High affinity Grb2-SH3 domain ligand incorporating Cβ-substituted prolines in a Sos-derived decapeptide

Article abstract of DOI:10.1016/j.bmc.2006.11.002

Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has been replaced by a cis C^β-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis C^β-methyl-L-proline restored the affinity. Other cis C^β-proline substituents led to a complete loss of binding potency. Combining the best modifications: a cis C^β-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhancement compared to the wild-type VPPPVPPRRR sequence. This study shows that C^β-substituted prolines may constitute a new alternative for PPII ligands, combining entropy and enthalpy beneficial effects.

Full text of DOI:10.1016/j.bmc.2006.11.002

Bioorganic & Medicinal Chemistry 15 (2007) 1439–1447
High affinity Grb2-SH3 domain ligand incorporating
Cb-substituted prolines in a Sos-derived decapeptide
Yves Jacquot,a,* Isabelle Broutin,b Emeric Miclet,a Magali Nicaise,c Olivier Lequin,a
a
a
a
c
´
Nicole Goasdoue, Charlotte Joss, Philippe Karoyan, Michel Desmadril,
Arnaud Ducruixb and Solange Laviellea
a
`
Universite´ Pierre et Marie Curie-Paris 6, UMR 7613, Paris, France; CNRS, UMR 7613, ‘‘Synthese, Structure et Fonction de
Mole´cules Bioactives’’, Paris, France; FR 2769, UMR 7613, Paris, France; Case courrier 45, 4, place Jussieu, 75005 Paris, France
bUniversite´ Rene´ Descartes, UMR 8015, Paris, France; CNRS, UMR 8015, Laboratoire de Cristallographie et RMN biologique,
Paris, France; 4, avenue de l’Observatoire, 75270 Paris Cedex 06, France
cUniversite´ de Paris-Sud, UMR 8619, Orsay, France; CNRS, UMR 8619, Laboratoire de Mode´lisation et d’Inge´nierie des Prote´ines,
ˆ
Orsay, France; Bat. 430, 91405 Orsay Cedex, France
Received 10 July 2006; revised 30 October 2006; accepted 2 November 2006
Available online 6 November 2006
Abstract—Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades
and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the
upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with
the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or
peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has
been replaced by a cis Cb-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these
substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis
Cb-methyl-L-proline restored the affinity. Other cis Cb-proline substituents led to a complete loss of binding potency. Combining the
best modifications: a cis Cb-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhance-
ment compared to the wild-type VPPPVPPRRR sequence. This study shows that Cb-substituted prolines may constitute a new alter-
native for PPII ligands, combining entropy and enthalpy beneficial effects.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: MAPK, mitogen-activated protein kinase; Grb2, gro-
wth factor receptor-bound protein 2; SH3, Src homology 3; PPII, p-
olyproline II; Sos, son of sevenless; CD, circular dichroism; TKR,
Growth factors are involved in many biological process-
es such as differentiation, survival, apoptosis as well as
tyrosine kinase receptor; NMR, nuclear magnetic resonance; Boc, tert-
cytoskeleton reorganisation via interactions with their
butyloxycarbonyl; Fmoc, 9-fluorenylmethoxycarbonyl; ITC, isother-
specific tyrosine kinase receptor (TKR), which activates
the Ras/MAPK cascade.1–3 This signalling pathway has
mal titration calorimetry; Tos, tosylate; PAM, phenylacetamidomethyl
resin; MBHA, 4-methylbenzhydrylamine resin; DCC, dicyclohexylcar-
been highlighted as mutations in the Ras protein have
been found in human carcinoma suggesting a crucial
role of Ras in cell degenerative processes and cancer
emergence.4 Stimulation by a growth factor of TKR
induces first dimerization followed by autophosphoryla-
tion of the receptor and the recruitment of the Grb2/Sos
complex. This latter complex is stabilized mainly by
hydrophobic interactions between the two SH3 domains
of Grb2 and the decapeptide Val1149-Arg1158
(VPPPVPPRRR) from Sos.5,6 NMR structure of the
N-terminal Grb2-SH3 domain/VPPPVPPRRR com-
plex7,8 showed that this decapeptide adopts a polypro-
bodiimide; HOBt, hydroxybenzotriazole; HBTU, O-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DMSO,
dimethylsulfoxide; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-
pyrimidinone; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic
acid; RP-HPLC, reverse-phase high performance liquid chromatogra-
phy; MALDI-TOF, matrix-assisted laser desorption ionization time-
of-flight; TFA, trifluoroacetic acid; NMP, N-methylpyrrolidone;
DIEA, N,N-diisopropylethylamine; Standard IUPAC single and triple
letter codes for amino acids are used.
Keywords: Grb2-SH3 domain; Cb-substituted prolines; Fluorescence;
ITC.
