First experimental identification of Ras-inhibitor binding interface using a water-soluble Ras ligand

Article abstract of DOI:10.1016/j.bmcl.2009.05.107

By combining in the same molecule Ras-interacting aromatic moieties and a sugar, we prepared a water-soluble Ras ligand that binds Ras and inhibits guanine nucleotide exchange. With this compound it was possible to determine experimentally by a ¹⁵

Full text of DOI:10.1016/j.bmcl.2009.05.107

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4217–4222
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
First experimental identification of Ras-inhibitor binding interface using a
water-soluble Ras ligand
Alessandro Palmioli a, Elena Sacco a, Sherwin Abraham c, Celestine J. Thomas b, Alessandro Di Domizio a,
Luca De Gioia a, Vadim Gaponenko c, Marco Vanoni a, Francesco Peri a,
*
a Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2, 20126 Milano, Italy
b Center for Biomolecular Structure and Dynamics (CBSD), Chem-218, 32 Campus Dr, University of Montana, Missoula, MT 59812, United States
c Department of Biochemistry and Molecular Genetics University of Illinois at Chicago, 900 S. Ashland Ave. Chicago, IL 60607, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
By combining in the same molecule Ras-interacting aromatic moieties and a sugar, we prepared a water-
soluble Ras ligand that binds Ras and inhibits guanine nucleotide exchange. With this compound it was
possible to determine experimentally by a 15N-edited HSQC NMR experiment the ligand-Ras binding
interface.
Received 10 March 2009
Revised 26 May 2009
Accepted 27 May 2009
Available online 30 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ras protein
Sugars
Synthesis
Anticancer agents
NMR
Molecular modeling
Ras, a key member in the super-family of small GTPases, is an
essential component of signal transduction pathways that regulate
cell growth, proliferation, differentiation and apoptosis.1–3 Operat-
ing as molecular switches, Ras GTPases, assisted by guanine
exchange factors (GEFs), undergo nucleotide exchange, allowing
them to rapidly cycle from the inactive GDP4 bound state to the
activated GTP bound state. The active Ras, aided by GTPase activat-
ing proteins (GAPs), hydrolyses the bound GTP to GDP and returns
to the inactive GDP bound state.5,6 Alteration of this deactivating
reaction is a common biochemical defect associated with onco-
genic Ras mutants.7–9 Oncogenic Ras mutants exhibit decreased
hydrolytic efficiency due to reduced affinities towards GAP
proteins.10
Identifying small drug molecules that selectively interact with
oncogenic Ras proteins and inhibit constitutive Ras activation is a
challenging and a still largely unsolved task. The prolonged life-
times of activated oncogenic Ras mutants can, in principle, be
reduced by the action of drugs that are capable of converting the
oncogenic states to behave like normal phenotypes. This can be
achieved by molecules that either inhibit the process of GEF-cata-
lyzed GTP loading on inactive RasÁGDP11,12 or by simply accelerat-
ing the GTP hydrolysis in the active RasÁGTP complex.13–15 While
several efforts have been made in developing agents that acceler-
ate the rate of GTP hydrolysis (self-hydrolyzing GTP analogues),
very limited strategies directed at inhibiting GEF-catalyzed nucle-
otide exchange have been undertaken. To address this issue, our
group has been actively engaged in developing sugar-derived
ligands that can inhibit GEF-promoted guanine nucleotide
exchange on H-Ras. Several generations of compounds, composed
of a sugar moiety or a linear spacer to which aromatic pharmaco-
phore groups are covalently linked, were synthesized and evalu-
ated for Ras inhibitory activity.16–20 These small organic
molecules (whose MW ranges from 300 to 500 Da) bind to H-
RasÁGDP with micromolar affinity and inhibit GEF-Ras interaction
thereby preventing guanine nucleotide exchange. All of these
inhibitors seem to recognize a common binding site on Ras. In sil-
ico docking studies indicated that amino acid residues Y96, G60,
Q61 and E62 of H-Ras exhibit strong interactions with these inhib-
itors. Hence a binding interface that partially overlaps with the
Switch II region (residues 60–76) was identified to be a plausible
binding pocket.17,18 Our earlier experiments using Surface Plasmon
Resonance (SPR) studies indicated that arabinose and glucose-de-
rived compounds interfere with the interaction of GEF molecules
with H-Ras, reinforcing our hypothesis that these inhibitors bind
to a site proximal to the Switch II region.20 However, the exact
mode of action of these nucleotide exchange inhibitors is currently
unknown since no structure of an inhibitor bound RasÁGDP
* Corresponding author.
E-mail address: francesco.peri@unimib.it (F. Peri).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.107

