed a K_i value of 7.10 ± 1.48 nM in a fluorescence polarization assay that monitors the strong association of CaM and its peptide ligand mastoparan X. (¹H,¹⁵N)-HSQC NMR spectroscopy experiments suggested that 1 binds to CaM in an analogous fashion to that of smMLCK.

Full text of DOI:10.1021/ol052478j

ORGANIC
LETTERS
2006
Vol. 8, No. 2
223-225
Arylamide Derivatives as Peptidomimetic
Inhibitors of Calmodulin
Hang Yin,† Kendra K. Frederick,† Dahui Liu,¶ A. Joshua Wand,† and
William F. DeGrado*,†,‡
Department of Biochemistry and Biophysics, UniVersity of PennsylVania School of
Medicine, Philadelphia, PennsylVania 19104
wdegrado@mail.med.upenn.edu
Received October 12, 2005
ABSTRACT
Many peptides bind to calmodulin (CaM) in a helical conformation. Here we describe a group of synthetic inhibitors of CaM based on an
arylamide scaffold that is intended to mimic smMLCK, a CaM-binding helical peptide. Compound 1 showed a Ki value of 7.10
± 1.48 nM in a
fluorescence polarization assay that monitors the strong association of CaM and its peptide ligand mastoparan X. (1H,15N)-HSQC NMR
spectroscopy experiments suggested that 1 binds to CaM in an analogous fashion to that of smMLCK.
Calmodulin (CaM) is a calcium-modulated protein that is
abundant in the cytoplasm of all higher cells and has been
highly conserved through evolution.1 CaM is a small
dumbbell-shaped protein composed of two globular domains
connected by a flexible linker.2 It mediates the intracellular
Ca2+ level to the degree of activation of a large number of
regulatory proteins, including kinases, phosphatases, and ion
channels. Thus it is of much current interest to develop highly
potent and specific inhibitors of CaM as it plays important
roles in many critical biological processes, such as inflam-
mation, metabolism, apoptosis, muscle contraction, intracel-
lular movement, and short-term and long-term memory.3
Many small molecules have been previously reported that
bind to CaM with high affinity;4 however, a substantial
number of them tend to do so with low specificity and
uncertain stoichiometry.5
Previous work has shown that CaM binds many targets
in helical conformations, such as an R-helical domain of
smooth muscle myosin light-chain kinase (smMLCK, Kd )
40.4 ( 13.7 nM),7 although some peptides, such as the
(3) Taules, M.; Rius, E.; Talaya, D.; Lopez-Girona, A.; Bachs, O.; Agell,
N. J. Biol. Chem. 1998, 273, 33279.
(4) For examples, see: (a) Orner, B. P.; Ernst, J. T.; Hamilton, A. D. J.
Am. Chem. Soc. 2001, 123, 5382. (b) Barnes-Seeman, D.; Park, S. B.;
Koehler, A. N.; Schreiber, S. L. Angew. Chem., Int. Ed. 2003, 42, 2376.
(c) Yamniuk, A. P.; Vogel, H. J. Mol. Biotechnol. 2004, 27, 33. (d) Sharma,
B.; Deo, S. K.; Bachas, L. G.; Daunert, S. Bioconjugate Chem. 2005, 16,
1257.
(5) (a) Vandonselaar, M.; Hickie, R. A.; Quail, W.; Delbaere, L. T. J.
Nat. Struct. Biol. 1994, 1, 795. (b) Harmat, V.; Bocskei, Z. S.; Na´ray-
Szabo´, G.; Bata, I.; Csutor, A. S.; Hermecz, I.; Ara´nyi, P.; Szabo´, B.; Liliom,
K.; Ve´rtessy, B. G.; Ova´di, J. J. Mol. Biol. 2000, 297, 747.
† Department of Biochemistry and Biophysics, University of Pennsyl-
vania.
‡ Department of Chemistry, University of Pennsylvania.
¶ Polymedix, Inc.
(1) Crivici, A.; Ikura, M. Annu. ReV. Biochem. Biomol. Struct. 1995,
24, 85.
(2) Liddington, R. C. Nature 2002, 415, 373.