*
Corresponding author. Tel.: +33 (0)1 44 27 26 78; fax: +33 01 44 27
38 43; e-mail: jacquot@ccr.jussieu.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.002

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Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
line II (PPII) helical conformation, i.e. a left-handed he-
lix with three residues per turn (/ = ꢀ78ꢁ, w = +146ꢁ).9
This VPPPVPPRRR sequence belongs to the proline-
rich domains superfamily, which represents the most
important protein interaction modules whose prominent
feature is their ability to form weak complexes with spe-
cific recognizing domains, their dissociation constants
being typically in the micromolar range.10,11
have been replaced by either an alanine or a sarcosine
by Nguyen et al.10 Since N-alkylation (sarcosine and
peptoids) is advantageously accepted in position 6,
we reasoned that (i) a Cb-substituted proline in posi-
tion 5 might project the b-substituent in the same
direction as an N-alkylated residue in position 6
(enthalpy gain) and (ii) the /-constraint of the proline
ring in position 5 will stabilize and thus increase the
concentration of the necessary PPII structure (entropy
gain). Both gains should lead to high affinity binders
of the Grb2 protein (Fig. 3). Thus, in the present
study, Val5 has been replaced by various cis Cb-substi-
Selective disruption of the Grb2/Sos association has
been considered as an attractive strategy for an up-
stream control of the Ras/MAPK cascade.3,12 Two
families of ligands have emerged to provide inhibitors
of this protein/protein interaction: (i) PPII ligand di-
mers, which allow simultaneous interaction with both
Grb2-SH3 domains13 and (ii) peptoids, which establish
new stabilizing interactions with the Grb2 protein.10,14
However, the selectivity of these ligands remains an
important question to be addressed,11,15,16 since more
than two hundred SH3 domains have been identified
or suspected in mammals.17 In that context, we have
designed analogues of the decapeptide VPPPVPPRRR
incorporating a Cb-substituted proline in place of the
crucial valine 5 residue, as an application of the chi-
meric prolinoamino acids (Fig. 1) previously de-
scribed.18–24 Indeed, close disclosure of the Grb2/
SH3 binding surface showed that the central valine
18–24
tuted-L-prolines.
We report herein the syntheses of: (1) N-Fmoc or N-Boc
aromatic, aliphatic-alkyl and polar cis Cb-substituted
prolines, and (2) the corresponding peptides. The affinity
of each peptide for recombinant Grb2 has been deter-
mined using fluorescence spectroscopy and/or ITC stud-
ies. Far-UV CD spectra have also been recorded for
some representative peptides.
2. Results
2.1. Cb-substituted prolines and peptide syntheses
1
side chain (Val5) of VPPPVPPRRR10 is located in
Syntheses of N-Fmoc or N-Boc cis Cb-substituted-L-
prolines (Table 1) were based on the asymmetric ami-
no-zinc-enolate carbometallation strategy, as we previ-
ously reported (Figs. 4 and 5).18–24
a hydrophobic groove (Fig. 2), and constitutes a stra-
tegic position to anchor the decapeptide within the
Grb2 protein. Furthermore, this Val5 residue is locat-
ed between the two prolines Pro3 and Pro6, which
Most of the monomeric peptides reported herein are
free amino and free carboxylic derivatives to allow di-
rect comparison with previously reported studies.13,25
They were prepared starting from preloaded Arg(-
Tos)-PAM resin by manual or automatic solid phase
peptide strategy with either DCC/HOBt or HBTU
for the coupling of the hindered modified prolines
(residue 5) and the following amino acid (residue 4).
For the C-terminal carboxamide peptide 20 a MBHA
resin and the Boc strategy were used, while the
peptide dimer 21 was obtained from a preloaded
Boc-Lys(Fmoc)-MBHA resin. Acetylation of the N-
terminal residue of peptides 20 and 21 was achieved
with acetic anhydride.
R
H N
2
HN
HN
COOH
COOH
COOH
Figure 1. Structures of L-valine, L-proline and of the chimeric cis
prolinoamino acid.
2.2. Monomers binding assays
Dissociation constants (Kd) of Grb2-SH3 domain/pep-
tide complex were measured for each peptide by fluores-
cence spectroscopy and are listed in Table 2. For the
wild-type Sos decapeptide VPPPVPPRRR (peptide 12)
a Kd of 38.7 1.5 lM was determined. No significant
binding affinity was detected for VPPPPPPRRR (pep-
tide 13). However, introduction of a cis Cb-methyl sub-
stituent on proline 5, VPPPP(CH3)PPRRR (peptide 14),
restored the binding potency (Kd = 16.8 0.7 lM), with
a slight affinity enhancement when compared to peptide
12 (Fig. 6 and Table 2). Surprisingly, none of the other
cis Cb-substituted monomeric peptides (peptides 15–19)
were able to interact with detectable affinity to the
Grb2 protein (Table 2).
