Development of a Nucleotide Exchange Inhibitor That Impairs Ras Oncogenic Signaling

Article abstract of DOI:10.1002/chem.201604905

Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras–guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switch II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.

Full text of DOI:10.1002/chem.201604905

A Journal of
Accepted Article
Title: Development of a nucleotide exchange inhibitor that impairs Ras
oncogenic signaling
Authors: Maria Luz Lopez-Rodriguez, Nagore I. Marin-Ramos, Carmen
Piñar, Henar Vazquez-Villa, Mar Martín-Fontecha, Angel
Gonzalez, Angeles Canales, Sergio Algar, Paloma P.
Mayo, Jesús Jimenez-Barbero, Consuelo Gajate, Faustino
Mollinedo, Leonardo Pardo, Silvia Ortega-Gutierrez, and Alma
Viso
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To be cited as: Chem. Eur. J. 10.1002/chem.201604905
Link to VoR: http://dx.doi.org/10.1002/chem.201604905
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Development of a nucleotide exchange inhibitor that impairs Ras
oncogenic signaling
Nagore I. Marín-Ramos,#[a, b] Carmen Piñar,#[a] Henar Vázquez-Villa,[a] Mar Martín-Fontecha,[a] Ángel
González,[c] Angeles Canales,[a] Sergio Algar,[a] Paloma P. Mayo,[a] Jesús Jiménez-Barbero,[d,e]
Consuelo Gajate,[f] Faustino Mollinedo,[f] Leonardo Pardo,[c] Silvia Ortega-Gutiérrez,[a] Alma Viso,[g] and
María L. López-Rodríguez*[a]
Abstract: Despite more than three decades of intense effort, no
anti-Ras therapies have reached clinical application. Contributing to
Introduction
this failure has been an underestimation of Ras complexity and a
dearth of structural information. In this regard, recent studies have
revealed the highly dynamic character of Ras surface and the
existence of transient pockets suitable for small-molecule binding,
opening new possibilities for the development of Ras modulators. In
this work we describe a novel Ras inhibitor (compound 12) that
selectively impairs mutated Ras activity in a reversible manner
without significantly affecting wild-type Ras, reduces the Ras-GTP
levels, inhibits the activation of the MAPK pathway, and exhibits a
remarkable cytotoxic activity in Ras-driven cellular models. The use
of molecular dynamics simulations and nuclear magnetic resonance
experiments has enabled to explore the molecular bases for the
interaction between compound 12 and Ras protein. The new Ras
inhibitor binds partially to the GTP-binding region and extends into
the adjacent hydrophobic pocket delimited by switch II. Hence, Ras
inhibitor 12 could represent a new compound for the development of
more efficacious drugs to target Ras-driven cancers, a currently
unmet clinical need.
Ras proteins are small GTPases that regulate signaling
pathways critical for proliferation and cell survival. Ras has three
isoforms, H-Ras, N-Ras and K-Ras. All of them oscillate
between an active GTP-bound and an inactive GDP-bound state.
This equilibrium must be tightly controlled, as the signaling
becomes oncogenic when it is deregulated.[1] It is known that
Ras over-activation in tumors is usually associated with a single
mutation typically at codon 12, 13, or 61. Mutation at these
conserved sites favors GTP binding and produces constitutive
activation of Ras. Aberrant Ras function is associated with
around 20 to 30% of all human cancers,[2] fact that makes Ras a
therapeutic target of remarkable interest in oncology. However,
after three decades of intense efforts in pharmaceutical industry
and academia, no direct inhibitors of Ras have been marketed,
circumstance that has reinforced Ras as the paradigmatic
example of undruggable protein. Nonetheless, the interest in
Ras inhibition has not decreased with time and the last years
have witnessed important advances in the field.[3] In general, two
approaches have been explored for inhibiting Ras activity: (i) the
direct inhibition of Ras and (ii) the indirect blockade of Ras
activity by interfering with the enzymes involved in its
posttranslational modifications.[4] In addition, direct Ras inhibitors
can be further divided into compounds that act by impairing the
Ras catalytic activity or molecules that block the activation of the
Ras-induced signaling pathway by hampering Ras interaction
with its effector proteins (protein-protein interaction inhibitors).[5]
During these years, the different strategies have raised hopes
that have been in general followed by profound
disappointments,[3d] but they have undoubtedly contributed to
increase the understanding of Ras structure and function, and
have set the basis for the current research directions. In this
context, it is generally considered that targeting Ras activity by
interfering with the enzymes involved in its posttranslational
modifications (such as farnesyl transferase) or blocking the
interaction between Ras and its effectors (such as the Raf
serine/threonine kinases, phosphoinositide 3-kinase, Ral or Rac
proteins) can have drawbacks such as the existence of
alternative prenylation,[6] a lack of selectivity, or the appearance
of resistances.[4a, 7] On the contrary, the direct inhibition of Ras
has generally faced with a lack of efficacy due to the high affinity
of Ras towards GDP and GTP and the high intracellular
nucleotide concentrations together with the lack of evident
druggable hydrophobic pockets in the flat surface of Ras.[3c, 5a]
However, recent results have challenged this idea. Ras surface
[a]
Dr. N.I. Marín-Ramos, Dr. C. Piñar, Dr. H. Vázquez-Villa, Prof.