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A. Palmioli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4217–4222
complex is available. The low aqueous solubility of these
compounds hampered meaningful NMR and crystallography
studies and therefore most insights were derived only from
computational analysis. Indeed numerous efforts by our group to
co-crystallize the hydroxylamine-containing inhibitors with Ras
were unsuccessful.21 The extremely low water solubility of these
compounds necessitated the addition of at least 10% methanol that
resulted in aggregation and precipitation of H-Ras, leading to a
generalized shift of cross-peaks in 2D NMR spectra.
provides, over traditional chemical glycosylation reactions, is the
use of unprotected and non-activated reducing sugar moieties as
glycosyl donors. Additionally, the final neoglycoside product is
obtained stereoselectively (at least in the case of glucose, galactose
and glucosamine-N-acetyl) in the b-anomeric configuration. This
highly convergent and efficient synthetic strategy can be effec-
tively employed for the preparation of an array of potential Ras
ligands, in which different reducing mono- or oligosaccarides are
combined with N,O-disubstituted hydroxylamines through a b-N
glycosidic linkage.
For the synthesis of compound 1, commercially available 3,4-
dihydroxybenzaldehyde was reacted with O-benzylhydroxylamine
hydrochloride in pyridine at rt, 12 h, affording the corresponding
O-benzyl oxime 2 with 98% yield. The oxime was reduced to the
4-[(benzyloxyamino)methyl]benzene-1,2-diol 3 by treatment with
NaCNBH3 in acetic acid at r.t. for 2 h (80% yield). The hydroxyl-
O
OH
OH
O
O
HO
HO
O
N
O
HO
HO
N
S
OH
O
OH
amine 3 and D-glucose were then reacted in DMF/AcOH/aqueous
acetate buffer pH 4.5 (1:1:1) at 50 °C overnight affording com-
pound 1 in 80% yield.
HOHN
In contrast to all Ras inhibitors (including SCH-54292) synthe-
sized and tested by our group, compound 1 was clearly soluble in
water or TRIS buffers to a concentration of several mM, and was sta-
ble at neutral pH for months (as assessed by TLC and HPLC analyses).
Isothermal titration calorimetric (ITC) studies were performed
to quantify the affinity of compound 1 towards H-Ras (1–166). This
OH
HO
1
SCH-54292
Structures of compounds SCH-54292 and 1.
In this Letter we present the first experimental characterization
of the H-Ras binding site using a rationally designed water-soluble
H-Ras ligand. The design of compound 1 was inspired by the N-glu-
cosylated sulphonamide SCH-54292 previously synthesized by
Schering Plough laboratories and used in NMR experiments to
evaluate binding to H-Ras.12 We re-synthesized compound SCH-
54292 and found it to be almost completely insoluble at the
concentrations required for NMR-based binding experiments.