Figure 2. Interaction of the VPPPVPPRRR sequence of Sos (blue)
with the N-terminal SH3 domain of Grb2 (red). Distances between the
Val5 residue of Sos and the residues that constitute the aromatic-rich
second pocket of the N-terminal SH3 domain of Grb2 are specified
(PDB code 1GBQ8).

Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
1441
a
b
c
O
O
O
O
O
O
N
HN
HN
N
N
N
R
R
Figure 3. (a) The Val5-Pro6 pair of the Sos wild-type decapeptide and corresponding structures for (b) the peptoid ligands developed by Nguyen
et al.,10 and for (c) the Cb-substituted Pro5 peptides described herein.
Table 1. N-Boc or N-Fmoc cis Cb-substituted-L-prolines used for the
synthesis of the VPPPVPPRRR-derived peptides
The affinity of dimeric peptide 21, obtained from the
best monomeric ligand (peptide 14), was determined
by fluorescence spectroscopy and ITC. Compared to
peptides 12 and 14, peptide 21 presented a dramatic in-
crease in affinity. By fluorescence, the Kd of 21 was esti-
mated between 9 and 520 nM, the dissociation constant
being quantitatively determined by ITC as being in the
nanomolar range, Kd = 68 5 nM (Fig. 8).
Cb-substituted Substituent
Protecting Compound Ref
group P
proline
R
H
CH3
Boc
Fmoc
Fmoc
5
6
R
22
26, 28
27
CH(CH3)2
CH2OCH2C6H5 Boc
7
CO H
2
N
P
8
CH2SCH3
CH2CN
CH2SO2C6H5
Fmoc
Fmoc
Boc
9
22, 23
25, 27
26
10
11
3. Discussion
2
The interacting surface (400 A ) delineated within each
˚
2.3. Far-UV CD analysis
Grb2-SH3 domain plays a crucial role in the modulation
of the MAPK-dependent intracellular mechanisms. The
Sos decapeptide, VPPPVPPRRR, that contains the spe-
cific PXXP signature of SH3 binding motifs (where X is
any amino acid), interacts within three grooves (Fig. 2)
located at either surface of the N- or C-terminal do-
mains of the Grb2 protein. Binding mode of this peptide
belongs to the type II, that is, with residues Pro2 and
Pro3 fitting within the first shallow pocket, while resi-
dues Val5 and Pro6 are located within the second, and
the charged Arg8 residue interacting with an aspartate
residue of the third pocket (Asp33 and Asp190 for the
Grb2 N-terminal and C-terminal SH3 domains, respec-
tively). Briefly, when the basic residues are located at the
N-terminus of the PPII peptide, the latter adopts a type-
I binding mode, whereas it is considered as a type-II
binding mode when the basic residues are located at
the C-terminus.27,28 The second pocket is an aromatic-
rich region delimited by residues Phe9, Trp36, Pro49
and Tyr52 for the N-terminal Grb2-SH3 domain,8 and
residues Phe167, Trp193, Pro206 and Tyr209 for the
C-terminal domain.29 The third pocket confers specific-
ity to the complex.5
Far-UV CD spectra were recorded to monitor the con-
formational changes on the PPII conformation induced
by substitution of Val5 by various cis Cb-substituted-L-
prolines of the active peptides. CD spectrum of
VPPPPPPRRR (peptide 13) revealed a characteristic
PPII signature with a strong minimum at 201 nm and
a weak maximum at 227 nm.26 The absence of maxi-
mum at 227 nm for VPPPVPPRRR (peptide 12) sug-
gests that the PPII conformation was less pronounced
(Fig. 7), although still present. Far-UV CD spectrum
of VPPPP(CH3)PPRRR (peptide 14) is almost superim-
posable with the spectrum of peptide 13.
2.4. Peptide optimization
To evaluate the effects of the N- and C-terminal charges
on the binding to Grb2, the N-acetyl, C-carboxamide
analogue of peptide 12 was prepared (peptide 20). These
two modifications induced a 10-fold increase in the
binding potency (Kd = 3.7 0.2 lM), when compared
to peptide 12 (Table 2). Then, peptide dimerization
was explored, as peptide dimers, which allow simulta-
neous interaction with both Grb2-SH3 domains, have
presented significant increase in binding potency.13,25
In their elegant work, Nguyen et al.,10 have shown that:
(i) prolines of the central PXXP sequence could actually
#
Br
Zn
ZnBr
O
COOBn
N
NH
NH
N
COOBn
N
2
i)
ii)
iii)
BnO
1
2
3
4
Figure 4. Amino-zinc-ene-enolate carbometallation reaction. Reagents and conditions: (i) 4-bromobutene, K2CO3, NaI, DMF, 80 ꢁC; (ii) benzyl
bromoacetate, K2CO3, anhydrous THF/DMPU, rt; (iii) anhydrous diethyl ether, LDA (ꢀ78 ꢁC), ZnBr2 (ꢀ78 ꢁC to rt).

