A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.9b00145

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC₅₀ = 2 μM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Full text of DOI:10.1021/acs.jmedchem.9b00145

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Article
A potent isoprenylcysteine carboxylmethyltransferase (ICMT)
inhibitor improves survival in Ras-driven acute myeloid leukemia
Nagore I. Marin-Ramos, Moisés Balabasquer, Francisco J. Ortega-Nogales, Iván R. Torrecillas,
Ana Gil-Ordoñez, Beatriz Marcos-Ramiro, Pedro Aguilar-Garrido, Ian Cushman, Antonio Romero,
Francisco J. Medrano, Consuelo Gajate, Faustino Mollinedo, Mark R. Philips, Mercedes Campillo,
Miguel Gallardo, Mar Martín-Fontecha, María L. López-Rodríguez, and Silvia Ortega-Gutierrez
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00145 • Publication Date (Web): 10 Jun 2019
Downloaded from http://pubs.acs.org on June 12, 2019
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A potent isoprenylcysteine
carboxylmethyltransferase (ICMT) inhibitor
improves survival in Ras-driven acute myeloid
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leukemia
Nagore I. Marín-Ramos,1,2,# Moisés Balabasquer, Francisco J. Ortega-
1,#
1
,#
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Nogales, Iván R. Torrecillas, Ana Gil-Ordóñez, Beatriz Marcos-Ramiro,
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Pedro Aguilar-Garrido, Ian Cushman, Antonio Romero, Francisco J.
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Medrano, Consuelo Gajate, Faustino Mollinedo, Mark R. Philips, Mercedes
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Campillo, Miguel Gallardo, Mar Martín-Fontecha, María L. López-
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,*
1,*
Rodríguez and Silvia Ortega-Gutiérrez
1Departamento de Química Orgánica I, Facultad de Ciencias Químicas,
Universidad Complutense de Madrid, E-28040 Madrid, Spain
2CEI Campus Moncloa, UCM-UPM and CSIC, E-28040 Madrid, Spain
3Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de
Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona,
Spain
4H12O-CNIO Haematological Malignancies Clinical Research Unit, Centro
Nacional de Investigaciones Oncológicas (CNIO), E-28029 Madrid, Spain
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5Department of Pharmacology and Cancer Biology, Duke University Medical
Center, Durham, NC 27710, USA
6Centro de Investigaciones Biológicas, CSIC, E-28040 Madrid, Spain
7Perlmutter Cancer Center, New York University School of Medicine, New
York, NY 10016, USA
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#These authors contributed equally to this work
*Correspondence: mluzlr@ucm.es, siortega@ucm.es
ABSTRACT
Blockade of Ras activity by inhibiting its post-translational methylation
catalysed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been
suggested as a promising antitumor strategy. However, the paucity of
inhibitors has precluded the clinical validation of this approach. In this work
we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM],
which is selective against the other enzymes involved in the post-translational
modifications of Ras. Compound 3 significantly impairs the membrane
association of the four Ras isoforms, leading to a decrease of Ras activity and to
inhibition of Ras downstream signaling pathways. In addition, it induces cell
death in a variety of Ras-mutated tumor cell lines and increases survival in an
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in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the
activity of the four Ras isoforms regardless of its activating mutation,
compound 3 surmounts many of the common limitations of available Ras
inhibitors described so far. In addition, these results validate ICMT as a
valuable target for the treatment of Ras-driven tumors.