Dr. M. Martín-Fontecha, Prof. Dr. A. Canales, MSc. S. Algar,
P.P. Mayo, Prof. Dr. S. Ortega-Gutiérrez, Prof. Dr. M. L. López-
Rodríguez,
Departamento de Química Orgánica I, Facultad de Ciencias
Químicas, Universidad Complutense de Madrid
E-28040 Madrid, Spain
E-mail: mluzlr@ucm.es
[b]
[c]
[d]
Dr. N.I. Marín-Ramos
CEI Campus Moncloa, UCM-UPM and CSIC
E-28040 Madrid, Spain
Dr. A. González, Prof. Dr. L. Pardo
Universitat Autònoma de Barcelona
E-08193 Bellaterra, Barcelona, Spain
Dr. J. Jiménez-Barbero
CIC bioGUNE, Parque Tecnológico de Bizkaia, Edif. 801A
48160 Derio, Spain
[e]
[f]
Ikerbasque, Basque Foundation for Science
48103 Bilbao, Spain
Dr. C. Gajate, Dr. F. Mollinedo
Department of Cellular and Molecular Medicine
Centro de Investigaciones Biológicas, CSIC
E-28040 Madrid, Spain
[g]
Dr. A. Viso
Instituto de Química Orgánica General, IQOG-CSIC
Juan de la Cierva, 3, E-28006 Madrid, Spain
These authors contributed equally to this work
[#]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201604905
Chemistry - A European Journal
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is highly dynamic and the protein exists in different
conformational states where transient pockets suitable for small-
molecule ligands are created.[8] These alternative sites, different
from the canonical nucleotide-binding site, are being explored
and structurally characterized, leading to the identification of
new compounds that behave as direct Ras inhibitors either in a
reversible[9] or irreversible[10] manner. These results clearly
support the notion that small molecules can indeed successfully
modulate Ras activity, although the compounds described up to
date still require further optimization in terms of activity, efficacy,
and potential toxicity liabilities.[3b] Hence, the ideal Ras direct
inhibitor should selectively inactivate mutant Ras to minimize
effects on wild-type Ras in order to reduce toxicity, and
decrease Ras-GTP levels so as to block the signaling pathways
that promote cell proliferation. With the aim of contributing to
such endeavor, in this work we describe the identification of a
new Ras direct inhibitor which selectively impairs Ras mutated
activity in a reversible manner without significantly affecting wild-
type Ras, reduces the Ras-GTP levels, inhibits the activation of
the mitogen-activated protein kinase (MAPK) and AKT pathways,
and exhibits a remarkable cytotoxic activity in Ras-driven cellular
models. This new compound adds to the scarce efficient direct
reversible Ras inhibitors described up to date and should help
developing K-Ras inhibitors suitable for treatment of specific
tumors with current bad prognosis, either by themselves or in
combination therapies in order to increase efficacy or to
decrease the appearance of resistances.
approach shown in Figure 1 as a possibility to improve biological
activity. In the initial design, the envisioned modification would
yield an aniline, but taking into account that this moiety has
raised structural alerts associated to potential side toxicities,[11]
we considered instead its isostere ether 3 (Figure 1, R’ = H).
This compound, synthesized as depicted in Scheme 1, showed
good cytotoxicity values in all K-Ras mutated cells (IC50 values
between 22 and 15 μM, see Table 2). Hence, it was selected for
further optimization.
Table 1. Cytotoxicity of compounds 1 and 2 in four mutated K-Ras
cancer cell lines
1, 2
Cpd
R
Cytotoxicity (IC50, μM)[a]
NIH-3T3
(G12V)
HCT-116
H-460
MDA-MB-231
1
-OCOPh
-OH
43
23
44
14
40
18
47
32
2
[a] Cytotoxicity was evaluated by the MTT assay (see Experimental
Section for details). IC50 values are the means from two to three
independent experiments performed in triplicate. In all cases, the SEM
is within 10% of the mean value.
Results and Discussion
Hit identification
The ExonHit proprietary library (1000 compounds) was screened
for its capacity to induce cytotoxicity in a panel of several cancer
cell lines characterized by the presence of mutated K-Ras,
which is the most frequently mutated Ras isoform. The cell lines
HCT-116 (G13D), H-460 (Q61H), and MDA-MB-231 (G13D)
were selected as representative of colon, lung, and breast
carcinomas with mutated K-Ras as indicated by the American
type cell collection (ATCC). Taking into account that up to 80%
of K-Ras mutations occur at codon 12 (G12X, being X different
amino acids such as aspartic acid, valine, or cysteine),[2] NIH-
3T3 cells transfected with the G12V mutant K-Ras were also
included in the panel. These assays led to the identification of
compound 1, with moderate values in the four cell lines, as an
initial hit (Table 1). Aimed at improving its biological activity, we
carried out some structural modifications in the ester group,
including its replacement by hydroxyl, ether, carbamate, and
amine groups (Supporting Table S1). The new derivatives were
synthesized and screened for cytotoxicity in the mutated K-Ras
cellular panel (Scheme S1, Table S1). They were analyzed
sequentially in the four cell lines and only compounds with IC50
values below 25 μM proceeded along the assays. Among them,
derivative 2 turned out to be the best one within the series
Figure 1. Design of compound 3 by the disconnection carried out in indole 2.
Optimization of compound 3
Through the optimization process, different substituents were
included in the phenyl ring of compound 3. We focused our
attention in the introduction of halogen atoms such as fluorine
and chlorine. This decision was based on the positive role of
fluorine and chlorine atoms in the pharmacokinetic properties,
metabolic stability, and bioavailability.[12] Hence, compounds 4-
12 were synthesized as shown in Scheme 1. Williamson
reaction of kojic acid with 1,5-dibromopentane, followed by
alkylation of intermediate 13 with the corresponding phenol
derivative afforded target final compounds. Non-commercial
intermediate phenols 14-17 were obtained by Claisen
rearrangement under microwave conditions of allyl aryl ethers
regarding cytotoxicity (Table 1) and hydrophobicity (cLogP = 2.4). 18-20 and subsequent catalytic hydrogenation of allyl derivatives
The fact that the rest of the analyzed indoles (see Table S1)
were poorly cytotoxic, led us to consider the disconnection
21-24. Compounds 3-12 were screened for cytotoxicity at the
four mutated K-Ras cell lines (Table 2). Within this series, the
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best results were obtained for compound 12, with IC50 values
equal or less than 15 μM in all analyzed cell lines. Table 2 also
includes the corresponding data for 2-(4,6-dichloro-2-methyl-1H-
indol-3-yl)ethanamine (DCAI),[9b] one of the scarce Ras direct
inhibitors described to date, as comparison. Finally, we
assessed the importance of the two chlorine atoms as well as
the influence of the spacer (see Table S2 and Scheme S2) but
none of these modifications improved the activity of compound
12, so it was selected for further biological studies focused on
confirming whether 12 was in fact affecting Ras activity in a
direct manner. To carry out these studies, MDA-MB-231 cell line
was chosen as an appropriate cellular model considering that
compound 12 showed the best IC50 value in this cell line (Table
2).