1442
Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
SO2Ph
ii)
COOH
N
P
11
SO2Ph
COOH
N
P
6
COOBn
N
N
N
vi)
COOH
i) ii)
vii)
N
7
P
ZnBr
CuCNZnBr
CN
iv) v)
COOBn
COOBn
COOBn
N
N
CN
iii)
viii)
ix)
COOH
4
N
P
10
SCH3
x)
COOBn
SCH3
xi)
xiii)
COOH
xii)
N
P
9
O
O
N
O
xiv)
OH
N
P
O
8
Figure 5. Synthesis of the cis Cb-substituted-L-prolines 6–11. Reagents and conditions: (i) NH4Cl, H2O; (ii) H2, Pd/C, Boc2O, MeOH or Fmoc-O-
succinimide, pH 9; (iii) CuCN/2LiCl; (iv) PhSSO2Ph; (v) CH2Cl2, THF, mCpBA; (vi) THF/DMPU, ꢀ78 ꢁC, LDA, CH3I and then LDEA, CH3I; (vii)
MeOH, Na/Hg, KH2PO4 and then H2, Pd/C, Boc2O, MeOH; (viii) TsCN; (ix) H2, Pd/C, Boc2O, MeOH; (x) CH3SSO2CH3 at 0 ꢁC; (xi) VocCl,
(CH2Cl)2; (xii) HCl gaz, dioxane and then Boc2O or Fmoc-O-succinimide; (xiii) O2; (xiv) H2, Pd/C, Boc2O, MeOH and then excess of KOH, BnBr.
main. The authors suggested that proline was not cho-
sen during evolution for its conformation properties,
Table 2. Kd measurements of synthetic peptides 12–21 for Grb2-SH3
domains by intrinsic tryptophane fluorescence spectroscopy
but because proline was the only naturally occurring
Synthetic peptide
Compound
Kd (lM)
N-substituted amino acid.10 Nguyen et al. also hypothe-
sized that N-substitution of the penultimate residue X in
the PX(X = Val)P sequence could not be tolerated by
H-VPPPVPPRRR-OH
H-VPPPPPPRRR-OH
12
13
14
15
16
17
18
19
20
21
38.15
nb
H-VPPPP(CH3)PPRRR-OH
H-VPPPP(CH(CH3)2)PPRRR-OH
H-VPPPP(CH2OH)PPRRR-OH
H-VPPPP(CH2SOCH3)PPRRR-OH
H-VPPPP(CH2COOH)PPRRR-OH
H-VPPPP(CH2SO2C6H5)PPRRR-OH
Ac-VPPPVPPRRR-NH2
11.60
nb
the SH3 domains, due to the narrowness of second
10
˚
˚
SH3 groove (25 A long and 10 A wide). Apparently
in contradiction with this enthalpy-driven hypothesis,
Ferreon et al.30 proposed an entropy-driven hypothesis
for the Grb2/Sos interaction, since they showed that de-
creased affinities for SH3 domain were observed when
the PPII conformation of Sos-derived peptides was
destabilized.
nb
nba
nbb
nb
3.67
<0.52
(Ac-VPPPP(CH3)PPRRR)2K-NH2
Proline substituent is oxidized into sulfoxide (a) or carboxylic acid (b),
respectively.
nb, no binding.
In the present study, far-UV CD spectrum of the
VPPPPPPRRR peptide 13 (substitution of Val5 by
Pro5) unambiguously showed that the PPII concentra-
tion is increased compared to that observed for
VPPPVPPRRR. However, peptide 13 has no affinity
for Grb2. Inspection of the published NMR structure
of the N-terminal Grb2-SH3 domain/VPPPVPPRRR
(PDB code: 1GBQ)8 shows the conformation of the
bound peptide and yields for Val5 the following values:
x = ꢀ177ꢁ, / = ꢀ69ꢁ, w = 139ꢁ with the N-Ca-Cb-Cc2
torsion angle equal to ꢀ33ꢁ. These values correspond
be replaced by non-natural N-substituted residues with-
out hampering complex formation and (ii) substitution
of Val5 in the VPPPVPPRRR decapeptide by sarcosine
was associated to a lower affinity for Grb2. Replacement
of Pro6, the last proline in the PXXP sequence, by, for
example, N-(S)-phenylethylglycine within the Sos deca-
peptide, was found to noticeably enhance (ꢁ100-fold)
the affinity for the isolated N-terminal Grb2-SH3 do-

Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
1443
Figure 6. Fluorescence change of Grb2 by (j) peptide 12 and (n) peptide 14 binding. Experimental datapoints have been fitted using a Monte Carlo-
based program.34
Figure 7. CD spectra of derivatives 12 (n), 13 (j) and 14 (d) recorded at 5 ꢁC. For each peptide, concentration sample was 300 lM in phosphate
buffer 15 mM, pH 7, NaCl 40 mM.
