A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf

Article abstract of DOI:10.1002/chem.201903129

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein–protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.

Full text of DOI:10.1002/chem.201903129

A Journal of
Accepted Article
Title: A Guanidyl-based Bivalent Peptidomimetic Inhibits K-Ras
Prenylation and Association with c-Raf
Authors: Junko Ohkanda, Mai Tsubamoto, Toan Khanh Le, Minghua
Li, Taku Watanabe, Chiemi Matsumi, Prakash Parvatkar,
Hiroshi Fujii, Nobuo Kato, and Jiazhi Sun
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201903129
Link to VoR: http://dx.doi.org/10.1002/chem.201903129
Supported by

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
A Guanidyl-based Bivalent Peptidomimetic Inhibits K-Ras
Prenylation and Association with c-Raf
Mai Tsubamoto,[b] Toan Khanh Le,[a] Minghua Li,[e] Taku Watanabe,[c] Chiemi Matsumi,[c] Prakash
Parvatkar,[d] Hiroshi Fujii,[a] Nobuo Kato,[b] Jiazhi Sun,[e] Junko Ohkanda*[a]
Abstract: Unusual lipid modification of K-Ras makes Ras-directed
cancer therapy a challenging task. Aiming to disrupt electrostatic-
driven protein-protein interactions (PPIs) of K-Ras with FTase and
GGTase I, a series of bivalent dual inhibitors that recognize the active
pocket and the common acidic surface of FTase and GGTase I were
designed. The structure-activity-relationship study resulted in 8b, in
which a biphenyl-based peptidomimetic FTI-277 was attached to a
guanidyl-containing gallate moiety through an alkyl linker. Cell-based
evaluation demonstrated that 8b exhibited substantial inhibition of K-
Ras processing without apparent interference with Rap-1A
processing. Fluorescent imaging showed that 8b disrupts localization
of K-Ras to the plasma membrane and impairs interaction with c-Raf,
whereas only FTI-277 was found to be inactive. These results suggest
that targeting the PPI interface of K-Ras may provide an alternative
method of inhibiting K-Ras.
efforts have largely relied on library screenings. Complementary
structure-based approaches may therefore lead to the
development and identification of K-Ras inhibitors.
Earlier efforts have focused on protein prenylation[9], as
posttranslational modification followed by association with the
plasma membrane is an essential step for Ras function. Many
inhibitors[10] and chemical probes[11] for the protein
farnesyltransferase (FTase) have been developed. However, the
clinical trials of FTase inhibitors (FTIs) as cancer therapies
eventually failed, shedding light on their ineffectiveness against
K-Ras-mutated cancers.
This obstacle has been explained by the intrinsically higher
affinity of K-Ras to FTase than H-Ras (20–50 times)[12,13] and the
prenylation
of
K-Ras
by
the
related
Type
I
geranylgeranyltransferase (GGTase I) under FTI treatment
conditions, after which the resulting geranylgeranylated K-Ras
retains full biological function.[14,15] The unusual prenylation
makes FTI-based approaches a difficult means of inactivating K-
Ras. Studies have revealed that the alternative prenylation of K-
Ras is triggered by electrostatic attraction between the basic C-
terminal of K-Ras and the acidic surface of the common -subunit
of FTase and GGTase I.[12–16] This suggests that mimicking
structural features of the cationic C-terminus of K-Ras may result
in a dual inhibitor of K-Ras farnesylation and geranylgeranylation.
