Cyclic isoDGR peptidomimetics as low-nanomolar αvβ 3 integrin ligands

Article abstract of DOI:10.1002/chem.201204639

A perfect fit: Shown here is the docking best pose of a low-nanomolar α_vβ₃ integrin ligand cyclo[DKP-isoDGR] (atom color tube representation; see figure) into the receptor binding pocket, overlaid on the bound conformation of Cilengitide (green tube representation). The extended isoDGR conformation permits all the important interactions with the extracellular domain of the α_vβ₃ integrin. The metal ion in the MIDAS region is represented by a magenta sphere. Copyright

Full text of DOI:10.1002/chem.201204639

COMMUNICATION
DOI: 10.1002/chem.201204639
Cyclic isoDGR Peptidomimetics as Low-Nanomolar avb3 Integrin Ligands
Michele Mingozzi,[a] Alberto Dal Corso,[a] Mattia Marchini,[a] Ileana Guzzetti,[a]
Monica Civera,[a] Umberto Piarulli,*[b] Daniela Arosio,[c] Laura Belvisi,[a, c]
Donatella Potenza,[a, c] Luca Pignataro,[a, c] and Cesare Gennari*[a, c]
Integrins are a large family of heterodimeric transmem-
brane glycoprotein receptors, composed of two noncovalent-
ly associated a and b subunits.[1] Many integrins, including
avb3, avb5, aIIbb3 and a5b1 recognize the tripeptide Arg-Gly-
Asp (RGD) motif in their ligands. In particular, integrin
avb3 was the first integrin to be characterized by X-ray struc-
tural determination and was shown to be one of the key reg-
ulators of angiogenesis.[2] Integrins avb3 and avb5 have been
found to be overexpressed on blood vessels in human
tumors, but not on vessels in normal human tissues. For this
reason, these integrins have become attractive targets for
pharmacological studies mainly in the oncology area. Many
peptide and peptidomimetic integrin ligands have been de-
veloped that contain the RGD tripeptide sequence with dif-
ferent flanking residues and three-dimensional presenta-
tion.[3] Recently, our group reported a new class of cyclic
RGD peptidomimetics (Figure 1), containing bifunctional
diketopiperazine (DKP) scaffolds, formally derived from
2,3-diaminopropionic acid and aspartic acid and differing in
the configuration of the two DKP stereocenters and in the
substitution at the DKP nitrogen atoms.[4] The trans-cyclo-
Figure 1. Cyclic RGD peptidomimetics containing the bifunctional DKP
scaffold.
the protein backbone NH on the primary amide side chain
of asparagine and ring opening of the succinimide, is a
known side reaction normally leading to loss of biological
activity.[6] In this case, however, the result is a gain of pro-
tein function and the creation of a new adhesion binding
site for integrins.[7] Subsequent biochemical, spectroscopic
and computational investigations have shown that the
isoDGR sequence can fit into the RGD-binding pocket of
avb3 integrin, establishing the same electrostatic clamp as
well as additional polar interactions.[8] Based on these obser-
vations, a few conformationally constrained cyclopeptides
containing the isoDGR sequence have been synthesized.[9]
However, these ligands show a moderate affinity for avb3 in-
tegrin in competitive binding assays.[9b]
Prompted by these observations, we synthesized two
cyclic isoDGR peptidomimetics (1, 2; Figure 2) containing
the bifunctional diketopiperazine scaffolds mentioned
above, and investigated their conformation in solution, and
their ability to compete with biotinylated vitronectin for
binding to the purified avb3 and avb5 receptors.
ACHTUNGTRENNUNG[DKP-RGD] (Figure 1) are low nanomolar ligands of integ-
rins avb3 and avb5.
Recent biochemical studies have shown that a spontane-
ous post-translational modification, occurring at the Asn-
Gly-Arg (NGR) motif of the extracellular matrix protein, fi-
bronectin, leads to the isoAsp-Gly-Arg (isoDGR) se-
quence.[5] This rearrangement, which involves the attack of
[a] M. Mingozzi, A. Dal Corso, Dr. M. Marchini, I. Guzzetti,
Dr. M. Civera, Dr. L. Belvisi, Dr. D. Potenza, Dr. L. Pignataro,
Prof. Dr. C. Gennari
Universitꢀ degli Studi di Milano, Dipartimento di Chimica
Via C. Golgi, 19, 20133, Milan (Italy)
E-mail: cesare.gennari@unimi.it
[b] Prof. Dr. U. Piarulli
Universitꢀ degli Studi dell’Insubria
Dipartimento di Scienza e Alta Tecnologia
Via Valleggio 11, 22100, Como (Italy)
E-mail: umberto.piarulli@uninsubria.it
The synthesis was conveniently performed on solid phase
(Fmoc strategy) using SASRINTM resin, following the strat-
egy depicted in Scheme 1. Fmoc-glycine was loaded onto
the resin and Fmoc-aspartic acid a-tert-butyl ester was cou-
pled. The trans DKP scaffolds (either 3R,6S or 3S,6R) were
then coupled to the supported dipeptide. The DKP scaffolds
are normally obtained as N-Boc protected amino acids,[4]
and as such are unsuitable for solid phase synthetic applica-
[c] Dr. D. Arosio, Dr. L. Belvisi, Dr. D. Potenza, Dr. L. Pignataro,
Prof. Dr. C. Gennari
CNR, Istituto di Scienze e Tecnologie Molecolari (ISTM)
Via G. Venezian, 21, 20133, Milan (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204639.