to typical angles for a proline within a PPII structure,
with an up-puckering (v1 = ꢀ27ꢁ) for the pyrrolidine
ring.31 Furthermore, the c1 and c2 methyls of Val5 in
the decapeptide interact strongly with the hydrophobic
residues located in the second pocket (distances mea-
sured between Cc1 and Pro49 Cb; Pro49 Cc; Phe9 Cf;
which were described as the prominent feature of
SH3 ligands.14 In the present study, cis-configuration
of the Cb-substituent on the proline scaffold was cho-
sen to explore the external face of the second groove.
trans-Configuration with bulky substituents might lead
to steric clashes with Phe9, Trp36 and Pro49 of the N-
terminal domain or Phe167, Trp193 and Pro206 of the
C-terminal domain, respectively. Among compounds
14–19, only peptide 14, bearing a cis Cb-methyl-L-pro-
line, P(CH3), exhibits an enhanced affinity for the
Grb2 protein. This improvement remains marginal
(factor 3), when compared to the wild-type peptide.
Unexpectedly, the phenyl-containing peptide 19 has
no affinity at all for Grb2, despite the presence of
numerous aromatic residues in the second SH3 pocket
Trp36 Cg2; Trp36 Cf2 are 3.75, 3.53, 3.91, 3.42, 3.61 A,
˚
respectively, and distances measured between Cc2 and
Tyr52 Cf; Tyr52 Ce1; Phe9 Ce1 are 3.90, 3.54, 3.61 A,
˚
respectively, Fig. 2).
Herein, we speculated that a Cb-substituted Pro5 (X)
followed by a Pro6 residue in the PXXP sequence
could constitute an amino acid combination compara-
ble to the sequential Ca- and N-substituted residues,

1444
Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
Figure 8. Grb2-binding curve obtained at 18 ꢁC with increasing concentrations of peptide 21. Dissociation constants were measured either by (a)
fluorescence spectroscopy (0,009 lM < Kd < 0.520 lM) or (b) by ITC (Kd = 68 5 nM, stoichiometry N = 1).
and the high affinity of some N-alkylated peptoids,
described by Nguyen et al.10 Compared to peptide
13, the affinity enhancement obtained for the peptide
14 (incorporating a cis Cb-methyl-L-proline) might
be promoted by local hydrophobic interactions involv-
ing the methyl substituent. Indeed, the CD spectrum
of peptide 14 is almost completely superimposable
with the CD spectrum of peptide 13, proving that
the methyl substituent neither increases nor decreases
significantly the PPII conformation (Fig. 7). Minimi-
zations of the complex containing peptide 14 and
the N-terminal Grb2-SH3 domain have been carried
out to correlate observed affinities and ligand/protein
interactions. Interestingly, each of the two P(CH3)
ring conformations (up- or down-puckering) was
found to share specific structural homologies with
the Val5 residue of the wild-type sequence (Fig. 9). In-
deed, superimposing the N, Ca, Cb, Cc2, C0 and O
atoms of Val5 (coordinates taken from the minimized
1GBQ structure8) with the corresponding N, Ca, Cb,
Cc, C0 and O atoms of the up-puckered and down-
puckered P(CH3) ring gave a rms value of 0.26 and
˚
0.65 A, respectively. These results are to be related
to the previous finding that Val5 torsion angles of
the bound peptide are almost identical to those found
in trans up-puckered prolines. Thus, the Cc-methylene
group of an up-puckered Pro5 could nicely mimic the
valine Cc2-methyl of the wild-type ligand and allows
the corresponding hydrophobic interactions with the
protein to take place. However, no new hydrophobic
interaction was detected between the Cc-methylene
group of the up-puckered 3-substituted Pro5 and the
protein when compared with those detected in the
wild-type complex, that is, between the c2-methyl of
Val5 and the protein. In contrast, based on these
structural alignments, the cis Cb-methyl group of the
˚
P(CH3) residue is found 2.01 A away from the valine
Cc1-methyl for the up-puckering form of Pro5 and
˚
only 0.54 A away for the down conformation
(Fig. 9). It is thus highlighted that the cis Cb-methyl

Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
1445
4. Conclusion
We report the synthesis of new Grb2 ligands starting
from the Sos decapeptide, VPPPVPPRRR substituted
in position 5. The key residue valine 5 has been replaced
by various cis Cb-substituted prolines. Data clearly show
that PPII conformation of the substituted peptide is sta-
bilized by introduction of an additional pyrrolidine ring
in position 5. But, an increase of PPII conformation is
not sufficient to yield high affinity Grb2 ligand. Howev-
er, an additional cis Cb-methyl on the pyrrolidine ring
slightly increases the binding potency to Grb2 of the
resulting peptide by mimicking partially the Cc1-methyl
of the valine 5. Other cis Cb-proline substituents, such as