FTase and GGTase I are heterodimeric zinc metalloenzymes,
and they transfer a prenyl group on the C-terminal cysteine
residue of substrate proteins. In general, FTase and GGTase I
recognize a consensus tetrapeptide sequence CAAX, where C is
a cysteine, AA is any aliphatic dipeptide, and X is Ser, Met, Ala or
Gln for FTase and hydrophobic Leu or Phe for GGTase I. Since
K-Ras possesses a CVIM sequence, it normally receives
farnesylation (Figure 1, path (i)). However, when FTase is
inhibited by FTIs, it in turn undergoes geranylgeranylation (Figure
1, path (ii)).[14,15] Biological studies[12–15] and X-ray crystal
structures of FTase bound to a K-Ras C-terminal peptide[16] have
demonstrated that the electrostatic interaction between the oligo
lysine domain of K-Ras and a local acidic surface area of the
common -subunit of FTase and GGTase I containing a number
of acidic amino acids (i.e. Glu125, Glu160, Glu161, Glu187,
Asp191, Glu229, Asp230) are responsible for increasing the
affinity for both enzymes.
Introduction
Members of the Ras family of 21-kDa GTPases play essential
roles as molecular switches of signalling pathways that regulate
many cellular events, including cell survival and proliferation. K-
Ras is the most frequently mutated Ras isoform in human cancers,
and it promotes aggressive tumorigenesis, leading to drug
resistance and poor clinical outcomes.[1,2] Thus, synthetic
molecules that suppress K-Ras-mediated signals are urgently
needed.[3] Recent success in library screening-based approaches
have identified low-molecular-weight agents[4–7] and cyclic
peptides[8] that allosterically or irreversibly inhibit K-Ras function,
highlighting the clinical promise of Ras-directed cancer therapy.
However, because spherical structural features of Ras lack
apparent binding cavities for small molecules, substantial medical
[a]
[b]
Toan Khanh Le, Prof. Dr. Hiroshi Fujii, Prof. Dr. J. Ohkanda
Academic Assembly, Institute of Agriculture
Shinshu University
8304 Minami-Minowa, Kami-Ina, Nagano 399-4598 (Japan)
E-mail: johkanda@shinshu-u.ac.jp
M. Tsubamoto, Prof. Dr. N. Kato
The Institute of Scientific Industrial Research
Osaka University
8-1 Mihogaoka, Ibaraki, Osaka 567-0047 (Japan)
Dr. T. Watanabe, Dr. C. Matsumi
Ina Laboratory, Medical & Biological Laboratories, CO., Ltd., Ina,
Nagano 396-0002 (Japan)
Dr. Prakash Parvatkar
Institute for Chemical Research, Kyoto University, Gokasho, Uji,
Kyoto 611-0011 (Japan)
Dr. Minghua Li, Prof. Dr. Jiazhi Sun
Department of Pharmaceutical Science, University of South Florida,
Tampa, Florida 33612 (USA)
We envisioned that an agent capable of disrupting electrostatic-
driven protein–protein interactions (PPIs) would be produced by
covalent linking of a CVIM or its equivalent moiety and a
compound mimicking the basic oligo lysine domain. The resulting
hybrid compound would anchor to the active pocket and deliver
the cationic moiety to the acidic PPI interfaces of FTase and
GGTase I (Figure 1, (iii)), thereby acting as a dual inhibitor of K-
Ras farnesylation and geranylgeranylation (Figure 1, (iv)).
Here, we report the first cell-permeable K-Ras C-terminal
mimetics in which a conventional FTI is linked to a branched
[c]
[d]
[e]
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
Figure 1. Schematic presentation of alternative prenylation of K-Ras in cells and the dual inhibition strategy for both farnesylation and geranylgeranylation of K-
Ras using a bivalent agent mimicking the structure of the K-Ras C-terminus.
R2
cationic element that binds to the acidic surface of these enzymes.
The compound is shown to inhibit cellular processing of K-Ras
more effectively than conventional FTIs and to disrupt localization
of K-Ras to the plasma membrane, thereby interacting with c-Raf,
a downstream effector.