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were performed by incubating the immobilized integrins
avb3, and avb5 with increasing concentrations (10À12–10À5 m)
of the two isoDGR ligands in the presence of biotinylated
vitronectin (1 mgmLÀ1), and measuring the concentration of
bound vitronectin in the presence of the competitive li-
gands.
The IC50 values of compounds 1 and 2 reported in Table 1
(entries 1, 2) compare favorably with other isoDGR ligands,
such as the cyclopentapeptide cyclo[GisoDGRphg] (entry 3)
recently published by Kessler and co-workers,[9b,10] as well as
with known RGD ligands, such as the cyclic peptidomimetic
cyclo
AHCTUNGTRENNUNG
[(3S,6R)DKP-RGD],[4]
cyclopentapeptides
cyclo-
(RGDfV)[11] and Cilengitide[11d] (entries 4–6). In particular,
Figure 2. Cyclic isoDGR peptidomimetics containing the bifunctional di-
ketopiperazine scaffolds.
the new isoDGR ligand 2 is ten-times more potent in bind-
ing integrin avb3 than cyclo[GisoDGRphg], only 2–3-times
tions. In order to avoid an exchange of the nitrogen protect-
ing group, involving a deprotection–reprotection sequence,
an intermediate of DKP synthesis, namely the azido-acids 3
and 4 were used in this case. The azide reduction
on resin was performed by using trimethylphos-
less potent than the RGD ligands cyclo
and cyclo[RGDfV], and fifteen-times less potent than the
powerful RGD ligand Cilengitide.
ACHTUNGTREN[NUGN (3S,6R)DKP-RGD]
AHCTUNGTRENNUNG
Table 1. Inhibition of biotinylated vitronectin binding to avb3 and avb5 receptors.
phine in dioxane/water and Cbz-ArgACTHUNRTGNE(GNU Mtr) was fi-
Entry Ligand
IC50 [nm][a]
avb5
IC50 ratio
avb5/avb3
nally coupled. The linear precursors were cleaved
from the resin under controlled acidic conditions
(to avoid deprotection of the tert-butyl ester and
the Mtr group) and, after hydrogenolysis of the
benzyloxycarbonyl group, were subjected to macro-
lactamization. Final side chain deprotection and pu-
rification by RP-HPLC afforded the cyclic isoDGR
peptidomimetics 1 and 2 ready for biological studies
and conformational analysis.
avb3
1
2
3
4
5
6
cyclo
cyclo
U
46.7Æ18.2
9.2Æ1.1
89Æ19[b]
4.5Æ1.1
3.2Æ1.3
0.6Æ0.1[c]
220Æ84
312Æ21
n.d.
4.7
33.9
n.d.
33.1
2.3
cyclo[GisoDGRphg]
cyclo
cyclo
cyclo
149Æ25
7.5Æ4.8
N
11.7Æ1.5[c] 19.5
[a] IC50 values were calculated as the concentration of compound required for 50% in-
hibition of biotinylated vitronectin binding as estimated by GraphPad Prism software;
all values are the arithmetic mean ÆSD of triplicate determinations. [b] Determined
by a solid phase binding assay by using supported vitronectin, soluble avb3 integrin,
specific primary and secondary antibodies (see ref. [9b] and the note in ref. [10]).