phenyl, substituted-phenyl, bulky aliphatic groups and
polar chains, unexpectedly led to complete loss of bind-
ing potency for Grb2. Finally, the cis Cb-methyl-proline
5, N-acetylated, C-carboxamide dimer 21 yielded a 560-
fold affinity enhancement compared to the wild-type
VPPPVPPRRR sequence. Interestingly, this compound
is one of the most potent Grb2 SH3-domain peptide li-
gands described so far when compared to the wild-type
monomer H-VPPPVPPRRR-OH. Indeed, the best
peptide ligands reported in the literature are a peptide
dimer13,25 and a peptoid10,14 that gave a 450- or a 125-
fold affinity increase, respectively (referring to the
wild-type monomer). This study shows that Cb-substi-
tuted proline may constitute a new alternative for PPII
ligands, combining entropy and enthalpy beneficial
effects.
Figure 9. Superimposition of the down-puckered (purple) and the up-
puckered (orange) cis 3-methylproline in position 5 of the peptide
ligand with the Val5 (in white) of the wild-type peptide after
minimization of the complexes.
substituent of Pro5 could fairly mimic the wild-type
valine Cc1-methyl, provided that the pyrrolidine ring
adopts a down-puckered conformation. In conclusion,
the cis 3-methylproline residue fits well with Val5 res-
idue and therefore allows the peptide to be well
accommodated within the binding pocket. The addi-
tional Cd-methylene group in the pyrrolidine ring is
oriented away from the protein surface and therefore
does not contribute significantly to van der Waals
interactions with the protein.
For peptide optimization the N- and C-terminal
charges of peptide 12 were first removed, since the
decapeptide of Sos is included in a protein sequence.
The resulting peptide 20 presented a 10-fold increase
in affinity for Grb2. Both peptides 12 and 20 exhibited
the same CD spectrum profile suggesting that the dif-
ferent affinities are only related to electrostatic effects
(data not shown). Then, with these N- and C-terminal
modifications, the best monomeric ligand, peptide 14,
has been dimerized with a lysine linker, as previously
published.13,25 The dimer dissociation constant was
too tight to be measured by fluorescence and was
therefore quantified by ITC (Kd = 68 5 nM). Inter-
estingly, ITC analysis demonstrated a stoechiometry
very close to N = 1 for the Grb2/ligand complex, in
good agreement with the model where one dimeric li-
gand simultaneously interacts with both Grb2-SH3
domains (Fig. 8).
5. Experimental
5.1. Chemistry
Automatic peptide syntheses were achieved on a ABI
433 A peptide synthesizer (Applied Biosystems, Foster
City, USA). Boc-protected natural amino acids, HBTU,
Arg(Tos), preloaded PAM resin (substitution
0.57 mmol/g)
and
MBHA
resin
(substitution
1.26 mmol/g) were purchased from Senn Chemicals
Int. (Cachan, France). Solutions in NMP of DCC
(1.0 M), HOBt (1.0 M) and DMSO were obtained from
Applied Biosystems (Courtaboeuf, France). NMP,
DIEA and dichloromethane were purchased from SDS
(Peypin, France). Boc or Fmoc N-protected modified
prolines 6–11 were prepared according to already
reported procedures.18–24 otherwise stated 10 equiva-
lents of the protected-amino acids was used. Peptides
were cleaved from the support by HF, as already de-
scribed.32 After lyophilisation, peptides were purified
by RP-HPLC with appropriate gradient (30 min) of sol-
vent B (60% acetonitrile in 0.1% (v/v) TFA/H2O). Pre-
parative RP-HPLC was achieved on a Dionex HPLC
system (Dionex P580 Pump, Dionex AD25 Absorbance
Detector at 200 nm, Dionex 4400 Integrator), with a
flow rate of 6 ml/min, using a SymmetryPrepꢂ C8 RP-
HPLC column (Waters 7.8 mm · 300 mm, 7 lm particle
Thus, the modest three-fold affinity gain achieved by
replacement of Val5 combined with neutralization of
the N- and C-terminal charges, as carboxamide and N-
acetyl forms, and finally dimerization leads to one of
the most potent Grb2-SH3-domain ligand described so
far with a 560-fold increase of affinity when compared
to the wild-type monomer H-VPPPVPPRRR-OH. To
date, the best ligands reported in the literature were a
peptide dimer13,25 and a peptoid10,14 and gave a 450-
and a 125-fold affinity increase, respectively (referring
to the wild-type monomer). Interestingly, our peptide
might be further optimized by Cc pyrrolidine ring func-
tionalization. Cd or trans Cb bulky substituents are
precluded because of steric hindrance within both the N-
and C-terminal domains of Grb2. However, introduction
of a trans Cb-methyl could be beneficial, since it could
mimic the Cc1 of Val5 in an up-puckered state of the pyr-
rolidine ring, this conformation being also required for
observing the Pro5 Cc–Val5 Cc2 structural homology.