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INTRODUCTION
Constitutively activated Ras is present in about one-third of the human
cancers, including those among the most aggressive ones such as
pancreatic, lung, or colon cancers or some leukemias. However, after
several decades of continuous research efforts, Ras direct inhibition with
small molecules still remains a current challenge.1-7 Instead, interference
with the enzymes involved in the post-translational modification of Ras
could be an alternative, considering that in absence of any of its post-
translational modifications Ras losses its capacity to promote tumor
transformation.8 Ras post-translational modification involves three
consecutive steps: prenylation, catalysed by farnesyltransferase (FTase) or
geranylgeranyltransferase (GGTase), proteolysis, mediated by the
endoprotease Ras-converting enzyme 1 (Rce1), and final methylation of
the carboxy terminal amino acid by the enzyme isoprenylcysteine
carboxylmethyltransferase (ICMT). Though FTase has been the target of a
variety of drug discovery programs, its inhibitors failed to show
satisfactory activity in clinical trials, probably because of the alternative
prenylation by GGTase. Consequently, more recent efforts have been
dedicated to target the post-prenylation enzymes Rce1 and ICMT.9
Whereas Rce1 inhibition has raised some toxicity concerns, ICMT
blockade seems more promising given that its genetic inactivation blocks
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Ras proper localization and activity, ameliorates phenotypes of K-Ras-
induced malignancies in vivo,11 and is critical for malignant
transformation and tumor maintenance by all Ras isoforms in humans.12
These findings provide a compelling rationale for the development of
ICMT inhibitors as a promising approach to anticancer drug development
for treating challenging Ras-driven tumors. However, its clinical
validation has been hampered by the lack of synthetic inhibitors and to
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date only one inhibitor, cysmethynil (Figure 1A), has been thoroughly
characterized. This compound effectively blocks the anchorage
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independent growth in a human colon cancer cell line and impairs
tumor progress in mice.14, 15 However, its advancement towards clinical
phases has been hampered by its limited potency in vitro in some cancer
cell lines14, 16 and in vivo models, where doses between 100 and 200
mg/kg are usually required to observe inhibition of tumor growth.14, 15
Further efforts in the development of more efficacious ICMT inhibitors
have led to the identification of several structural scaffolds able to inhibit
in some extent ICMT,17-20 including indole derivatives,21, 22 some of them
structurally similar to cysmethynil but with improved cellular
antiproliferative activities,16, 23 farnesyl cysteine analogs,24-26 and a series
of tetrahydropyrane derivatives, being one of these compounds the most
potent ICMT inhibitor described to date, with a IC value of 1.3 nM.
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However, this excellent in vitro value led only to a modest reduction in
cell viability in different tumor cell lines.27 In this context, new
compounds able to block ICMT activity would be highly desirable. In this
work we describe a new potent and selective ICMT inhibitor, compound 3,
which significantly impairs the membrane association of the four Ras isoforms,
leading to a remarkable reduction in Ras activity, to the inhibition of the
mitogen-activated protein kinase (MAPK) signaling pathway, and finally to cell
death by autophagy and apoptosis. In addition, compound 3 significantly
reduces cell viability in a variety of Ras-mutated tumor cell lines and,
importantly, shows in vivo activity, as it increases survival in the Ras-driven
model of acute myeloid leukemia at a dose of 25 mg/kg without causing any
toxic side effect.
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RESULTS AND DISCUSSION
Hit identification
In order to identify new ICMT inhibitors, we carried out a screening of our in-
house library that led to the identification of compound 1 as an initial hit
(Figure 1A). This compound blocked ICMT activity, albeit with moderate
potency (25% inhibition of the ICMT activity at 50 µM), making necessary
further optimization of the compound. As a first step in that direction, we
decided to perform docking studies. For this, we built a homology model of the
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human enzyme (h-ICMT) using as templates the Methanosarcina acetivorans
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ICMT (Ma-ICMT), the only structure available when this work was started
(Supporting Figure S1), and the recently disclosed Tribolium castaneum enzyme
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(
Tc-ICMT), the only eukaryotic ICMT described up to date (Supporting Figure
S2). Analysis of the complexes between the Ma-ICMT derived h-ICMT with N-
acetyl-S-farnesyl-L-cysteine (AFC), the minimal structural element recognized
by ICMT (Supporting Figure S1, panel B), or with the inhibitor cysmethynil
(Supporting Figure S1, panel C) showed the possibility of adding, in compound
1, a hydrophobic tail to expand toward the membrane through the substrate
tunnel, similarly to the farnesyl moiety of AFC or the octyl chain of
cysmethynil. An analogous situation was observed in the models of the h-ICMT
derived from Tc-ICMT in complex with cysmethynil (Figure 1B) or with AFC
(Figure 1C), fact that prompted us to design and synthesize derivative 2 (Figure
2
A). Remarkably, this compound displayed an enhanced capacity to inhibit
ICMT activity (55% inhibition at 50 µM).