[c] Calculated as the concentration of compound required for 50% inhibition of bioti-
Cyclic isoDGR peptidomimetics 1 and 2 were ex-
amined in vitro for their abilities to compete with
biotinylated vitronectin for binding to the purified
avb3 and avb5 receptors (Table 1). Screening assays nylated vitronectin binding.[11d]
Scheme 1. a) Fmoc-Gly-OH, DIC, cat. DMAP, DMF, 2 h; b) 20% piperidine in DMF; c) Fmoc-Asp(OH)-OtBu, DIC, HOAt, DMF; d) 2% piperidine
and 2% DBU in DMF; e) 3 or 4, DIC, HOAt, DMF, 18 h; f) Me3P, dioxane/water 4:1; g) Cbz-Arg
ACHTUNGTRENN(UNG Mtr)-OH, DIC, HOAt, DMF; h) 1% TFA in DCM;
i) H2, 10% Pd/C, THF/water 1:1; j) HATU, HOAt, DIPEA, 1.4 mM in DMF; k) TFA/thioanisole/EDT/anisole 90:5:3:2.
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Cyclic isoDGR Peptidomimetics
COMMUNICATION
À
The structure and connectivity of compounds 1 and 2
were unambiguously assigned from the mono- and bidimen-
sional 1H and 13C NMR spectra. The preferred conforma-
À(Asp) average distance of 8.6 ꢂ was
O. A CbACTHGNUTRENNU(G Arg) COO ACHTNUGTRENNUNG
obtained during the MC/SD calculations. A representative
energy minimized conformation featuring the 11-membered
ring hydrogen bond between Gly-NH and C(5)=O is shown
in Figure 3 (structure on the right).
1
tions of 1 and 2 were investigated by H NMR spectroscopy
of dilute H2O/D2O 9:1 solutions and by computational
methods, with the aim of rationalizing the affinity of these
compounds for the avb3 receptor at a molecular level. One-
dimensional 1H NMR spectroscopy experiments were con-
ducted to detect intramolecular hydrogen bonds, by measur-
NMR spectroscopy experiments performed on the
isoDGR peptidomimetic 2 suggest a different conformation-
al preference. In fact, compound 2 exists in two preferred
conformations (Figure 4a and b). The NOESY cross-peak
between DKP-NH10 and Arg-NH (strong) is indicative of a
b-turn conformation stabilized by a hydrogen bond between
DKP-NH10 and isoAsp-C=O (Figure 4a), whereas the
NOESY cross-peak between Gly-NH and isoAsp-NH
(weak) is indicative of a pseudo-b-turn motif (Figure 4b)
similar to that observed for compound 1.
À
ing the chemical shift of the amide N H protons, their tem-
perature coefficients (Dd/DT) and their chemical exchange
rate in the presence of D2O. The results are reported in the
Supporting Information. NOESY spectra were also recorded
to investigate both sequential and long-range NOE signals
that provide evidences of preferred conformations. Three-di-
mensional structures satisfying long-range NOE contacts
were generated by restrained mixed-mode Metropolis
Monte Carlo/stochastic dynamics (MC/SD) simulations,[12]
using the implicit water GB/SA solvation model.[13]
The data obtained from the NMR experiments (see the
Supporting Information) show that compound 1 (Figure 3)
is characterized by a high conformational flexibility. In fact,
Figure 4. Preferred intramolecular hydrogen-bonded patterns proposed
for the cyclic isoDGR peptidomimetic 2, on the basis of spectroscopic
data. The arrows indicate the NOE contacts. a) b-Turn at Gly-Arg;
b) pseudo-b-turn at DKP-isoAsp.
Only the distance restraint corresponding to the strong
NOE contact between DKP-NH10 and Arg-NH (Figure 4a)
was applied in the MC/SD simulations of compound 2. It is
worth noting that approximately 80% of the conformations
sampled during the simulations adopted an extended ar-
rangement of the isoDGR sequence, characterized by a
pseudo-b-turn at DKP-isoAsp and the formation of the cor-
responding hydrogen bond between Gly-NH and C(5)=O
(Figure 4b). In addition, the formation of a distorted b-turn
at Gly-Arg (Figure 4a), characterized by hydrogen bond dis-
tances of about 3.5 ꢂ between DKP-NH10 and isoAsp-C=
À
Figure 3. Left: preferred intramolecular hydrogen-bonded pattern pro-
posed for the cyclic isoDGR peptidomimetic 1, on the basis of spectro-
scopic data. The arrow indicates the NOE contact. Right: 3D structure of
1 as obtained by restrained MC/SD simulations based on experimental
distance information, after energy minimization [pseudo-b-turn at DKP-
À
isoAsp, CbACHTUNGTRENNUNG(Arg) COO ACHTUNGTRENNUNG
À(Asp) distance=8.8 ꢂ].
the Dd/DT values range from À4.7 ppbKÀ1 (Gly-NH) to
À8.5 ppbKÀ1 (DKP-NH10) and the N H chemical shifts in-
À
dicate that the amide protons of 1 are all solvent exposed.