˚
size, 300 A pore size). Analytical RP-HPLC was
achieved on a Waters HPLC system (Waters 600 Pump
and Controller, Waters 2487 Dual k Detector, Waters
746 Data Module), with a flow-rate of 1.5 mL/min,

1446
Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
using
a
C8
RP-HPLC
column
(Waters
acetylated by an excess of acetic anhydride RP-HPLC,
Rt = 13.2 min (40 min gradient 0–60% solvent B). MAL-
DI-TOF (M+H+) 2557.1 (calcd 2556.6).
˚
4.6 mm · 300 mm, 5 lm particle size, 300 A pore size).
MALDI-TOF spectra were recorded with a PerSeptive
Biosystems Voyager Elite mass spectrometer (Applied
Biosystems Framingham, USA).
5.2. Modelling
5.1.1. Synthesis of peptides 12 and 13. Peptides 12 and 13
were obtained starting from a Boc-Arg(Tos)-preloaded
PAM resin (0.1 mmol).
Calculations were performed on Silicon Graphics O2
workstations, using Discover software (Accelrys, Inc,
San Diego, USA) and cff91 forcefield. The complex be-
tween the N-terminal Grb2-SH3 domain and the Ac-
VPPPVPPRRR-NH2 peptide was obtained from the
Protein Data Bank (PDB code: 1GBQ)8 and was used
in all calculations. Visualisation and chemical modifica-
tions of the PDB structure were carried out using Insight
II and Biopolymer (Accelrys). A minimization protocol
was applied on the wild-type complex as well as on the
complex incorporating peptide 14, by considering sepa-
rately an up- or down-puckering of the P(CH3). Non-
Peptide 12: RP-HPLC, Rt = 10.3 min (30 min gradient
from 9% to 48% solvent B). MALDI-TOF (M+H+)
1170.7 (calcd 1169.7). Peptide 13: RP-HPLC,
Rt = 12.7 min (30 min gradient from 0% to 24% solvent
B). MALDI-TOF (M+H+) 1168.9 (calcd 1167.7).
5.1.2. Synthesis of peptides 14–19. Peptides 14–19 were
obtained starting from
a Boc-Arg(Tos)-preloaded
PAM resin (0.1 mmol). The cis Cb-substituted Pro5
and the next Pro4 residues were manually coupled, 5
equiv for the substituted Pro5 and 10 equiv for Pro4,
respectively, the activation being performed with HBTU
in the presence of DIEA in NMP. The Boc or Fmoc pro-
tecting groups were removed from the modified prolines
with either TFA (20% v/v in dichloromethane) or piper-
idine (20% v/v in dichloromethane), respectively.
bond interactions were calculated using a 8 A cutoff.
˚
The electrostatic potential was calculated in vacuo with
a distance-dependent dielectric e = 4r. The minimization
protocol consisted of four stages in which constraints on
atom positions were gradually removed: (i) relaxation of
peptide ligand side chains, (ii) relaxation of the whole
peptide, (iii) relaxation of the entire complex except
the protein backbone and (iv) relaxation of the entire
complex. Each minimization step consisted of a total
of 5000 iterations (10,000 for the final one) using steep-
Peptide 14: RP-HPLC, Rt = 18.7 min (30 min gradient
from 0% to 18% solvent B). MALDI-TOF (M+H+)
1182.9 (calcd 1181.7). Peptide 15: RP-HPLC,
Rt = 11.3 min (30 min gradient 0–18% solvent B). MAL-
DI-TOF (M+H+) 1210.5 (calcd 1209.8). Peptide 16: RP-
HPLC, Rt = 16.5 min (30 min gradient 0–18% solvent
B). MALDI-TOF (M+H+) 1198.8 (calcd 1197.7). Pep-
tide 17: RP-HPLC, Rt = 20.8 min (gradient 0–30% of
solvent B over 30 min). MALDI-TOF (M+H+) 1244.9
(calcd 1243.7). Peptide 18: RP-HPLC, Rt = 19.2 min
(30 min gradient 0–18% solvent B). MALDI-TOF
(M+) 1225.3 (calcd 1225.7). Peptide 19: RP-HPLC,
Rt = 21.5 min (30 min gradient 0–18% solvent B). MAL-
DI-TOF (M+H+) 1322.6 (calcd 1321.7).
est descent and conjugate gradient algoritꢀh1ms until the
gradient was less than 0.001 kcal molꢀ1
˚
A .