R
R3
R4
R1
S
1
2
3
4
1
2
3
4
5
6
A
G
G
H
G
G
H
H
G
G
H
G
A
G
H
H
H
G
H
H
H
G
G
H
O
OR5
O
NH
N
H
H
N
O
5
N
H
O
SH
Results and Discussion
A = O(CH)3NH2
1-6 R5
G = O(CH2)3NHC(NH)NH2
a
b
H
Me
Design and synthesis of 1st generation of bivalent
compounds
NH
We previously prepared a peptide-based bivalent agent, in which
a CVIM tetrapeptide was covalently attached via an alkyl spacer
to a gallate derivative containing six primary amino groups.[17] An
in vitro enzyme assay demonstrated that the resulting hybrid
compound shows substantially improved inhibition activity against
farnesylation and geranylgeranylation of the C-terminal K-Ras
peptide relative to the CVIM tetrapeptide alone, indicating the
potential of the bivalent strategy for disrupting the PPIs.[17]
However, the compound largely lacks cell permeability,
hampering further cell-based evaluation.[17,18] These results
prompted us to modify the chemical structure to improve its cell-
based activity.
H2N
NH
NH
O
S
H2N
HN
H2N
NH
NH
O
OH
N
O
O
H
H2N
O
N
H
SH
O
OH
FTI-249
7
Figure 2. Chemical structures of bivalent compounds (1–6) and module
compounds, FTI-249 and guanidyl-containing gallate 7.
Arginine-rich peptides are widely used for intracellular delivery
of bioactive compounds. The bidentate conjugated guanidinium
cation in an arginine residue interacts with lipid phosphate anions
and induces membrane curvature alteration,[19] while the C3 side
chain is thought to interact with the hydrophobic portion of lipids
and trigger membrane penetration of the peptide.[19,20] Thus, C3-
alkylated guanidine moiety may be a useful building block for
improving the compound’s permeation of cells.
Over the last few decades, Ras CAAX peptidomimetics have
been intensively studied in development of pharmaceuticals for
cancers[10] and parasitic diseases.[21] Hamilton and Sebti
developed a series of 4-amino benzoic acid derivatives that mimic
an extended conformation of a CVIM tetrapeptide.[22] Their early
examples include FTI-249[23] (Figure 2), which selectively inhibits
FTase over GGTase I in vitro (for FTase, IC50 = 0.03 M; for
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
GGTase I, IC50 = 4.4M),[23] and the corresponding methyl ester-
prodrug, which suppressed H-Ras processing in cells.[23]
When an alkyl gruanidyl group in 2a was repositioned from R3
to R2, the resulting 3a exhibited significantly lower activity for
GGTase I, indicating that R2 substitution is unfavorble for binding
GGTase I. This was further supported by 5a, which has a
substitutent at the R1 position, showing better activity for GGTase
I than 4a, which is structually identical to 5a except for the
substituent occuring at R2, not R1. As seen in 2a and 5a,
introduction of the substitutent at R4 reduced activity for FTase
approximately 9-fold, presumably owing to a steric repulsion with
the protein.
We then introduced an additional substituent to obtain a
trisubstituted compound, and the resulting 6a showed
substantially improved activity for FTase compared to 2a,
presumably owing to the gain in electrostatic force at the acidic
surface of the protein. In contrast, gallate derivative 7 showed no
inhibitory activity, validating that the strategy of anchoring and
delivering the small cationic moiety onto the common acidic
surface enables dual inhibition of FTase and GGTase I.
Thus, we first decided to incorporate C3-alkylated guanidyl
groups and a FTI-249 unit into our new inhibitor design and then
prepared a series of bivalent compounds 1a–6a, in which guanidyl
or amino groups were introduced to various positions of the
benzoyl scaffold (Figure 2). The cationic benzoic acid was linked
to FTI-249 through a C5 alkyl spacer, as this length was found to
be optimal in previous work[17] (for synthesis, see SI). The
corresponding methyl esters of each compound were also
synthesized for cell-based evaluation (1b–6b).
In vitro inhibition of FTase and GGTase I activities
Inhibitory activities of 1a–6a against recombinant FTase
andGGTase I were performed with a previously reported
fluorescent spectroscopic assay[18] using dansyl-labelled K-Ras
C-terminal peptide (K)6SK(Dans)TKCVIM (Dans = -N-dansyl) as
a substrate, and the results are summarized in Table 1.