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Figure 1. (A) Structures of cysmethynil, AFC, and compound 1. (B) A Tc-ICMT-
derived homology model of h-ICMT in complex with cysmethynil (orange) and S-
adenosylmethionine (SAM) cofactor (cyan). The indol ring is positioned in front of
Trp216, the amide group is located between Arg274 and Asp122, and the octyl tail
expands towards Tyr236. (C) A Tc-ICMT-derived homology model of h-ICMT in
complex with AFC (orange) and SAM cofactor (cyan). AFC positions the carboxyl
group between conserved Arg174, His187, Arg247, Trp216 and the methyl group of
SAM. The prenyl moiety expands between TM5 and TM8 helices towards Tyr236. Tc-
ICMT PDB code: 5V7P.
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A closer look to the interaction model between compound 2 and the protein
(Figure 2A) highlighted the presence of a cluster of aromatic amino acids in the
binding site formed by Trp216 of TM7 and Phe243 and Phe244 of TM8. Hence,
it would be conceivable that the replacement of the methyl and cyclohexyl
substituents of compound 2 by phenyl rings, able to establish π-π stacking
interactions, allowed for an improvement of affinity towards the protein.
Consequently, we designed compound 3, which fitted perfectly in the model
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(Figure 2B). One phenyl ring can fill the pocket formed by Arg174 of TM6,
Tyr213 and Trp216 of TM7 and Phe243 and Phe244 of TM8. The amide group is
hydrogen bonding Arg247 of TM8 and the octyl chain of the ligand fills the
lipid site. The other phenyl group is placed between the TM5-TM8 crevice,
while the adjacent amide group is able to establish a hydrogen bond with
Ser129 on the top of TM5 (Figure 2B). It is worthy to mention that the h-ICMT
model based on the Tc-ICMT structure, as well as all the mutagenesis data
published to date, confirm the conserved interactions of ICMT ligands with
Arg247 below the cofactor binding site. Hence, compound 3 was synthesized
and tested for ICMT activity, showing, to our delight, an excellent 93% enzyme
activity inhibition at 50 µM and an IC value of 2 µM (see Supporting Figure S3
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Figure 2. Tc-ICMT-derived homology models of h-ICMT in complex with compounds
2
and 3. (A) Compound 2 (in orange) positions the octyl chain toward the membrane in
a similar manner as the hydrophobic tail of cysmethynil and AFC. S-
adenosylmethionine (SAM) cofactor is shown in cyan. (B) Extracellular view of the
homology model of the h-ICMT in complex with compound 3 (orange) and SAM
(cyan). Tc ICMT PDB code: 5V7P.
The importance of the structural elements suggested by the model was
confirmed with the synthesis of several sets of compounds. In this regard, we
verified that replacement of one or the two phenyl rings by non-aromatic
moieties (cpds 4-10, Figure 3) led to a decrease in activity (Figure 3 and Table 1).
Also, we validated the importance of the n-octyl chain because its substitution
by shorter or oxygen-containing chains (cpds 11-16, Figure 3 and Table 2)
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dramatically diminishes activity when compared to the 93% inhibition of
compound 3.
O
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R
N
N
N
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H
H
(
)7
25-34: R= o,m,p-F; o,m,p-CF3, p-NHCOCH3, p-CN, p-OCH3, p-CON(CH3)2
ICMT inh. (50 M) = 23-84%
O
O
O
O
X
Y
N
N
N
N
R
N
N
R
H
H
N
N
N
( )7
4
H
H
( )7
(
)7
3
1
9-24: X,Y=CO, SO2; R=H, Me
ICMT inh. (50 M) = 55%
ICMT inh. (50 M) = 93%
ICMT inh. (50 M) = 10-68%
O
O
O
O
H
H
N
( )
N
N
N
R
n N
( )7
H
N
N
R
N
(
)7
H
H
O
O
5
-10
1
1-16
17,18: n=1,2
ICMT inh. (50 M) = 62%, 68%
( )5
( ) ( )
3 O
N
H
3
N
N
H
( )
6
( ) ( )
3 O 2O
H
R
R
O
(
O
N
)
( )
N
N
N
2 O 2O
H
ICMT inh. (50 M) = 0-45%
ICMT inh. (50 M) = 3-82%
Figure 3. Synthesized compounds 3-34.