However, the strong NOE contact between isoAsp-NH and
Gly-NH (Figure 3, structure on the left) indicates a prefer-
red folding through the formation of a pseudo-b-turn stabi-
lized by an intramolecular hydrogen bond between Gly-NH
and DKP-C(5)=O.
O, was observed for 10% of the simulations. A CbACHTUNGTRENNUNG(Arg)
COOÀ
ACHTNUTRGNEUNG(Asp) average distance of 10.7 ꢂ was obtained during
the MC/SD calculations of compound 2. Two representative
energy-minimized conformations featuring the two hydro-
gen bond patterns compatible with the strong NOE contact
between DKP-NH10 and Arg-NH are shown in Figure 5.
In order to rationalize, on a molecular basis, the affinity
of compounds 1 and 2 for the avb3 receptor, docking studies
were performed starting from the representative conforma-
tions obtained from the restrained MC/SD simulations. The
crystal structure of the extracellular segment of integrin avb3
complexed with the cyclic pentapeptide Cilengitide (PDB
The distance restraint corresponding to the NOE contact
between isoAsp-NH and Gly-NH was applied in the MC/SD
simulations of compound 1. Approximately 50% of the con-
formations sampled during the simulations adopted an ex-
tended arrangement of the isoDGR sequence characterized
by a pseudo-b-turn at DKP-isoAsp and the formation of the
corresponding hydrogen bond between Gly-NH and C(5)=
Chem. Eur. J. 2013, 19, 3563 – 3567
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3565

U. Piarulli, C. Gennari et al.
Figure 5. The 3D structures of compound 2 as obtained by restrained
MC/SD simulations based on experimental distance information, after
energy minimization. a) Distorted b-turn at Gly-Arg, populated for 10%
À
Figure 6. Best pose of compound 2 (atom color tube representation,
pseudo-b-turn at DKP-isoAsp) into the crystal structure of the extracellu-
lar domain of avb3 integrin (a unit red and b unit blue wire representa-
tion) overlaid on the bound conformation of Cilengitide (green tube rep-
resentation). Only selected integrin residues involved in the interactions
with the ligand are shown. The metal ion in the MIDAS region is repre-
sented by a magenta sphere. Nonpolar hydrogen atoms were removed
for clarity.
À(Asp) distance=10.7 ꢂ; b) pseudo-b-
of the simulation, CbACHTUNGTRENNUNG(Arg) COO CAHTUNGTRENNUGN
À
turn at DKP-isoAsp, populated for 80% of the simulation, CbACTHNUTRGNEU(GN Arg)
COO ACHTUNGTRENNUNG
À(Asp) distance=10.8 ꢂ.
ID: 1L5G) was taken as a reference model for the interpre-
tation of the docking results in terms of ligand–protein inter-
actions.[14] In the X-ray complex, Cilengitide binds to the in-
terface of the a and b units forming specific electrostatic in-
teractions. The acid and basic pharmacophoric groups and
their orientation are essential for binding to avb3, because
they act like an electrostatic clamp and interact with charg-
ed regions of the receptor binding site.
binding pocket of avb3 integrin, establishing the same elec-
trostatic clamp as well as additional key interactions.
Acknowledgements
Docking calculations starting from the pseudo-b-turn
DKP-isoAsp geometries of compound 1 (Figure 3, right)
and from the distorted b-turn Gly-Arg conformations of
compound 2 (Figure 5a), produced top-ranked poses con-
serving the key electrostatic interactions but lacking further
stabilizing hydrogen bond interactions with the b subunit
(e.g., hydrogen bonds of ligand carboxylate oxygen with the
backbone amides of Asn215 and Tyr122, and hydrogen
We thank Milan University for PhD Fellowships (to M.M., A.D.C., M.M.
and I.G.). We also gratefully acknowledge Ministero dell’Universitꢀ e
della Ricerca for financial support (PRIN project 2010NRREPL: Synthe-
sis and Biomedical Applications of Tumor-Targeting Peptidomimetics).
U.P. thanks Fondazione CARIPLO for a research grant (Project: Red-
Drug-Train).
Keywords: conformational analysis · diketopiperazines
molecular modeling · peptides · peptidomimetics
·
À
bonds between ligand backbone N H and protein backbone
carbonyl groups).
On the contrary, docking calculations starting from the
pseudo-b-turn DKP-isoAsp geometries of compound 2 (Fig-
ure 5b) produced top-ranked poses conserving all the impor-
tant interactions of the X-ray complex (Figure 6).
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ligand interacts with the negatively charged carboxylates of
Asp218 and Asp150 in the a unit, one carboxylate oxygen of
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Cyclic isoDGR Peptidomimetics
COMMUNICATION
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Received: December 31, 2012
that is, c
G
G
Published online: February 19, 2013
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