Table 2. Cytotoxicity of compounds 3-12 in four mutated K-Ras cell lines.
3-13
Cpd
Ar
Cytotoxicity (IC50, μM)[a]
bNIH-3T3
(G12V)
HCT-116 H-460
MDA-MB-231
15
O
O
O
3
4
5
6
7
8
9
22
50
19
ND[b]
ND
15
15
HO
Br
O
a
( )
5
OH
OH
O
ND
ND
12
ND
ND
27
13
Rn
OH
b
>50
20
14-17 or
commercially available
O
O
O
O
( )
5
Rn
OH
3-12
R = Pr, F, Cl, CF3
>50
>50
10
ND
ND
10
ND
ND
20
ND
ND
25
n = 1 or 2
Pr
c
d
Cln
Cln
Cln
OH
OH
21-24
O
18-20
14-17
Scheme 1. Synthesis of compounds 3-12: a) 1,5-dibromopentane, K2CO3, NaI,
DMF, 80 ºC, 24 h, 54%; b) K2CO3, NaI, 2-butanone, 80 ºC, 24 h, 53-91%; c)
MW, 210 ºC, 45 min, 20-66%; d) H2, 10% Pd/C (H-Cube reactor), EtOH, rt, 92-
98%.
27
17
8
ND
15
15
ND
30
7
ND
ND
14
10
11
Inhibition of Ras activity by compound 12
Oncogenic mutant Ras is locked in the active GTP-bound state,
leading to constitutive stimulation of its downstream signaling
pathways, which involve a plethora of pro-tumor effects including
a sustained proliferative signaling and an increase of the cell
migration. Hence, a Ras inhibitor should reduce all these effects.
Notably, compound 12 induced a remarkable decrease in the
GTP-bound (active) form of Ras in MDA-MB-231 cells (Figure 2),
significantly larger than that induced by DCAI, used for
comparison (Supporting Figure S1). As expected, this reduction
in the active form of Ras translated into an inhibition of the main
Ras-induced signaling pathways, including the downstream
kinases AKT, MEK, and ERK, all of them related with cellular
proliferation (Figure 3).
12
DCAI
14
ND
25
ND
[a] Cytotoxicity was evaluated by the MTT assay (see Experimental Section for
details). IC50 values are the means from two to three independent experiments
performed in triplicate. In all cases, the SEM is within 10% of the mean value.
[b] ND, not determined.
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show that in control conditions (Figure 4A) MDA-MB-231 cells
can readily migrate and only 37% and 6% of the initial wound
area remains free of cells after 24 h and 72 h, respectively.
However, in the presence of 25 μM of compound 12, cell
migration was considerably blocked and after 24 h and 72 h, the
71% and the 59%, respectively, of the initial wound area still
remained free of cells (Figure 4B). In order to rule out general
cytotoxicity and to study the reversibility of the effect, we
performed an additional experiment where the compound was
removed after the first 24 h and the cells were incubated for an
additional 48 h. Under these conditions (Figure 4C), the cells
recovered their ability to migrate and only the 14% of the initial
wound area remained free of cells (Figure 4C, right panel), being
this value comparable to the 6% obtained under the control
conditions (Figure 4A, right panel). In further support of these
results, direct quantification of the number of migrated cells was
also determined using a Boyden chamber, and after 24 h, cell
migration was inhibited in about a 40% (Figure 4E). Importantly,
compound 12 also affected the ability of MDA-MB-231 cells to
grow in the absence of anchorage (Figure 5A) and their invasive
capacity (Figure 5B) in a dose-dependent manner. In summary,
all these results suggest that the new Ras inhibitor 12 can
efficiently prevent the metastatic activity induced by mutated
Ras.
Figure 2. Compound 12 significantly reduces the levels of the Ras-GTP
complex (active form). MDA-MB-231 cells were treated with compound 12 (25
μM), lysed, and Ras-GTP complex was immunoprecipitated and then
visualized by western blot. The bar graph shows the ratio Ras-GTP/total Ras
(T-Ras), expressed as percentage relative to DMSO after band intensity
quantification. Images are representative of four independent experiments. **,
P<0.01 vs DMSO (Student’s t test).
Specificity of Ras inhibition
Another important aspect was to confirm the generality of these
results in other tumor cell lines as well as the specificity of Ras
inhibition. Hence, we selected a broader tumor cell panel (Table
3) that included cells with mutant K-Ras (such as the PANC-1
and MIA PaCa-2 pancreatic carcinoma cell lines, or the colon
SW-620 cell line) but also with wild type K-Ras (BxPC-3 or MCF-
7 cell lines). This fact is important because it is considered that
compounds that selectively inhibit mutant K-Ras but not its wild
type counterpart are less prone to induce side cellular toxicity.