Table 1. Inhibition of ICMT activity of synthesized compounds 5-10
Cpd
R
ICMT
inhibition (%)[a]
82±3
Cpd
R
ICMT
inhibition (%)[a]
48±6
5
6
8
9
[b]
(12±3)
-CH3
30±5
Inactive[c]
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
20±2
10
29±5
[a]
The values are the mean±standard error of the mean (SEM) from two to three
independent experiments performed in triplicate. ICMT inhibition values were
determined at 50 μM.
parentheses. Data correspond to the mean±SEM from two independent experiments
carried out in duplicate. Compounds inhibiting less than 10% of the ICMT activity at
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
b]
IC50 values for ICMT inhibition are shown between
[
c]
50 μM were considered inactive.
Table 2. Inhibition of ICMT activity of synthesized compounds 11-16
Cpd
R
ICMT
Cpd
R
ICMT
inhibition
inhibition
%)[a]
(%)[a]
(
11
12
-CH3
Inactive[b] 14
Inactive[b] 15
30±8
45±6
15±3
1
3
14±4
16
[a]
The values are the mean±SEM from two to three independent experiments performed
[b]
in triplicate. ICMT inhibition values were determined at 50 μM.
inhibiting less than 10% of the ICMT activity at 50 μM were considered inactive.
Compounds
In addition, the adequate distance between the two carbonyl groups was
confirmed with the synthesis of derivatives 17 and 18, which also showed a
decrease in activity (Figure 3 and Table 3). The exploration of different
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hydrogen acceptor groups such as N-methyl amides and sulphonamides (cpds
19-24, Figure 3 and Table 3) did not yield any enhancement in activity. Finally,
we also studied whether the inhibitory capacity of the compound could be
improved by the introduction of different substituents in the phenyl ring (cpds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5-34, Figure 3). The obtained results suggest that only the introduction of small
substituents in the para position keeps the ability to inhibit ICMT (Table 4)
being the p-fluor derivative 27, with 84% inhibition at 50 µM and an IC value
5
0
of 3 µM the best inhibitor within this series. Although none of the synthesized
analogues was able to improve the activity of inhibitor 3, compounds 5 and 27
showed comparable values to derivative 3 in terms of ICMT inhibitory potency
(maximal inhibition values between 82% and 93% at 50 µM). Accordingly, they
were evaluated for their IC value at ICMT, for in vitro cellular activity, and for
5
0
their ADME profile (Table 5). Taken together, these data showed that
compound 3 not only exhibited the best value in terms of ICMT inhibition (93%
of maximal activity and IC value of 2 µM vs 82% of maximal activity and 12
5
0
µM of compound 5 and 84% and 3 µM of compound 27) but also the lowest
clogP (4.1 for compound 3 vs 5.1 and 4.5 for derivatives 5 and 27, respectively)
and the highest cellular activity (IC value of 5 µM for compound 3, whereas
5
0
the IC values for compounds 5 and 27 were 29.1 and 13.7 µM, respectively).
5
0
These results, together with the higher permeability (68% for compound 3 vs
0% and 54% for compounds 5 and 27, respectively), stability in mouse serum
5
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2
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3
3
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3
3
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3
3
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5
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5
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5
5
5
6
(
171 min for compound 3 compared to 118 and 55 min for compounds 5 and 27,
respectively) and in human serum (>24 h), and the slightly lower human serum
albumin (HSA) binding, prompted us to select compound 3 for an in-depth
cellular and in vivo characterization aimed at validating ICMT as a target of
interest for Ras inhibition. Towards this objective we sought to confirm that the
inhibition of ICMT by compound 3 (i) delocalizes Ras from the membrane; (ii)
inactivates Ras and its downstream signaling pathways; (iii) induces cell death
in Ras-dependent tumor cell lines; and (iv) has in vivo efficacy in Ras-
dependent tumors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Inhibition of ICMT activity of synthesized compounds 17-24
Cpd
ICMT inhibition (%)
[a]
1
1
7
8
62±7
68±5
68±7
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65±4
22±5
10±5
12±6
34±5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22
2
2
3
4
[
a] The values are the mean±SEM from two to three independent experiments
performed in triplicate. ICMT inhibition values were determined at 50 μM.