Compound 1 was tailored specifically to overcome this insolubility
issue associated with previously developed H-Ras inhibitors. Our
molecule is composed of an O-Benzyl-N-(3,4-dihydroxybenzyl)
molecule was able to bind to H-Ras with an affinity of 37 lM and
with an enthalpy of binding of À1964 40 cal/mol (error was cal-
culated on the basis of three independent titrations).
Compound 1 exhibited exothermic heats in ITC profile. Interest-
ingly the stoichiometric value of the compound 1: H-Ras interac-
tion was 1:1 (equimolar). Figure 1S (Supplementary data)
indicates the binding to H-Ras to compound 1.
Compound 1 was then tested for its ability to bind H-RasÁGDP
(1–166) by NMR experiments in solution. The titration of com-
pound 1 into a solution of 15N H-Ras (1–166) was followed by
15N-edited HSQC experiments that allowed delineation of the
Ras–ligand binding interface. A series of spectra was collected at
different Ras:ligand molar ratios. The results of these experiments
are depicted in Figure 1. The residues exhibiting statistically signif-
icant chemical shift perturbations are marked on the graph. Upon
titration with 1, several cross-peak chemical shifts changed, while
most signals did not show significant perturbations (Fig. 1A). The
changes in chemical shifts were concentration-dependent as
shown in Figure 1B–D. The presence of fast exchange on the
NMR time-scale evident in Figure 1B–D is in agreement with low
micromolar binding affinity of 1 for H-RasÁGDP as observed in
ITC studies. The statistically significant chemical shift perturba-
tions were observed in the b-3 strand (residues I55, T58, A59,
hydroxylamine moiety that is N-glycosylated with D-glucose. To
ensure water-solubility, the hydrophylic sugar and dihydroxyben-
zyl moieties were included in the design of 1, and the hydrophobic
naphthyl of SCH-54292 was replaced with a benzyl group. The key
step in the synthesis of 1 is the chemoselective glucosylation of the
O-benzyl-N-(3,4-dihydroxybenzyl) hydroxylamine 3 (Scheme 1).
Our group pioneered the chemoselective formation of glycosidic
bonds between reducing sugars and a N,O-disubstituted hydroxyl-
amines to form ‘neoglycosides’ and this method is now extensively
used by other research groups for the preparation of libraries of
differently glycosylated bioactive compounds ranging from
neoglycopeptides, neooligosacchardies to neoglycosteroids and
antibiotics.22–27 The notable advantage that this approach
and Y64) of the central b-sheet and
a-2 helix (residues M67, R68,
OH
O
O
NH2
OH
O
HO
HO
O
H
O
NH
OH
O
N
b
OH
a
O
HO
HO
N
+
c
OH
OH
OH
OH
OH
OH
OH
HO
OH
2
3
1
Scheme 1. Chemoselective synthesis of compound 1. Reagents and conditions: (a) dry pyridine, 98%; (b) NaCNBH3, glacial AcOH, 80%; (c) DMF/AcOH/aqueous acetate buffer
at pH 4.5, 80%.