10.1021/ol052478j CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/24/2005

MARKS peptide, are bound in nonhelical conformations.8
Mutagenesis studies have established a key role for three
residues within smMLCK (Trp800, Thr803, and Val807),
at the i, i+3, and i+7 positions, respectively, in binding to
the C-terminal domain of CaM in a complex that also
involves the collapsed N-terminal region.9
We therefore designed a series of CaM inhibitors based
on a thioether-substituted arylamide scaffold, which previ-
ously has been shown to be a good template for mimicking
membrane-binding helical peptides.10 The thioether substit-
uents in this scaffold help rigidify the conformation through
the formation of intramolecular hydrogen bonds, while also
providing a site to introduce groups required for molecular
recognition. Additional critical substituents can be added to
both the amino groups as well as the isophthalic acid ring.11
Molecular modeling indicated that the tert-butyl groups in
1 would match well with hydrophobic side chains in
smMLCK, while the two aromatic side chains from the Phe
residues would be able to access a deep pocket in each of
calmodulin’s globular domains (Figure 1).
Arylamide derivatives were prepared using a previously
reported modular synthesis (Supporting Information).12 To
prevent proteolytic degradation, D-amino acids with aromatic
side chains were used to append to either end of the
arylamide backbone.13 Compound 4 with L-2-naphthylalanine
was prepared to compare with 3 to demonstrate the effects
of the introduced chiral centers within the amino acid
residues. The monocoupled product 5 was obtained as a
byproduct in the preparation of 2 due to the incomplete
coupling with the pyridylalanine.
A fluorescence polarization (FP) assay has been developed
to evaluate the inhibitory effects of these arylamide deriva-
tives in disrupting the CaM-ligand interaction. We have
selected mastoparan X (MaX) as the fluorescence probe of
the FP assay. MaX (INWKGIAAMAKKLLX) is a tetrade-
capeptide from the vespid wasp having exceptional affinity
for CaM (Kd ) 0.3 nM).10 By monitoring the dissociation
of MaX and CaM induced by the arylamide derivatives, the
inhibitory potency of these compounds can be determined.
We monitored the intrinsic fluorescence derived from the
Trp residue within MaX. The wavelength scanning experi-
ments indicate that the optimal excitation and emission
wavelengths are at 292 and 341 nm, respectively. The affinity
and 1:1 stoichiometry between CaM and mastoparan X was
Figure 1. Arylamide 1 designed as a peptidomimetic of the CaM-
binding smMLCK helix. Overlay of arylamide 1 (stick) and
smMLCK (red ribbon) complexed with CaM (purple cartoon).6
confirmed in an experiment in which CaM was titrated into
a solution of MaX (Supporting Information Figure S1). Upon
titration of CaM into the MaX solution, the maximum
emission wavelength of the intrinsic fluorescence shifts from
341 to 327 nm, suggesting that the peptide binding site is
either hydrophobic and/or rigid within the protein’s interior.14
With the addition of the arylamide inhibitor, the maximum
fluorescence emission shifts back to the original wavelength
as the bound peptide is released.
The potency of the arylamide inhibitors was determined
in a competition assay in which the 1:1 CaM/MaX complex
(0.5 µM) was titrated with increasing concentrations of
inhibitors. The dissociation of the CaM/MaX complex was
monitored by the increase in polarization as well as the shift
in emission maximum. Quantitative analysis of the data
showed compound 1, which has two D-Phe residues, strongly
inhibited with an apparent Ki of 7.10 ( 1.48 nM. The
naphthylalanine derivatives (3, 4) are less potent than 1 and
the D-pyridylalanine derivative 2, suggesting that single six-
membered aromatic rings provide better spatial complemen-
tarity than naphthylene groups. The D-2-naphthylalanine
analogue 3 gave a Ki of 83.4 ( 6.2 nM, while the L-naphthyl
enantiomer 4 was at least 1.4-fold less potent of an inhibitor.
The limited solubility of this compound precluded more
detailed studies. This result also suggested that the CaM has
some stereochemical selectivity in the recognition of these
ligands. The control compound 5, which only displayed half
(6) Molecular modeling studies were conducted using MacPyMOL v0.98
(http://www.pymol.org).
(7) Edwards, R. A.; Walsh, M. P.; Sutherland, C.; Vogel, H. J. Biochem.