Remarkably, compound 12 exhibited increased cytotoxicity
toward human cancer cells that featured a mutated oncogenic K-
Ras versus those that are not K-Ras mutated. For example, the
IC50 values for PANC-1 and MIA PaCa-2 were 17.3 and 10.2 μM,
respectively, whereas in the case of BxPC-3 cells, the
compound showed an IC50>50 μM (Table 3). Similarly, for breast
cancer, the IC50 value for MDA-MB-231 cells was 7 μM and for
MCF-7 was 50 μM. To further confirm the mechanism of action,
we checked that in the highly aggressive PANC-1 the compound
was inhibiting the activation of K-Ras (Supporting Figure S2)
and we also compared the cytotoxicity values showed by
inhibitor 12 with those of doxorubicin, a marketed anticancer
drug used for treating different types of cancer with a different
mechanism of action (doxorubicin is an intercalating DNA agent).
As expected, doxorubicin shows similar IC50 values regardless
Ras status (Table 3). In addition, to further rule out general
cytotoxicity, we evaluated the specificity of compound 12 in four
different non-tumor cell lines: NIH-3T3 fibroblasts, lung MRC-5
cells, breast MCF-10A cells, and HPNE pancreatic cells.
Importantly, Ras inhibitor 12 did not induce significant
cytotoxicity (IC50>50 μM) in these cells. Furthermore, in
agreement with the dependence on the presence of mutated
Figure 3. Compound 12 significantly inhibits the phosphorylation (activation)
of the Ras effector kinases AKT, MEK, and ERK. Lysates were obtained from
MDA-MB-231 cells treated with vehicle (DMSO) or compound 12 (25 M).
Representative western blots of phosphorylated (p-AKT) and total AKT (T-
AKT), phosphorylated (p-MEK1/2) and total MEK1/2 (T-MEK1/2), and
phosphorylated (p-ERK1/2) and total ERK1/2 (T-ERK1/2) are shown. The bar
graphs represent the optical density of the immunoreactive phosphorylated
protein normalised to the total corresponding protein and expressed as the
percentage relative to DMSO. Data correspond to the average ± SEM of three
to four independent experiments performed in triplicate. **, P<0.01; ***,
P<0.001 vs DMSO (Student’s t test).
Inhibitory effects of compound 12 on the growth of cancer
cell cultures
We next tested the effect of compound 12 on migration,
invasiveness, and anchorage-independent growth, hallmarks of
transformed cells related to their metastatic potential.
Remarkably, compound 12 profoundly impaired migration
(Figure 4) and growth of MDA-MB-231 cells in matrigel (Figure
5A), and invasive capacity of these cells (Figure 5B). Our results
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Ras, compound 12 efficiently reduced colony formation of
RasG12V–transfected NIH-3T3 cells in a dose-dependent manner
without significantly affecting empty vector-transformed cells
(Figure 6).
Figure 5. Compound 12 inhibits the anchorage-independent proliferation and
the invasiveness of MDA-MB-231 cells in a dose-dependent manner. (A) Cells
were seeded and grown in matrigel for 7 days in the presence of increasing
concentrations of compound 12. (B) Cells were treated overnight with the
indicated concentration of the compound and placed in the upper chamber of
a Boyden chamber whose membranes have been previously coated with
matrigel. Medium containing 10% of FBS plus the appropriate concentration of
compound was placed in the lower chamber as a chemoattractant. After 6 h at
37 ºC, the cells were fixed and stained and those on the underside of the
filters were visualised and counted under light microscope. Data represent the
mean ± SEM of three independent experiments performed in triplicate. *,
P<0.05; ***, P<0.001 (vs DMSO-treated cells, Student’s t test).
Table 3. Cytotoxicity of compound 12 and doxorubicin (Dox) in different cell
lines
12
Dox
(IC50, μM)[a]
Pancreatic cells
Ras status
(IC50, μM)[a]
PANC-1
MIA PaCa-2
BxPC-3
Mutated K-Ras (G12D)
Mutated K-Ras (G12C)
Wild type Ras
17.3
10.2
>50
2.3
0.12
3
HPNE
Non-tumor cell
>50
0.8
Breast cells
MDA-MB-231
MCF-7
Mutated K-Ras (G13D)
Wild type Ras
7
0.3
50
0.17
ND[b]
MCF-10A
Lung cells
H-460
Non-tumor cell
>50
Figure 4. Compound 12 impairs cell migration without inducing general
cytotoxicity. (A-C) Compound 12 prevents cell migration, but cells recover their
ability to migrate in the absence of compound. In vitro scratches (wounds)
were made by scraping confluent MDA-MB-231 cell monolayers with a sterile
pipet tip and were visualized by phase contrast microscopy. After the indicated
times, remaining wound area was quantified in cells treated with (A) vehicle,
(B) compound 12 (25 μM) during 72 h, or (C) compound 12 (25 μM) during 24
h and vehicle for additional 48 h. Bar, 250 μm. (D) Percentage of the
remaining wound area. (E) Percentage of migrating cells quantified with a
Boyden chamber. Bar graphs represent the mean ± SEM of the remaining
wound area of three independent experiments performed in triplicate in which
three different fields were analyzed. **, P<0.01; ***, P<0.001 (vs DMSO-
treated cells, Student’s t test); ###, P<0.001 (vs cells treated with 25 μM of
compound 12 during 72 h, Student’s t test).
Mutated K-Ras (Q61H)
Non-tumor cell
15
ND
ND
MRC-5
>50
Colon cells
SW-620
Mutated K-Ras (G12V)
Mutated K-Ras (G13D)
37.1
8
3.7
HCT-116
Fibroblasts
NIH-3T3
0.73
Non-tumor cell
>50
ND
[a] Cytotoxicity was evaluated by the MTT assay except for pancreatic cell
lines. In this case, the XTT assay was employed (see Experimental Section for
details). IC50 values are the means from two to three independent experiments
performed in triplicate. In all cases, the SEM is within 10% of the mean value.
[b] ND, not determined.
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Figure 6. Inhibition colony formation in RasG12V-transfected cells by compound
12. Empty vector (white columns) or RasG12V-transfected NIH-3T3 cells (black
columns) were incubated with the indicated concentration of compound.