5.3. Fluorescence-based binding assays
The recombinant Grb2 protein has been produced and
purified according to already reported protocol.33 Affin-
ity of the peptides for the recombinant Grb2 protein was
evaluated using fluorescence-based titration assays.
Experiments were conducted at 18 ꢁC in a stirred 1 cm
pathlength cell using a Jasco FP-6200 spectrofluorimeter
(Jasco, Essex, United Kingdom). Excitation and fluores-
cence emission were monitored at 280 and 350 nm,
respectively. In all cases, concentration of Grb2 was
kept at 1 lM in 50 mM of Tris buffer adjusted at pH
8.0. Peptide concentrations varied typically from
50 nM to 250 lM. Dissociation constants Kd were deter-
mined by monitoring changes of fluorescence upon addi-
tion of the peptide at defined concentrations.
Experimental curves were fitted using the Monte Car-
lo-based MC-Fit program.34 Dissociation constants
(Kd) were calculated according to the following equa-
5.1.3. Synthesis of the N-acetylated and C-carboxamide
peptide monomer 20. Peptide 20 was obtained starting
from a MBHA resin. Fmoc cis Cb-methyl proline was
coupled as described for peptide 14. After removal of
the last Boc-protecting group, the amino terminal was
acetylated by an excess of acetic anhydride. RP-HPLC,
Rt = 15.9 min (30 min gradient 6–30% solvent B). MAL-
DI-TOF (M+H+) 1211.9 (calcd 1210.7).
tion:
Fb = (Kd + P + L ((Kd + P + L)2ꢀ4PL)1/2)/2P,
5.1.4. Synthesis of the N-acetylated and C-carboxamide
peptide dimer 21. Peptide 21 was obtained starting from
a MBHA resin preloaded with Boc-Lys(Fmoc)-OH
(0.1 mmol). After sequential removal of the Boc and
the Fmoc protecting groups, both amino functions of
the lysine residue were simultaneously elongated by dou-
ble couplings (each time 10 equiv). Fmoc cis Cb-methyl
proline (residue 5) as well as the following amino acid
(Pro4) were introduced manually by single coupling (5
and 10 equiv, respectively) after activation with HBTU
in the presence of DIEA in NMP. After removal of
the last Boc-protecting group, the amino terminal was
where P and L are the protein and peptide concentra-
tions, respectively, and Fb the fraction of the bound pep-
tide. For high affinities, Kd ꢁ P.
5.4. Isothermal titration calorimetry
ITC experiments were carried out on a VP-ITC isother-
mal titration calorimeter (Microcal, Northampton,
USA) at 18 ꢁC. The same buffer (50 mM Tris, pH 8.0)
was used for compound 21 and Grb2. Grb2 concentra-
tion was determined by UV spectroscopy and initially
adjusted at 10.4 lM in the microcalorimeter cell (2.1

Y. Jacquot et al. / Bioorg. Med. Chem. 15 (2007) 1439–1447
1447
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mL). A 5 or 10 lL volume of peptide 21 (stock solution
at 125 lM) was added from a computer-controlled
300 lL microsyringe at intervals of 4 min. Heat dilution
of the peptide was taken into account from peaks mea-
sured after full saturation of Grb2 by the peptide. A the-
oretical titration curve was fitted to the experimental
data using the ORIGIN software (Microcal). This soft-
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each injection and DH ꢁ (enthalpy change in
kcal moleꢀ1), KA (association binding constant in
M
ꢀ1), n (number of binding sites per monomer), total
protein concentration and free and total ligand
concentrations.
5.5. Circular dichroism
CD spectra were acquired on a Jobin Yvon CD6 dichro-
ism spectropolarimeter at 5 ꢁC using a 1 mm pathlength
quartz cell. Spectra were recorded from 185 to 260 nm
using a 300 lM solution of peptide in phosphate buffer
15 mM, pH 7, NaCl 40 mM, and were averaged over
four scans after baseline correction.
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Acknowledgments
We are grateful to Dr. Fabienne Burlina for mass spec-
tra analysis and to Vanessa Point for experimental assis-
tance. We are also grateful to Drs. S. Fermandjian and
L. Zargarian (CNRS-UMR 8113, Institut Gustave
Roussy, Villejuif) for dichroism experiments.
27. Mayer, B. J. J. Cell Sci. 2001, 26, 1253.
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