Table 4. Inhibition of ICMT activity of synthesized compounds 25-34
Cpd
R
ICMT
Cpd
30
R
ICMT
inhibition (%)[a]
inhibition
(
%)[a]
2
5
o-F
23±5
p-CF3
63±7
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
6
m-F
p-F
23±4
84±7
31
32
p-NHCOCH3
40±5
68±3
27
p-CN
[
b]
(
3±1)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
8
9
o-CF3
30±6
35±4
33
34
p-OCH3
53±4
47±5
m-CF3
p-CON(CH3)2
[a]
The values are the mean±SEM from two to three independent experiments performed
in triplicate. ICMT inhibition values were determined at 50 μM. [b] IC50 values for
ICMT inhibition are shown between parentheses. Data correspond to the mean±SEM
from two independent experiments carried out in duplicate.
Table 5. Selected data for compounds 3, 5, and 27
Cpd ICMT
inhibition
clogP[c] Cellular
cytotoxicity (%)[e]
(IC ,
Permeability Mouse Human HSA
serum
t1/2
serum
binding[g]
%)[a]
[f]
(
t (h)
1/2
5
0
F (K , μM)
b
d
μM)[d]
(min)[f]
3
5
2
93±4
4.1
5.1
4.5
5.0±0.7
68±9
50±3
54±13
171±23 >24
95.4 (28)
99.5 (3)
(2±1)[b]
82±3
29.1±0.6
13.7±0.1
118±17 >24
(12±3)[b]
7
84±7
55±24
>24
98.7 (7.8)
(3±1)[b]
[a]
The values are the mean±SEM from two to three independent experiments performed
in triplicate. ICMT inhibition values were determined at 50 μM. [b] IC50 values for
ICMT inhibition are shown between parentheses. Data correspond to the mean±SEM
from two independent experiments carried out in duplicate.
calculated with ACDLabs Percepta Software (ACDlogP classic algorithm, version
[c]
clogP values were
[d]
2
018.1.1).
assay. In all cases, the percentage of viable cells after treatment with 50 μM of the
compound compared to vehicle was less than 5%. Data for IC values correspond to the
Cellular viability was determined in MDA-MB-231 cells by the MTT
50
[e]
mean±SEM from at least two independent experiments carried out in duplicate.
Permeability is expressed as the percentage of the compound that passes through a
parallel artificial membrane. Data correspond to the mean±SEM from at least two
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independent experiments carried out in duplicate. [f] Data correspond to the mean±SEM
[g]
from two or three independent experiments carried out in duplicate.
Binding to
human serum albumin (HSA) is expressed as the bound fraction (F ). Dissociation
b
constants (K ) are given between parentheses. Data represent the means from two
d
independent experiments performed in duplicate. SEM is in all cases within a 10% of
the mean value.
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0
Compound 3 induces mislocalization of endogenous Ras, decreases Ras
activation and induces cell death by autophagy and apoptosis
First, we checked the ability of compound 3 to produce mislocalization of
endogenous Ras in PC-3 cells, which were chosen because they have high levels
3
0
of ICMT and they have been described to be cysmethynil-sensitive, although
the direct effect of ICMT inhibition in endogenous Ras has not been ever
studied. Remarkably, treatment of PC-3 cells with 5 µM of compound 3
induced significant Ras mislocalization (Figure 4). The observed effects were
dose-dependent (Supporting Figure S4) and higher than the ones induced by
the same concentration of cysmethynil (Figure 4 and Supporting Figure S4).
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Figure 4. Compound 3 induces Ras mislocalization. Compound 3 (5 µM) induces
Ras mislocalization from the cellular membrane (left image, white arrows) to
intracellular locations in PC-3 cells in higher extent that cysmethynil (Cysm) at the
same concentration. Immunofluorescence images show Ras in green stained using an
anti-Ras primary antibody followed by the appropriate secondary FITC-labelled
antibody. Nuclei (in blue) were stained with Hoechst 33258. Images were obtained in a
Leica confocal microscope under the same conditions and are representative of 3 to 5
independent experiments. Bars, 30 µm. The bar graphs represent the optical density of
the membrane Ras signal quantified with ImageJ. White bar, DMSO; grey bar,
compound 3 (5 μM); black bar, cysmethynil (Cysm, 5 µM). Data correspond to the
average±SEM of 3 independent experiments and have been analysed with GraphPad
Prism 5. ns, not significant; **, P<0.01 vs DMSO (1-way ANOVA followed by the
Dunnett’s Multiple Comparison Test).