A. Palmioli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4217–4222
4219
Figure 1. NMR titration of H-RasÁGDP 1–166 with compound 1. (A) 15N HSQC spectra of H-RasÁGDP 1–166 with 0 (blue) and 20 M equiv of compound 1 (red) are
superimposed. Examples of chemical shift perturbations are marked. Spectral inserts for Y64 (B), R68 (C), and M72 (D) show addition of 0 (blue), 1, 2, 5, 10, and 20
(red) M equiv of compound 1. The arrows mark the direction of chemical shift changes. (E) normalized HN chemical shift perturbations. The boxes and arrows above the
graph represent helices and sheets in H-RasÁGDP 1–166.28 The horizontal line marks the average value of chemical shift perturbations plus one standard deviation.
Y71, M72, R73, T74 and G75) as shown in Figure 1E. Residues K5
and V103 are adjacent to the b-3/ -2 region and complete a
continuous binding site of the drug lead compound on the catalytic
domain of H-RasÁGDP.
a

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A. Palmioli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4217–4222
Figure 2. (A) Contact maps and (B) 3D representations of the two most representative lowest-energy protein–ligand complexes (termed as A and B) as obtained from MD
calculations starting from the best docking results of compound 1.
The crosspeak shifts observed in HSQC experiments are caused
by a variation in the electromagnetic environment of the amino
acids. This may indicate direct binding with ligand or structural
rearrangement of the protein due to binding. In the case of residues
E143 and K147 that do not belong to the continuous binding site,
the chemical shift variations outlined in Figure 1E may be due to
a remote or propagated structural change of Ras induced by ligand
binding.
Exchange assays were performed in the presence of increasing
concentrations of compound 1 (0, 50, 100, 250 and 500 M)
(Fig. 3A). The initial rate of each exchange reaction, calculated as
described in Supplementary data was plotted as a function of
compound concentration (dose–response curve, Fig. 3B). It is evi-
l
dent that at 100 lM compound 1 induce about a fifty percent of
inhibition of the GEF-catalyzed exchange rate on H-Ras. Circular
Dichroism experiments (data not shown) demonstrated that bind-
ing of compound 1 to Ras does not induce protein denaturation nor
damages the protein structure.
To verify the activity of 1 on cells, experiments that monitor the
rate of proliferation were performed using NIH3T3 mouse fibro-
blasts. Cells were plated at a density of 3000 cells/cm2 and left to
adhere onto plastic surface for 18 h. Then cells were treated with
The NMR binding data were complemented by molecular
dynamic (MD) and docking experiments. Assuming the cleft in the
vicinity of the Switch II region of H-Ras as the receptor binding site,
we docked compound
1 into the rigid structure of human
H-Ras derived from crystallographic coordinates (PDB code: 4q21).
Two principal clusters of ligand arrangements were obtained and
docking energies of the two lowest energy protein-ligand complexes
are À6.41 and À5.91 kcal/mol, respectively (Fig. 2).
1 (at concentrations 0, 50, 150 and 450 lM) supplemented in the
growth medium, and counted at 24, 48 and 72 h after treatment.
The obtained growth curves (Fig. 4A) and morphological analysis
Explicit solvent MD simulations were then carried out starting
from the best protein-ligand complexes obtained by docking calcu-
lations, with the aim of refining and improving docking results
including solvent effects and accounting for induced fit.29 The
MD refined structures, obtained starting from cluster A and B
geometries, were then analyzed, with the aim of highlighting
protein residues for which different chemical shifts are expected
to deviate from the unbound (H-RasÁGDP) to the bound (H-
RasÁGDPÁ1) structures. In particular, protein residues which drasti-
cally change their chemical environment are listed in Table 1S
(Supplementary data).
of treated cells (Fig. 4B) demonstrate that compound
150 M effectively inhibits cell proliferation. In fact the number
of cells treated with 150 M and 450 M 1 at 24 h from treatment
1 at
l
l
l
is lower than control and does not increase later in time. Accord-
ingly, in the culture medium of treated cells (Panel B) it is possible
to appreciate many dead cells in suspension (refrangent cells
circled in white).31,32
The data presented here point out that compound 1 binds to
H-Ras with low affinity but it is capable of inhibiting GEF-mediated
nucleotide exchange. The ITC experiments conclude that
In order to correlate binding data with biochemical activity, the
inhibitory effect of compound 1 on the GEF-catalyzed exchange of
H-Ras-bound GDP was tested. The catalytic domain of Ras-specific
GEF RasGRF1 was used to promote exchange as described earlier.30
compound 1 has a
Ras–ligand complex is formed.
NMR and MD based experimental results converge to indicate
that certain H-Ras residues are critical for ligand binding. These in-
l
M affinity towards H-RasÁGDP and that 1:1