Colonies were counted after staining with Fuschin 2% under the microscope.
All in sum, compound 12 is a potent K-Ras inhibitor as
demonstrated by the cytotoxicity values and the decrease of the
Ras-GTP complex levels with selectivity for tumor cells versus
non-tumor cells and specificity for mutant Ras versus wild type
Ras. Moreover, to rule out that the effects induced by compound
12 in Ras were due to regulation of other proteins upstream of
Ras or that the decrease in Ras-GTP complex levels was due to
an impairment on the Ras localization in the membrane, we
confirmed that compound 12 did not affect the activation of Shc
(the main Ras upstream activator) or the membrane localization
of Ras (Supporting Figure S3).
Figure 7. Cartoon representation of backbone superimposition of Ras proteins.
Ras is characterized by a prototype “G domain fold”, consisting of a mixed six-
stranded -sheets flanked by five -helices. The N-terminal effector (residues
1-86) and C-terminal allosteric (87-166) lobes are indicated as well as the
functional P-loop (10-16 in green), switch I (30-38 in red) and switch II (60-68
in blue). Three major sites (sites 1-3) for small molecule binding are indicated
as transparent surfaces in magenta, yellow and cyan. Structures displayed
correspond to the GppNHp-bound H-Ras (PDB ID: 4EFL), GDP-bound H-Ras
(PDB ID: 4Q21), GDP-bound K-Ras (PDB ID: 4OBE), GDP-bound K-Ras
G12V (PDB ID: 4TQ9), GDP-bound K-Ras G13D (PDB ID: 4TQA), GppNHp-
bound K-Ras Q61H (PDB ID: 3GFT), GppNHp-Kobe2601 H-Ras T35S
complex (PDB ID: 2LWI), GppCp-DCAI K-Ras G12D complex (PDB ID: 4DST),
GDP-IBLP K-Ras G12V complex (PDB ID: 4EPY), GDP-disulphide-based
compounds K-Ras G12C complexes (PDB IDs: 4LV6, 4LUC and 4M22) and
GppNHp-Zn2+cyclen H-Ras complex (PDB ID: 3LY8). Small molecules GDPs,
GTP analogs, allosteric inhibitors, and Mg2+ are displayed as sticks.
Molecular basis for the interaction of compound 12 with Ras
Ras proteins have been extensively studied by high-resolution
nuclear magnetic resonance (NMR) spectroscopy and X-Ray
crystallography methods (see[8c] for a revision). Comprehensive
information at the structural level has been disclosed for H-Ras,
K-Ras and N-Ras, for several well-known mutations, as well as
their complexes with small molecules and downstream effectors.
Altogether, these studies underscore the structural resemblance
among Ras isoforms, the dynamic nature of the functional
regions P-loop (residues 10-16), switch I (residues 30-38) and
switch II (residues 60-68), and the presence of several
accessible small ligand-binding sites (Figure 7).
These structures displayed
a considerable conformational
variability as revealed by their C root-mean-square deviations
(RMSDs) (Supporting Figure S4). Clearly, the functional P-loop
and switch regions I and II are more dynamic (average RMSD
values of 2.9 Å) than the entire proteins (1.5 Å). In addition, the
G12V, G13D, and Q61H mutants displayed increased
fluctuations in the functional regions relative to wild type K-Ras
(Supporting Figure S4), as it has been previously stated by other
computational studies.[14]
Compound 12 was docked to each of the 80 structures obtained
by MD simulations, using two overlapping grid boxes centered at
the N-terminal effector and C-terminal allosteric lobes,
respectively (see Supporting Figure S5 and Experimental
Section). The obtained docking solutions (20 runs per structure,
a total of 1,600 poses) were clustered and ranked according to
the predicted binding energy score. The best-scored docking
poses locate compound 12 within the “effector lobe” in close
The significant structural heterogeneity, together with the
dynamic nature of the accessible pockets on the Ras surface,
which depend on the conformational state of the protein or are
transiently induced by the interaction with ligands,[8a, 13] make the
estimation of the binding modes of small molecules
a
challenging task. To overcome this problem, we used molecular
dynamics (MD) simulations to increase the structural ensembles
of Ras for molecular docking experiments and, in a second step,
for testing the stability of the predicted protein-ligand complexes
(see Experimental Section). Twenty MD snapshots extracted
from 100 ns trajectories of the apo-forms of wild type K-Ras,
G12V, G13D, and Q61H oncogenic mutants were considered as
a flexible ensemble of protein coordinates (a total of 80
structures) for docking of compound 12.
proximity to the P-loop, switch
I and switch II regions
(Supporting Figure S5). The twenty lower docking energy
protein-ligand complexes were subjected to 100 ns of MD
simulations. Analysis of the trajectories revealed that one of
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these predicted complexes was highly stable, with average
ligand RMSD < 2.0 Å relative to the initial docking pose in the
G12V, G13D, and Q61H K-Ras simulations (Supporting Figure
S6). In this conformation, compound 12 binds partially to the
GTP-binding region and extends into the adjacent hydrophobic
pocket delimited by switch II (site 2 in Figure 7). In particular, the
pyranone ring of the ligand and its substituents overlap with the
based allosteric inhibitors.[10a] Clearly the halobenzene rings of
compound 12 and these allosteric inhibitors overlap (Figure 8).