Antiproliferative activity against cancer cells
Table 1. Inhibition of prenylation of the K-Ras C-terminal peptide by FTase
and GGTase I [a]
Next, we evaluated whether these compounds inhibit cell
proliferation as reported for FTIs.[26–28] Previous studies have
demonstrated that ester-prodrugs significantly improve cell-based
activity.[29] Therefore, methyl esters 1b, 2b, 6b and 7 were
Compound
Ki (M) [b]
Farnesylation
0.785 ± 0.008
0.00074
Geranylgeranylation
-
subjected to
a trypan blue assay using oncogenic Ras-
FTI-249[c]
transformed T24 human bladder carcinoma cells (Figure 3). All
tested compounds, except for 7, exhibited antiproliferation activity
in a concentration-dependent manner, in which their descending
order of effects (6b, 2b and 1b) corresponds with the descending
order of enzyme inhibition activities that were observed for their
corresonding free acids (6a, 2a and 1a) (Table 1). These results
demonstrated that the guanidyl-containing bivalent agents
penetrate through cell membranes and exhibit inhibitory activity
against cell growth. However, the activity remains moderate
across a high micromolar range, and only weak inhibition against
FTase was detected for 6b in cells (Figure S1), prompting us to
further modify the chemical structure of the agents.
FTI-276[d]
-
1a
2a
3a
4a
5a
6a
7
0.805 ± 0.009
0.156 ± 0.014
0.768 ± 0.011
1.46 ± 0.011
1.35 ± 0.011
0.026 ± 0.001
>500
0.856 ± 0.037
0.573 ± 0.054
8.67 ± 0.077
8.13 ± 0.049
1.87 ± 0.045
2.50 ± 0.046
-
8a
0.0006 ± 0.0002
0.710 ± 0.002
[a] Fluorescent in vitro assays were performed in a 96-well black plate using
KKKKKK(Dans)TKCVIM (1 M), farnesyl pyrophosphate (FPP, 5 M),
compounds (0–500 M) and enzyme (50 nM) in 50 mM Tris HCl (pH 7.5) at
30°C. The reaction was monitored at 520 nm (ex. 340 nm) for 5 min with a
plate reader. The standard deviation is given for n = 3. [b] Ki values were
obtained by conversion of IC50 values using the Cheng–Prusoff equation
(Ref. 24), Ki = IC50/{1+([S]/Km)}, where for FTase Km = 0.006 ± 0.002 M
and for GGTase Km = 0.033 ± 0.008 M (Ref. 17). [c] Ref. 18. [d] Ref. 25.
Figure 3. Antiproliferative activity for T24 cells (n = 3). Cells (1 × 104 cells) were
treated with 1b, 2b, 6b, 7 or DMSO and incubated at 37°C, 5% CO2 for 5 days
prior to a trypan blue assay. n ≥ 3.
Primary amino group–containing 1a had similar activity for
FTase and GGTase I, demonstrating that attachment of the
cationic moiety to FTI-249 converts the selective FTI into a dual
inhibitor, which is consistent with previously reported results.[17]
Compound 2a, in which the primary amino groups were replaced
by guanidyl groups, was approximately 5-fold more active than 1a,
suggesting that a guanidyl group is more capable of association
with acidic surfaces.
Second generation guanidyl-containing bivalent compounds
based on a biphenyl scaffold
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
To this end, we decided to synthesize 8, the second generation
of guanidyl-containing bivalent compound, by exploiting biphenyl-
based peptidomimetic FTI-276[30] (Figure 4). We envisioned that
exploiting the potent peptidomimetic (for FTase, IC50 = 0.61 nM;
for GGTase I, IC50 = 50 nM)[30,31] would increase in vitro and whole
cell activity of the resulting bivalent compounds 8a and 8b (Figure
4), thus allowing for a detailed evaluation of their effects on
cellular events.