A. Palmioli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4217–4222
4221
A 100
90
80
70
60
50
40
30
20
10
0
)
L
R
T
(C
0
500
1000
1500
2000
2500
3000
3500
4000
Time (s)
120
B
100
80
60
40
20
0
0
100
200
300
400
500
600
[ Compound 1 ]
Figure 3. Effect of compound 1 on GEF-catalyzed nucleotide exchange of H-Ras. (A) Stimulation of the GDP to mantGDP exchange reaction on H-Ras determined adding to
0.25 M RasGRF1 and different concentrations (0, 50, 100, 250 and 500 M) of compound 1. Fluorescence measurements were
M H-RasÁGDP, 1.25 mM mantGDP, 0.0625
l
l
l
made with a Perkin–Elmer LS-45 luminescence spectrometer, monitoring the fluorescence each second for at least 1500 sec. For graphical reason in figure only a point every
15 has been plotted. Each experimental curve was fitted to a non linear ‘growth-sigmoidal Hill’ curve (n = 1) with OriginPro 8.0 software, reported in graph as a thin line. In
the graph the maximum value of relative fluorescence (100 on Y-axis) represent the fully charged Ras status obtained as plateau of an exchange curve without compound. For
each reaction the initial exchange rate was calculated as first derivative at time 0 with OriginPro 8.0 software. (B) Dose–response curves of compound 1 on GEF-catalyzed
exchange of Ras. The initial exchange rate of each exchange reaction (mean of at least three independent experiments) was plotted as a function of the compound
concentration (dose–response curves). The exchange rate of control reaction (performed without compound) was normalized to 100.
clude amino acids T58, A59 and Y64 belonging to the b-3 strand
and R68, M72 on the -2 helix. The observed binding site covers
inhibitor binding to H-Ras, and how these may affect Ras-modu-
lated downstream signaling events.
a
a region that is essential for Ras interactions with the Ras–GEF
domain of RasGRF1, homologous to SOS, as described by the crystal
structure of the Ras–SOS complex.33 Therefore, it is possible that
compound 1 interferes with H-Ras–GEF interaction, as previously
outlined using SPR experiments.20 The affinity of binding to H-
Ras as calculated by ITC experiments (Fig. 1S) is in the same order
of magnitude of the affinity between RasÁGDP and GEF.34 This
means that compound 1, although having low affinity for Ras,
can compete effectively with GEF for Ras binding thus inhibiting
Ras activation.
In conclusion, because of the favorable solubility of the
designed ligand, we were able to determine experimentally the
Ras-inhibitor binding site as well as fundamental thermodynamic
binding parameters. The involvement of sections of the Switch II
region in inhibitor binding was previously hypothesized by us on
the base of NMR STD experiments and is now conclusively demon-
strated in this report. We are currently investigating whether
global or allosteric conformational changes can occur upon
We consider the development of soluble inhibitors against
mutant Ras super-family of GTPases as one of the strongest routes
to provide clinically effective cancer therapeutics. We propose that
our design and evaluation of small molecule inhibitors, such as
compound 1, may serve as templates on which future pharmaco-
logically relevant drug design studies can be attempted.
Acknowledgements
The authors wish to thank Annalisa D’urzo for technical assis-
tance. Dr. C. J. Thomas would like to thank Dr. Stephen R Sprang
(SRS), Director of CBSD, University of Montana for his support in
both space and instruments towards this project and also for the
salary support from National Institutes of Health Grants
DK46371. Dr. V. Gaponenko acknowledges the generous support
from the American Cancer Society Grant RGS-09-057-01-GMC.
Professor M. Vanoni acknowledges Creabilis and MIUR-FAR for
financial support.

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A. Palmioli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4217–4222
A
NIH-3T3 treated with compound 1
1000000
100000
0 µM (CTRL)
50 µM
150 µM
450 µM
10000
NIH-3T3 (48h)
compound 1
0
24
48
72
96
Time (h)
50µM
150µM
450µM
B
CTRL
Figure 4. (A) Proliferation curves of NIH3T3 fibroblasts grown in media supplemented with different concentrations of compound 1: 0
l
M (grey diamond; CTRL = control),
50 M (black square), 150 M (black triangle) and 450 lM (black circle). Cells were counted after 24, 48 and 72 h of treatment with compound 1. Plotted data are mean s.d.
l
l
computed from at least three independent experiments. (B) Morphological analysis of cells treated for 48 h with different concentrations of compound 1 (or buffer, CTRL).
White circles indicate floating dead cells detached from the plate.
15. Ahmadian, M. R.; Zor, T.; Vogt, D.; Kabsch, W.; Selinger, Z.; Wittinghofer, A.;
Supplementary data
Scheffzek, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7065.
16. Colombo, S.; Peri, F.; Tisi, R.; Nicotra, F.; Martegani, E. Ann. NY Acad. Sci. 2004,
Synthetic procedures, products characterization, biochemistry
experiments, ITC experiments, NMR experiments, cell biology.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.107.
1030, 52.
17. Peri, F.; Airoldi, C.; Colombo, S.; Martegani, E.; van Neuren, A. S.; Stein, M.;
Marinzi, C.; Nicotra, F. ChemBioChem 2005, 6, 1839.
18. Peri, F.; Airoldi, C.; Colombo, S.; Mari, S.; Jiménez-Barbero, J.; Martegani, E.;
Nicotra, F. Eur. J. Org. Chem. 2006, 3707.
19. Mueller, C.; Gomez-Zurita Frau, M. A.; Ballinari, D.; Colombo, S.; Bitto, A.;
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Peri, F. ChemMedChem 2009, 4, 524.
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