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Importantly, Ras mislocalization paralleled substantial decreases in active Ras
(GTP-bound complex) and in the activity of its downstream MEK/ERK and
PI3K/AKT signalling pathways (Figure 5). These effects led finally to cellular
death by autophagy, as indicated by the increase of the levels of microtubule-
associated protein 1A/1B-light chain 3 (LC3), and apoptosis, as shown by the
caspase-mediated cleavage of poly (ADP-ribose) polymerase (PARP) that leads
to the appearance of the specific 89 kDa fragment and by the increase in the
caspase 3 activity (Figure 6). Again, compound 3 induced superior effects than
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cysmethynil even when used at a lower concentration (10 µM and 25 µM,
respectively, Figure 6).
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0
Figure 5. Compound 3 significantly reduces the Ras-GTP (active form) levels and
its downstream MEK/ERK and PI3K/AKT signaling pathways. (A) Ras-GTP
complex from PC-3 cells treated with DMSO, cysmethynil (CYSM, 25 µM) or
compound 3 (10 μM) were immunoprecipitated and visualized by western blot. The bar
graph shows the ratio Ras-GTP/total Ras, expressed as percentage relative to DMSO.
(B) Representative western blots of phosphorylated MEK1/2 (pMEK1/2), ERK1/2 (p-
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ERK1/2), and AKT (p-AKT) and the corresponding total kinases (T-MEK1/2, T-
ERK1/2, and T-AKT). Lysates were obtained from PC-3 cells treated with DMSO,
cysmethynil (CYSM, 25 µM) or compound 3 (10 μM). 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. White bars,
DMSO; grey bars, cysmethynil (CYSM, 25 µM); black bars, compound 3 (10 μM).
Data correspond to the average±SEM of 3 to 5 independent experiments. ns, not
significant; *, P<0.05; **, P<0.01 vs DMSO (Student’s t test).
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Figure 6. Compound 3 induces autophagy and apoptosis in PC-3 cells. Cells treated
with DMSO, cysmethynil (CYSM, 25 µM) or compound 3 (10 μM) were lysed and
stained for (A) LC-3 expression; (B) PARP levels; and (C) caspase 3 activity. Images
are representative of at least three independent experiments and data in C correspond to
the average±SEM of three independent experiments. ***, P<0.001 vs DMSO (Student’s
t test).
Compound 3 induces mislocalization of the four Ras isoforms
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The four Ras isoforms (H-Ras, K-Ras4A and 4B, and N-Ras) contribute in
3
1
different extent to different tumors. For instance, K-Ras is the most frequently
mutated isoform in the majority of cancers, with 90% of pancreatic or 35% of
1
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colon tumours harbouring K-Ras mutations. Instead, N-Ras mutations have a
higher presence in melanoma and hematopoietic tumors.32,
33
Then, it is
essential to assess whether a compound affects to all the Ras isoforms. Notably,
compound 3 induces the mislocalization of the four Ras isoforms (Figure 7), as
shown when AD-293 cells were transfected with H-, N-, and K-Ras4A and 4B
isoforms. These effects are especially significant in the case of H-Ras (most
common isoform in cervix and urinary tract tumors) and K-Ras4A (necessary
33
for lung tumor initiation), where compound 3 is clearly more efficacious than
the same concentration of cysmethynil.
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Figure 7. Compound 3 impairs plasma localization of the four Ras isoforms.
Confocal images of live AD-293 cells that had been transiently transfected with H-Ras,
K-Ras4A, K-Ras4B, and N-Ras GFP fusion plasmids, and treated overnight with
vehicle (DMSO), cysmethynil (CYSM, 5 μM), or compound 3 (5 μM). Live cells were
imaged with an inverted Zeiss LSM 510 Meta laser scanning confocal microscope.