First, we evaluated the enzyme inhibition activity of 8a and
compared it with that of FTI-249-based 6a (Figure 5a). Thus, 8a
exhibited significant inhibition activity against FTase (Ki = 0.0006
± 0.0002M, red solid line in Figure 5a), which is approximately
50-fold greater than that of 6a (Ki = 0.026 ± 0.001 M, blue solid
line in Figure 5a). Notably, 8a maintains submicromolar activity
for GGTase I (Ki = 0.71 ± 0.002 M), although the improvement
remains only approximately 3-fold compared with that of 6a (Ki =
2.5 ± 0.046 M, red and blue dash lines in Figure 5a,
respectively). This somewhat moderate effect of the introduction
of FTI-276 on GGTase I inhibition might be explained by a
possible conformational distortion of the peptidomimetic unit
caused by the amide linkage to the gallate moiety, as previously
discussed in the literature.[32] The presence of the substituent at
the R4 position in 8a may also account for the relatively low activity
for GGTase I, as discussed above.
Figure 4. Chemical structures of the bivalent compound 8 and its module
compound, FTI-276.
Figure 6. (a) Effect of 8b on processing K-Ras (G12V) in NHH-3T3 cells stably
expressing mutated K-Ras. U = unprocessed, P = processed. (b) Effect of FTI-
277 and 8b on processing K-Ras and Rap-1A and on induction of p21. Anti-K-
Ras, -geranylgeranylated Rap-1A and -p21 were used, respectively. (c)
Confocal fluorescent imaging using anti-K-Ras antibody and DAPI staining of
Panc-1 cells treated with 8b (10 M).
Next, we evaluated the methyl ester form, 8b (MT-103), against
endogenous FTase in cells by monitoring the farnesylation level
of the 40-kDa heat-shock protein HDJ-2 by a similar procedure to
that reported in the literature.[29] Briefly, T24 cells were treated
with various concentrations of 8b, and cell lysates were analysed
by western blot using anti-HDJ-2 antibody, allowing the
differentiation of processed from unprocessed HDJ-2 protein[29]
(Figure 5b). In contrast to 6b (Figure S1), 8b supressed HDJ-2
processing, with an IC50 value of approximately 0.5 M, which is
more than two orders of magnitude greater than that of 6b,
supporting the remarkable inhibition efficacy of 8b against FTase
in cells.
Figure 5. (a) Dose-response curves for the inhibition of FTase (solid) and
GGTase I (dash) by 6a (blue triangles) and 8a (red circles). Fluorescent in vitro
assays were conducted using KKKKKKSK(Dans)TKCVIM (1 M) and FPP (5
M) in 50 mM Tris HCl (pH 7.5) at 30°C. n = 3. (b) Effect of 8b on processing of
HDJ-2 in T24 cells. Inhibition of farnesylated HDJ-2 is seen by the band shift
from processed (P) to unprocessed (U) protein. Imidazole-containing FTI-2153
(Ref. 33) and GGTI-2154 (Ref. 29) (10 M) were used as positive controls (Ref.
34). Their chemical structures are shown in Figure S2.
Bivalent compound inhibits prenylation processing of K-Ras
We then wanted to address a critical question: does the bivalent
8b disrupt K-Ras processing in cells? Thus, we treated NIH-3T3
cells stably expressing G12V K-Ras with various concentrations
of 8b and performed western blot analysis using anti-K-Ras
antibody. Notably, K-Ras processing was inhibited at low
micromolar concentrations in cells (Figure 6a), whereas only FTI-
277, the methyl ester form of FTI-276, was shown to be inactive
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
Figure 7. (a) Fluorescence images of cultured HEK293 cells stably expressing K-Ras (G12C) fused to Ash-tag and cRaf (RBD) fused to hAG (humanized Azami-
Green). Cells were treated with 50 M ARS-853, 30 M FTI-277 and 30 M 8b, respectively, and incubated for 4.5 h prior to fixation with paraformaldehyde. Scale
bars, 50 m. Arrows in magnified window indicate puncta accumulated at the plasma membrane. (b) Fluorescence intensities of puncta were segmented and
quantified using the ICY image analysis platform and then divided by the number of nuclei. At least 232 cells were analyzed for each image (n = 3).
at 10 M, as previously reported[35] (Figure 6b, upper row). These
results demonstrate that the ability to bind to the acidic surface of
FTase and/or GGTase I accounts for the potency of 8b against K-
Ras processing.