This mode of binding explains the experimental observation
(Table S2) that the optimal spacer requires five methylene units
(n=5) so that the halobenzene ring can reach, but not surpass,
the site II cavity. Moreover, the hydrophobic propyl chain and
chlorine
atoms
favour
the
interaction
with
the
triphosphate moiety of GTP in such
hydroxymethyl group mimics an oxygen atom of the
phosphate of GTP, the oxygen atom within the pyranone ring
mimics an oxygen atom of the  phosphate, and the carbonyl
group of the pyranone and the ether oxygen mimic two oxygen
atoms of the  phosphate (Figure 8).
a
manner that the
hydrophobic/aromatic site II cavity. The chlorine atoms of
compound 12 mimic the chlorine atoms of the irreversible
inhibitor 6H05 analog (Figure 8).[10a]

Thus, compound 12 overlays both the binding region of the
triphosphate group of GTP and the allosteric binding site of the
irreversible inhibitors. The MD simulations show that the more
flexible part of the ligand corresponds to the halobenzene ring,
whereas the binding of the pyranone ring is highly stabilized by
the polar/H-bonds interactions in the triphosphate-binding region
of the protein. Moreover, all the oncogenic assayed mutations
were located in close proximity to the ligand, suggesting a direct
role of these positions in binding. V12 in the G12V mutant forms
hydrophobic-hydrophobic interactions with the methylene chain
of compound 12 whereas H61 in the Q61H mutant forms
aromatic-aromatic interactions with the halobenzene ring (Figure
8). The role of D13 in the G13D mutant is not straightforward but
its interaction with K117 probably modifies the shape of the
binding cavity and the orientation of the backbone amide group
that is involved in the interaction with the ligand (see above).
Considering that all the obtained results were consistent with a
direct inhibition of Ras by compound 12, we used nuclear
magnetic resonance (NMR) spectroscopy to obtain direct
insights into the binding mode of compound 12 to Ras protein.
First, we studied the proton NMR signals of the compound upon
addition of Ras protein (Figure 9). NMR transverse relaxation
(described by the transverse relaxation time T2) directly depends
on the molecular size; then, an increase in T2 is related to an
increase in the size or apparent size of the system and can be
seen in NMR spectra as intensity lost due to line broadening.
Therefore, the enhancement of ligand relaxation is usually used
to detect binding. In this case, a clear line broadening of the
proton NMR signals of compound 12 was detected upon
addition of Ras, pointing out that the compound binds to the
protein. In particular, the intensity of H6 signal, located in the
less flexible pyranone ring, decreases to 33% of the initial
intensity in the presence of Ras (Figures 9A,B). The effect is
less pronounced in the more flexible propyl chain attached to the
halobenzene ring but can be also detected (CH315 signal
intensity decreases to 70% upon addition of Ras, Figures 9A,B
right panels). Addition of GTP to the NMR sample enables to
recover the signal intensity (Figure 9C), confirming the
competition of both molecules for partially overlapped binding
sites.
Figure 8. Backbone superimposition of energy minimized complexes between
compound 12 (yellow) and wild type K-Ras, G12V, G13D, and Q61H
oncogenic mutants (highlighted as sticks). P-loop and switch I/II regions are
colored in green, red, and blue, respectively. Figure inset shows a detailed
view of the interactions between compound 12 and wild type K-Ras. Structural
superimpositions with the GTP analog-bound K-Ras Q61H (PDBid: 3GFT) and
GDP-6H05 analog K-Ras G12C complex (PDB IDs: 4LV6) reveal the spatial
correspondence of the pyranone ring of compound 12 (in yellow) with the
triphosphate group of GTP (purple) and between the halobenzene ring of
compound 12 (yellow) and the disulphide irreversible inhibitor 6H05 (cyan).
Accordingly, the pyranone system of compound 12 reproduces
the interactions with the backbone amide group of G13 in the P-
loop, the protonated side chain of K16, and the side chains of
T35 and Y32 in switch I (Figure 8), also observed for GTP.
Furthermore, the alkyl chain expands through switch II
positioning the halobenzene ring into the hydrophobic/aromatic
site II cavity near Q61. This hydrophobic pocket was first
discovered as the binding site of K-Ras (G12C) disulphide-
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Figure 9. Binding of compound 12 to Ras protein assessed by NMR
spectroscopy. (A) 1H NMR spectrum of compound 12 acquired with 160 scans
in a 700 MHz NMR spectrometer equipped with a cryo probe. Ligand
concentration was 250 μM in buffer Tris 20 mM, NaCl 150 mM, MgCl2 2 mM
pH 7.4 in D2O. (B) 1H NMR spectrum of compound 12 acquired upon addition
of RasG12V-GST protein (compound:protein ratio, 40:1). (C) 1H NMR
spectrum of the same sample than in panel B upon addition of GTP to reach a
final concentration of 250 μM.
Figure 10. Binding of compound 12 to RasG12V using affinity mass
spectrometry. (A) RasG12V was incubated either with compound 5 or with
compound 12. The mixture was filtrated in a micro bio-spin chromatography
column to isolate ligand-bound complexes. The eluted complexes were
dissociated and the corresponding compound detected by liquid
chromatography coupled to mass spectrometry. (B) Compounds 5 and 12
(upper and lower chromatograms, respectively) added as internal standards
for comparison. Shown peaks correspond to the [M+H]+ ion of each compound
obtained by selected ion monitoring in positive mode. Chromatograms are
representative of two independent experiments carried out in duplicate.
To further confirm the direct interaction of compound 12 with
Ras, the analogous experiment with the less potent derivative 5
(Table 2) was carried out. In this case, a significantly smaller
perturbation of the NMR signals of the compound was observed
(Supporting Figure S7), strongly suggesting that 12 displays a
stronger interaction with Ras than compound 5. In addition, we
carried out a thermal shift assay for Ras in the presence of
compound 12, and a small increase in the melting temperature
(ΔTm) for Ras was observed (ΔTm=1 ºC).
Conclusions
Additional experiments were carried out to further confirm the
direct interaction between compound 12 and Ras. In particular,
affinity mass spectrometry methods were performed. RasG12V
was incubated in the presence of compound 5 or 12 and the
mixture filtered through a size exclusion chromatography unit.