Similar results were obtained with three independent transfections performed in
triplicate. Bars, 10 μm.
In order to corroborate that the observed mislocalization of the Ras isoforms
is caused by ICMT inhibition, and not by other non-specific mechanisms, we
corrobated that Fyn, a protein which belongs to the Src family of tyrosine
protein kinases that is located at the plasma membrane after myristoylation and
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palmitoylation processes, but which is not a substrate of ICMT, is not affected
by compound 3 nor cysmethynil (Supporting Figure S5). We also confirmed
that compound 3 did not affect geranylgeranylated proteins, because AAX
removal and subsequent methylation by ICMT takes place only for formerly
farnesylated Ras. The differential substrate recognition of FTase against GGTase
I is dictated by the amino acid situated in the X position of the CAAX
1
1
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tetrapeptide:
S
and
M
defines farnesylation whereas
L
indicates
geranylgeranylation.34 Using a K-Ras4B protein tagged with GFP which
contains a point mutation in the CAAX motif designed to replace farnesylation
by geranylgeranylation, we determined the influence of compound 3.
Consistent with an ICMT-dependent effect, geranylgeranylated K-Ras4B (K-
Ras4B-CVIL-GFP) was located in the plasma membrane with an identical
pattern in the presence or in the absence of derivative 3, while farnesylated K-
Ras4B-GFP was mislocalized to the cytosol after the incubation with compound
3
(Supporting Figure S6). To further verify the specificity of the compound’s
mechanism of action we tested its effects on cells where ICMT expression has
been blocked with a specific small interfering RNA as well as on the enzymes
involved in the prenylation pathway (FTase, GGTase, and Rce1). Our results
show that when ICMT expression is blocked with a specific small interfering
RNA, compound 3 does not induce significant cytotoxicity, suggesting that the
effects observed are mainly due to ICMT inhibition (Figure 8A). Moreover, this
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compound does not inhibit in a noticeable manner FTase, GGTase or Rce1
enzymes, as supported by the fact that specific substrates of these enzymes
remain unaffected in the presence of compound 3. For example, protein HDJ2 is
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specifically farnesylated by FTase. Accordingly, lonafarnib, a selective FTase
inhibitor, impairs its farnesylation as revealed by the appearance of a non-
farnesylated HDJ2 band in the immunoblot (Figure 8B). Conversely, neither
compound 3 nor a specific GGTase inhibitor (compound GGTI298) affect HDJ2
processing. In the case of Rap1A, a specific substrate of GGTase,35 neither
compound 3 nor lonafarnib have any influence in the geranylgeranylation of
this protein whereas the specific GGTase inhibitor GGTI298 clearly inhibits its
prenylation, as indicated by the appearance of the corresponding upper band
(Figure 8B). Similarly, compound 3 does not affect to the hydrolysis by Rce1 of
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the specific peptide substrate KSKTKC(farnesyl)VI (Supporting Figure S7).
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Figure 8. Compound 3 does not induce cytotoxicity in ICMT-depleted cells
nor inhibits FTase or GGTase enzymes. (A) Effect of siRNA-induced silencing
of ICMT gene expression on cytotoxicity induced by compound 3. Cytotoxicity
of compound 3 at different concentrations is eliminated by siRNA-induced
reduction of ICMT expression levels. PC-3 cells were transfected with siRNA-
targeting ICMT for 72 h. Equal amounts of total protein from PC-3 cells
transfected with siRNA-targeting ICMT or with the control were subjected to
immunoblotting analyses with antibodies against ICMT or GADPH as loading
control. Results shown in the lower panel are the average±SEM of three
different experiments. Shown gel is representative of those obtained in three
independent experiments. ns, not significant; ***p < 0.001 (Student’s t-test) vs
ICMT expression in siRNA control-transfected cells. (B) Effect of compound 3
on the prenylation state of HDJ2 and Rap1A proteins. PC-3 cells were treated with
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vehicle, compound 3, GGTase inhibitor GGTI298 (10 µM) or lonafarnib (1 μM), lysed
and immunoblotted with antibodies against HDJ2, Rap1A or GADPH (used as loading
control). Images are representative of three independent experiments.