Ras(G12C) fused to an assembly helper protein (Ash-K-
Ras(G12C)) and the Ras-binding domain of c-Raf (c-Raf(RBD))
fused to fluorescent Azami-Green (hAG-cRaf(RBD)) were stably
expressed in HEK239 cells, where Ash protein forms a oligomer
and hAG protein forms a tetramer. As prenylation of clustered
Ash-K-Ras(G12C) occurs and the tagged K-Ras proteins are
promoted to localise near cytoplasmic membranes, tetrameric
cRaf(RBD) are recruited to the membranes through PPIs with the
tagged K-Ras to form a large protein assembly. This results in the
formation of fluorescent puncta composed of these tagged
proteins near the cell periphery.[37] A selective covalent inhibitor
of K-Ras(G12C), ARS853,[38] induced no apparent formation of
puncta at 50 M (Figure 7a, left), indicating that AR853 inhibits K-
Ras/c-Raf interactions. In contrast, when cells were treated with
FTI-277 at 30 M, puncta were clearly observed near membranes
(Figure 7a, middle), indicating that K-Ras is recruited to the
plasma membrane for interactions with c-Raf. In contrast, 8b
reduced the degree of puncta formation (Figure 7a, right),
demonstrating that 8b inhibits K-Ras-c-Raf interaction by
approximately 25% compared to FTI-277 (Figure 7b). To our
knowledge, this is the first example of a dual inhibitor of FTase
and GGTase I disrupting K-Ras plasma membrane localization
and interaction with c-Raf, one of its downstream effectors.
Surprisingly, 8b did not interfere the native GGTase I
processing of Rap-1A (Figure 6, middle row). Rap-1A is a Ras-
related protein with a C-terminal CLLL sequence and exclusively
receives geranylgeranylation. The western blot analysis using
anti-geranylgeranylated Rap-1A antibody showed that FTI-277
partially inhibited Rap-1A processing as previously reported at
high concentrations (10–15 M),[34] whereas 8b did not show
apparent activity (Figure 6b, middle row). This result suggests that
covalent linking of the gallate moiety to FTI-277 reduced the
affinity of 8b for GGTase I, therefore 8b failed to inhibit
geranylgeranylation of the native substrate, which comprises the
C-terminal leucine residue that has higher affinity to the active site
of GGTase I than methionine residue does.
It has been reported that inhibition of FTase and GGTase I
causes induction of cyclin-dependent kinase inhibitor p21 protein
and thus G1 arrest.[33,36] As shown in Figure 6b (lower row), both
FTI-277 and 8b clearly induced p21 expression, further
supporting that FTase and/or GGTase I are inhibited in cells.
Furthermore, confocal fluorescent microscopy analysis of K-Ras-
transformed pancreatic carcinoma Panc-cells using anti-K-Ras
antibody showed that 8b promotes cytosolic delocalization of
endogenous K-Ras (Figure 6c), further supporting that K-Ras
prenylation was disrupted by 8b.
Conclusions
Based on the C-terminal structure of K-Ras, we developed a
series of bivalent dual inhibitors of FTase and GGTase I by
covalently linking a commercially available CVIM peptidomimetic
and various guanidyl-containing benzoyl moieties. The biological
evaluation demonstrated that 8b is capable of inhibiting HDJ-2
and K-Ras processing in cells at submicromolar to low micromolar
concentrations. Furthermore, fluorescent imaging revealed that
Bivalent compound inhibits K-Ras/c-Raf interaction on
cytoplasmic membranes
Finally, we examined whether 8b is capable of disrupting K-Ras-
Raf PPIs on plasma membranes. To address this issue, we used
a
previously developed fluorescent-based PPI-visualisation
system (Fluoppi)[37] to monitor interactions between the K-Ras
GTP-bound form (i.e. active form) and c-Raf in cells. Briefly, K-
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
[8]
[9]
P. Upadhyaya, Z. Qian, N. G. Selner, S. R. Clippinger, Z. Wu, R.
Briesewitz, D. Pei, Angew. Chem. Int. Ed. 2015, 54, 7602-7606.