Thus, the free compound is retained in the column whereas the
bound ligand elutes as Ras-compound complex. After
dissociation of the complex, the corresponding compounds are
detected by liquid chromatography coupled to mass
spectrometry (Figure 10). Again, the obtained results clearly
support the direct interaction of compound 12 with Ras, unlike
derivative 5, which does not bind Ras in a noticeable extent.
All in sum, in this work we describe a new Ras reversible
inhibitor that binds in an overlapping site that comprises the
GTP-binding region and extends into the adjacent hydrophobic
pocket delimited by switch II. Ras inhibitor 12 impairs selectively
the activity of mutated Ras isoforms, fact that leads to reduced
toxicity towards non-tumor cells, and decreases tumor hallmarks
related to cellular proliferation, such as the MAPK signaling
pathway, or to metastatic processes such as cell migration and
invasiveness. Inhibitor 12 is
a new compound for the
development of new antitumor agents suitable for the treatment
of specific Ras-driven tumors with current bad prognosis, either
by themselves or in combination therapies.
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dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium
Experimental Section
bromide (MTT) or the (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-
(2H)-tetrazolium-5-carboxanilide (XTT) salt by the viable cells as
previously described.[15]
Compound synthesis. Unless stated otherwise, starting
materials, reagents and solvents were purchased as high-grade
commercial products from Sigma-Aldrich, Acros, Fluka, Scharlab,
or Panreac, and were used without further purification. All non-
aqueous reactions were performed under an argon atmosphere
in oven-dried glassware. Tetrahydrofuran (THF) and
dichloromethane (DCM) were dried using a Pure SolvTM Micro
100 Liter solvent purification system. Reactions under
microwave (MW) irradiation were performed in a Biotage Initiator
2.5 reactor. Hydrogenation reactions were carried out using an
H-Cube continuous-flow hydrogenation reactor. Full details
regarding the synthetic procedures and characterization data of
all compounds are given in the Supporting Information.
Spectroscopic data of all described compounds were consistent
with the proposed structures. Elemental analyses (C, H, N) were
within 0.4% of the calculated values, confirming a purity of at
least 95% for all tested compounds. DCAI was purchased from
Aldrich. General Procedure for the Synthesis of Final
Compounds 3-12. A mixture of intermediate 13 (1.1 equiv, 1-2
mmol), the corresponding phenol (commercially available or 14-
17, 1 equiv), anhydrous K2CO3 (1.2 equiv), and a catalytic
amount of NaI in anhydrous 2-butanone (3 mL/mmol) was stirred
at 80 ºC for 24 h. After this time, the reaction mixture was filtered
and concentrated under reduced pressure. The residue was
dissolved in diethyl ether, washed with water, dried (anhydrous
Na2SO4) and evaporated under reduced pressure. The crude
was purified by column chromatography (hexane/ethyl acetate,
1:1) to afford the corresponding final compounds, which were
crystallized in the appropriate solvent. 5-{[5-(3,4-Dichloro-2-
propylphenoxy) pentyl]oxy}-2-(hydroxymethyl)-4H-pyran-4-
Active Ras pulldown assay. MDA-MB-231 cells were plated at
a density of 2 x 106 cells in 15 cm dishes and incubated in
DMEM with 10% FBS at 37 ºC and 5% of CO2. After 24 h,
medium was replaced by fresh DMEM with 1% FBS, containing
25 µM of compound 12 or an equivalent volume of DMSO for
vehicle control, and cells were incubated for 24 h at 37 ºC and
5% of CO2. After 5 min of stimulation with 10 ng/mL of EGF,
cells were washed with PBS and lysed with ice-cold lysis buffer.
Lysates were clarified by 15 min of centrifugation at 16000g and
protein concentration was measured using bicinchoninic acid
method (Thermo Scientific). Before performing the assay, 50 µL
of each lysate were separated to analyze the total Ras
expression. For the analysis of active Ras, a Ras-GTP pulldown
assay kit (Thermo Scientific) was used following manufacturer’s
instructions. Briefly, lysates were incubated with a glutathione S-
transferase fusion of the Ras binding domain (RBD) of Raf1
along with glutathione agarose resin to specifically pull down
active Ras. The entire samples obtained after the pulldown
assay were boiled for 5 min and loaded onto 4-20% SDS-PAGE
gels (Bio-Rad). Ras proteins were visualized by immunoblotting
on nitrocellulose membranes using mouse anti-Ras provided by
manufacturer. Blots were analyzed by densitometry using
ImageJ software (NIH).
Western blot analysis. The expression of the different proteins
was determined by immunoblot analysis following the conditions
16]
previously reported[15b,
Information.
as detailed in the Supporting
one, 12. 73% yield. Rf (EtOAc)
= 0.2; mp: 85-86 ºC
1
(EtOAc:diethyl ether); H NMR (300 MHz, CDCl3): δ 0.95 (t, J =
7.4, 3H, CH3), 1.46-1.58 (m, 2H, CH2),1.59-1.70 (m, 2H, CH2),
1.81-1.94 (m, 4H, 2CH2), 2.07 (br s, 1H, OH), 2.76-2.81 (m, 2H,
CH2Ar), 3.89 (t, J = 6.4, 2H, CH2O), 3.95 (t, J = 6.2, 2H, CH2O),
4.48 (s, 2H, CH2OH), 6.52 (s, 1H, H3), 6.67 (d, J = 8.9, 1H, CHAr),
7.21 (d, J = 8.9 Hz, 1H, CHAr), 7.56 (s, 1H, H6); 13C NMR (75
MHz, CDCl3,):  14.3 (CH3), 21.9, 22.7, 28.9, 29.1 (4CH2), 30.2
(CH2Ar), 60.9 (CH2OH), 68.3, 69.7 (2 CH2O), 110.4 (C3), 111.9,
124.0 (2CHAr), 127.5, 131.8, 132.0 (3CAr), 139.6 (C6), 147.9 (C5),
156.2 (C-O), 169.0 (C2), 175.1 (C=O); IR (KBr): 3300, 2945,
1647, 1607, 1452, 1259, 1207, 1151, 1082; MS (ESI, m/z): 414.1
[M]+; Elemental analysis calcd (%) for C20H24Cl2O5: C 57.84, H
5.82; found: C 57.96, H 5.72.