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Compound 3 induces cell death in a variety of Ras-mutated tumor cell lines
Taken together, all these data suggest that compound 3 should be able to
significantly impair cell proliferation in Ras-mutated tumor cells of different
origins and with distinct Ras mutations. To assess this, we have selected a panel
of cells bearing the most frequently mutated Ras isoform in the majority of
cancers (K-Ras) with the most abundant point mutations (G12C, G12D, G12V,
and G13D) and two cell lines with mutated N-Ras, the most important isoform
3
3
in acute myeloid leukemia (AML) and in melanoma. The obtained data (Table
6
) show that compound 3 significantly reduces cell proliferation in all tested
Ras mutant cell lines with IC50 values in the low micromolar range and
selectivity against non-tumor fibroblasts. Although these results are consistent
with a Ras-dependent cytotoxicity mediated by ICMT inhibition, the existence
of genetic alterations and differences between cell lines can influence sensitivity
to ICMT inhibitors and of course only a careful delineation of all the oncogenic
signalling pathways together with data on ICMT expression and activity will
enable the complete understanding of the ICMT contribution to cancer.
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Table 6. Cytotoxicity of compound 3 in a panel of Ras-mutated cancer cell lines
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Cell
Tissue
Origin
Ras isoform
and status
IC50
(μM)a
PANC-1
MIA PaCa-2
MDA-MB-231
SW620
Pancreas
Pancreas
Breast
K-Ras (G12D)
K-Ras (G12C)
K-Ras (G13D)
K-Ras (G12V)
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2
5
Colon
3
SK-Mel-173
HL60
Melanoma N-Ras (Q61K)
12
2
Blood
N-Ras (Q61K)
Non-tumor
NIH3T3
Fibroblast
Fibroblast
> 50
> 50
1
42BR
Non-tumor
a
Cytotoxicity was evaluated by the MTT or the XTT assay. IC values are the means
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from two to three independent experiments performed in triplicate. In all cases, the
SEM is within 10% of the mean value.
The dose-response analysis showed that compound 3 promoted cell death at
1
0 µM after 24 h treatment in MIA PaCa-2 and HuP-T4 as well as at 20 µM in
the rather resistant PANC-1, PK9, and Capan-2 cell lines whereas a
concentration of 50 µM was required to induce cell death with cysmethynil.
Furthermore, pancreatic cancer Capan-2 cells were even resistant to this
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concentration of cysmethynil (Supporting Figure S8). The analysis of the sub-
G /G region of cell cycle confirmed that compound 3 induced an apoptosis-like
0
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cell death (Supporting Figure S9).
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In vivo antitumor effect of compound 3
Finally, to evaluate the potential of the ICMT inhibitor in an in vivo setting,
we performed a xenograft mice model by transplanting HL-60 N-Ras mutated
AML cells into NSG mice. An initial pharmacokinetic profile was carried out in
mice prior to the in vivo efficacy testing. In vitro, the compound showed a half-
life in mouse serum of around 3 h and in human serum of more than 24 h. For
in vivo pharmacokinetic studies, mice received a single intraperitoneal injection
of 25 mg/kg of compound 3 and the plasma concentration of the compound was
measured at different time points (Supporting Figure S10). At this dose,
concentration of compound reached a maximal concentration in serum of 1700
nM 1 h post-injection and remained around 480 nM up to 6 h post-injection, so
we considered these values as satisfactory to allow the in vivo use of the
compound using these conditions. Treatment of mice with compound 3 (25
mg/kg, intraperitoneally) for 15 days (3 cycles of 5 days of treatment followed
by 2 days of rest) increased mice survival (Figure 9A, p = 0.0274) and reduced
the tumor burden in the bone marrow compared with vehicle (Figure 9B,C).
Before sacrifice, the serum levels of compound 3 were measured the last day of
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the treatment (2 h post-injection) and turned out to be 736 nM, in full
consistency with the pharmacokinetic curve. These results demonstrate the in
vivo efficacy of compound 3 against Ras-dependent malignancies.
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Figure 9. Antitumor effect of compound 3 in the Ras-driven model of acute
myeloid leukemia. (A) Kaplan-Meier curves indicating survival of HL-60 transplanted
NSG mice treated with vehicle (n=5) or with compound 3 (n=9). Statistical significance
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