F. L. Zhang, P. J. Casey, Annu. Rev. Biochem. 1996, 65, 241-269.
8b disrupts membrane localization of K-Ras and its association
with c-Raf.
Because K-Ras must be localized to plasma membranes to be
functional,[39,40] chemical disruption of the process of membrane
localization remains an attractive therapeutic approach, and
various inhibitors of Rce,[41] Icmt,[42,43] PDE[44] and others are
under development. Although the activity of 8b remains moderate,
the results obtained in this study suggest that a molecular strategy
for targeting PPI interfaces between K-Ras and FTase and
GGTase I may provide a solution for overcoming the obstacles of
FTIs in K-Ras-directed anticancer clinical applications. One
limitation of 8b is its sensitivity to oxidation caused by sulphur
atoms in the peptidomimetic, and at present, it is unclear whether
8b inhibits geranylgeranylation of K-Ras in cells or the degree to
which downstream signals of K-Ras were perturbed by 8b. These
issues will need to be addressed in order to better understand the
mode of action. To that end, more metabolically stable agents
based on non-thiol-containing peptidomimetics are therefore
desirable. Work is currently underway in our laboratory towards
these goals.
[10] Examples of recent reviews: a) C. C. Palsuledesai, M. D. Distefano, ACS
Chem. Biol. 2015, 10, 51-62; b) N. Berndt, A. D. Hamilton, S. M. Sebti,
Nat. Rev. Cancer 2011, 11, 775-791.
[11] A. T. Placzek, A. J. Krzysiak, R. A. Gibbs, Enzymes 2011, 30, 91-127.
[12] G. L. James, J. L. Goldstein, M. S. Brown, J. Biol. Chem. 1995, 270, 6221.
[13] F. L. Zhang, P. Kirschmeier, D. Carr, L. James, R. W. Bond, L. Wang, R.
Patton, W. T. Windosor, R. Syto, R. Zhang, W. R. Bishop, J. Biol. Chem.
1997, 272, 10232-10239.
[14] D. B. Whyte, P. Kirschmeier, T. N. Hockenberry, I. Nunez-Oliva, L.
James, J. J. Catino, W. R. Bishop, J. Pai, J. Biol. Chem. 1997, 272,
14459-14464.
[15] C. A. Rowell, J. J. Kowalczyk, M. D. Lewis, A. M. Garcia, J. Biol. Chem.
1997, 272, 14093-14097.
[16] S. B. Long, P. J. Casey, L. S. Beese, Structure 2000, 8, 209-222.
[17] S. Machida, N. Kato, K. Harada, J. Ohkanda, J. Am. Chem. Soc. 2011,
133, 958-963.
[18] S. Machida, M. Tsubamoto, N. Kato, K. Harada, J. Ohkanda, Bioorg.
Med. Chem. 2013, 21, 4004-4010.
[19] I. Nakase, T. Takeuchi, G. Tanaka, S. Futaki, Adv. Drug. Deliver Rev.
2008, 60, 598-607.
[20] E. G. Stanzl, B. M. Trantow, J. R. Vargas, P. A. Wender, Acc. Chem.
Res. 2013, 46, 2944-2954.
[21] Example of recent review: S. Fletcher, C. G. Cummings, K. Rivas, W. P.
Katt, C. Horney, F. S. Buckner, D. Chakrabarti, S. B. Sebti, M. H. Gelb,
W. C. Van Voorhis, A. D. Hamilton, J. Med. Chem. 2008, 51, 5176-5197.
[22] D. Knowles, J. Sun, S. Rosenberg, S. M. Sebti, A. D. Hamilton, in
Farnesyltransferase Inhibitors in Cancer Therapy (Eds. S. M. Sebti A. D.