J. 1998, 331, 149.
(8) (a) O’Neil, K. T.; DeGrado, W. F. TIBS 1990, 15, 59. (b) Rhoads,
A. R.; Friedberg, F. FASEB J. 1997, 11, 331.
(9) Meador, W. E.; Means, A. R.; Quiocho, F. A. Science 1992, 257,
1251.
(10) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll,
P. J.; Klein, M. L.; DeGrado, W. F. Proc. Natl. Aca. Sci. U.S.A. 2002, 99,
5110.
(11) Choi, S.; Clements, D. J.; Pophristic, V.; Ivanov, I.; Vemparala, S.;
Bennett, J. S.; Klein, M. L.; Winkler, J. D.; DeGrado, W. F. Angew. Chem.,
Int. Ed. 2005, 44, 6685.
(12) Liu, D.; Choi, S.; Chen, B.; Doerksen, R. J.; Clements, D. J.;
Winkler, J. D.; Klein, M. L.; DeGrado, W. F. Angew. Chem., Int. Ed. 2004,
43, 1158.
(13) Tryptophan was not selected because its emission wavelength
overlaps with that of the fluorescence probe peptide.
(14) (a) Malencik, D. A.; Anderson, S. R. Biochemistry 1984, 23, 2420.
(b) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic: New York, 1999; Chapter 1, p 17.
224
Org. Lett., Vol. 8, No. 2, 2006

of the functionality of 2, did not show significant inhibitory
effects at concentrations as high as 1 mM, confirming the
necessity of the intact arylamide scaffold. We have conducted
Table 1. Results of the Fluorescence Polarization Assay
entry
R1
R2
Ki (nM)
1
2
3
4
5
D-Phe
D-Phe
D-3-PyA
D-2-Nal
L-2-Nal
H
7.10 ( 1.48
25.7 ( 3.42
83.4 ( 6.2
>120
D-3-PyAa
D-2-Nalb
L-2-Nal
D-3-PyA
>1200
a PyA is pyridylalanine. b Nal is naphthylalanine.
2D-(1H,15N)-HSQC experiments for arylamide derivatives 1
and 2, which have confirmed that these compounds bind to
CaM in a similar way as the CaM-binding peptide smMLCK
does. In Figure 2, the spectrum of free CaM is shown in
red, CaM in complex with compound 1 is in purple, and
CaM in complex with smMLCK is in green. These results
show that titrations with 1 and smMLCK affect many of the
same residues as indicated by changes in the chemical shift
between the free and complexed forms of CaM. For the
CaM/1 complex, 9 residues had significant changes in amide
proton chemical shift and 15 residues had significant changes
in amide nitrogen chemical shift. Of the sites with perturbed
nitrogen chemical shifts, all but two are also significantly
perturbed in the CaM/smMLCK complex. This is in marked
contrast to the complexes of CaM with the MARCKS family
of peptides where available data indicate only one of these
sites is perturbed.15 The fact that many residues were affected
suggests that the protein undergoes global conformational
changes during the ligand/receptor association, which is
consistent with the maximum fluorescence emission changes
that occur during MaX binding to CaM. Comparison of the
chemical shift changes induced by 1, smMLCK, and
MARCKS confirmed that 1 binds CaM in a fashion similar
Figure 2. HSQC NMR spectra of the CaM protein (red), in the
presence of 2.0 equiv of 1 (purple) and of 1.2 equiv of smMLCK
(green).
to the helical CaM ligands (Supporting Information), indicat-
ing arylamide derivatives are good peptidomimetics of the
R-helical conformation.
In conclusion, we have developed a series of small
molecule inhibitors of protein-protein interaction.16 A group
of arylamide derivatives based on structure-based design that
disrupt the CaM-ligand association has been identified.
These arylamide derivatives have shown inhibitory effects
in the lower nanomolar level, and the binding mode of these
inhibitors was confirmed using HSQC NMR spectroscopy.
Acknowledgment. We thank the NIH for support of this
work. We also thank Dr. David J. Rys for his generous help
in the synthesis of the arylamide intermediates.
Supporting Information Available: Experimental details
for the FP binding assay, protein preparation, NMR param-
eters and results. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL052478J
(15) Porumb, T.; Crivici, A.; Blackshear, P. J.; Ikura, M. Biophys. J.
1997, 25, 239.
(16) Yin, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2005, 44, 4130.
Org. Lett., Vol. 8, No. 2, 2006
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