Thermal Shift Assay. The assay was carried out by Reaction
Biology Corp. using RasG12V and compound 12 at 25 μM in
Hepes buffer (pH 7.5).
Migration, anchorage-independent growth, and invasion
assays. Migration was determined by the wound-healing assay
as previously described[17] and with a Boyden chamber. To
evaluate the effect of the compound on the capacity of tumor
cells to grow without anchorage, cells were seeded in Matrigel
(BD, France). Chemoinvasion assay was performed in a Boyden
chamber. The experimental details are included in the
Supporting Information.
Ras-dependent colony formation assay. 50% confluent NIH-
3T3 cells in a 10 cm diameter plate were transfected with 0.5 μg
pSV2-RasV12 plasmid plus 100 ng pSV2-neomycin plasmid
using Lipofectamine-Plus reagent (Invitrogen) according to the
manufacturer’s instruction. After 48 h, cells were treated with
trypsin, splitted to 1/3, and incubated in the presence of
complete medium supplemented with 400 μg/mL geneticin
(Invitrogen) and different doses of compound until emerging
resistant clones appear (2-3 weeks). The medium was renewed
Cell culture and cytotoxicity assays. Human carcinoma cell
lines from colon (HCT-116 and SW-620), lung (H-460), breast
(MCF-7 and MDA-MB-231) and pancreas (PANC-1, MIA PaCa-2,
and BxPC-3); immortalized but not tumorigenic cell lines NIH-
3T3 mouse fibroblasts and human breast, lung, and pancreas-
derived cell lines, MCF-10A, MRC-5, and HPNE, respectively,
were purchased at ATCC and cultured according to their
recommendations. Cell viability was determined after 48 h of
incubation with the compound under study using standard
colorimetric assays based on the metabolism of the 3-(4,5-
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10.1002/chem.201604905
Chemistry - A European Journal
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every 3 days. Colonies with a minimum of 50 cells were counted
after staining with Fuschin 2%.
nucleotide was done by adding GTP to the compound:protein
sample to reach a final concentration of 250 μM.
Molecular dynamics simulations. Four structures of K-Ras
(PDB IDs: 4OBE, 4TQ9, 4TQA, and 3GFT) were obtained from
the Protein Data Bank (www.rcsb.org), representing the GDP-
bound wild type K-Ras; GDP-bound K-Ras G12V mutant, GDP-
bound K-Ras G13D mutant and GppNHp-bound K-Ras Q61H
mutant, respectively. The apo-forms of wild type K-Ras, G12V,
G13D, and Q61H oncogenic mutants were obtained by
removing either GDP or the GTP analog. These apo-forms and
20 docking poses of the complex between compound 12 and
Ras (see below) were solvated with explicit simple point charge
(SPC) water in a rhombic dodecahedron box. The distance
between the protein and the edge of the box was ∼1.5 nm, to
ensure that the minimum distance between periodic images was
satisfied. Na+ and Cl- ions were added to neutralize charges,
reaching a final concentration of 0.15 M. Model systems were
energy minimized and equilibrated with positional restraints on
the C atoms using the GROMACS software v5.0.6,[18] the
Amberff99SB-ILDN force field for the protein, and the general
Amber force field (GAFF) and HF/6-31G*-derived RESP atomic
charges for compound 12. The positional restraints were
gradually released by decreasing the force constant on the
protein in four distinct steps (1000, 500, 10, and 0 kJ/mol nm2) of
10 ns each. After relaxation, 100 ns of MD trajectories were
produced. All simulations were run at a constant temperature of
300 K using separate v-rescale thermostats for the protein and
solvent molecules. Lennard-Jones interactions were computed
using a cutoff of 10 ꢀ, and electrostatic interactions were treated
using PME with the same real-space cutoff. A time step of 2 fs
was used.
Acknowledgements
This work was supported by ExonHit Therapeutics and by grants
from the Spanish Ministerio de Economía y Competitividad
(MINECO, SAF2013-48271 and SAF2016-78792-R), and
Comunidad de Madrid (S2010/BMD-2353). The authors thank
Campus de Excelencia CEI Moncloa for a predoctoral fellowship
to N.I.M.-R., the UCM’s CAI Cytometry and Fluorescence
Microscopy for their assistance, and the computer resources
provided by the Barcelona Supercomputing Center-Centro
Nacional de Supercomputación.
Keywords: Medicinal chemistry • Cancer • Ras • Anticancer
compounds • Ras inhibitors
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10.1002/chem.201604905
Chemistry - A European Journal
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Blocking Ras! The oncogenic activity
of mutated Ras is impaired by the new
inhibitor 12, which reduces the Ras-
GTP levels, inhibits the MAPK
pathway, and is cytotoxic in Ras-
driven cellular models. Molecular
dynamics simulations, mass
N.I. Marín-Ramos, C. Piñar, H.
Vázquez-Villa, M. Martín-Fontecha, A.
González, A. Canales, S. Algar, P.P.
Mayo, J. Jiménez-Barbero, C. Gajate, F.
Mollinedo, L. Pardo, S. Ortega-
Gutiérrez, A. Viso, M.L. López-
Rodríguez*
spectrometry, and nuclear magnetic
resonance show that 12 binds Ras
and identify its binding site in the
protein. This new inhibitor adds to the
scarce number of compounds to fight
Ras-driven cancers, a currently unmet
clinical need.
Page No. – Page No.
Development of a nucleotide
exchange inhibitor that impairs Ras
oncogenic signaling
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