Hamilton), Humana Press, New Jersey, ch. 4, pp. 49-64.
[23] A. Vogt, Y. Qian, M. A. Blaskovich, R. D. Fossum, A. D. Hamilton, S. M.
Sebti, J. Biol. Chem. 1995, 270, 660-664.
Acknowledgements
This work was supported by JSPS (18H02106, 25288076,
26102727 to J.O.) and MEXT Grants (18H04394 to J.O.), and the
Nagase Science and Technology Foundation, Astellas
Foundation for Research on Metabolic Disorders, Naito
Foundation, and Uehara Foundation (J.O.). We thank the
Comprehensive Analysis Center at ISIR, Osaka University and A.
Oda of the Instrumental Center, Shinshu University for spectral
measurements, and K. Tanaka, F. Sugino, and S. Igaue for
characterization of compounds. J.O. sincerely thanks Profs. A. D.
Hamilton and S. M. Sebti for their valuable comments throughout
this work.
[24] Y. Cheng W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099-3108.
[25] Y. Qian, Ph. D. thesis, University of Pittsburgh (USA), 1996.
[26] A. D. Cox, C. J. Der, Biochim. Biophys. Acta 1997, 1333, F51-71.
[27] J. B. Gibbs, A. Oliff, Annu. Rev. Pharmacol. Toxicol. 1997, 37, 143.
[28] S. M. Sebti, C. J. Der, Nat. Rev. Cancer 2003, 3, 945-951.
[29] J. Sun, J. Ohkanda, D. Coppola, H. Yin, M. Kothare, B. Busciglio, A. D.
Hamilton, S. M. Sebti, Cancer Res. 2003, 15, 8922-8929.
[30] Y. Qian, J. J. Marugan, R. D. Fossum, A. Vogt, S. M. Sebti, A. D.
Hamilton, Bioorg. Med. Chem. 1999, 7, 3011-3024.
Keywords: K-Ras • prenylation • protein-protein interactions •
[31] E. C. Lerner, Y. Qian, M. A. Blaskovich, R. D. Fossum, A. Vogt, J. Sun,
A. D. Cox, C. J. Der, A. D. Hamilton, S. M. Sebti, J. Biol. Chem. 1995,
270, 26802-26806.
bivalent inhibitors • c-Raf
[1]
[2]
J. L. Bos, Cancer Res. 1989, 49, 4682-4689.
[32] S. Machida, K. Usuba, M. A. Blaskovich, A. Yano, K. Harada, S. M. Sebti,
N. Kato, J. Ohkanda, Chem. Eur. J. 2008, 14, 1392-1401.
[33] J. Sun, M. A. Blaskovich, D. Knowles, Y. Qian, J. Ohkanda, R. D. Bailey,
A. D. Hamilton, S. M. Sebti, Cancer Res. 1999, 59, 4919-4926.
[34] F. L. Delarue, J. Adnane, B. Joshi, M. A. Blaskovichi, D-A. Wang, J.
Hawker, F. Bizouarn, J. Ohkanda, K. Zhu, A. D. Hamilton, S. Chellappan,
S. M. Sebti, Oncogene 2007, 26, 633-637.
A. Lièvre, J-B. Bachet, D. Le Corre, V. Boige, B. Landi, J-F. Emile, J. F.
Côté, G. Tomasic, C. Penna, M. Ducreux, P. Rougier, F. Penault-Liorca,
P. Laurent-Puig, Cancer Res. 2006, 66, 3992-3995.
[3]
[4]
A. D. Cox, S. W. Fesik, A. C. Kimmelman, J. Luo, C. J. Der, Nat. Rev.
Drug Discov. 2014, 13, 828-851.
F. Shima, Y. Yoshikawa, M. Ye, M. Araki, S. Matsumoto, J. Liao, L. Hu,
T. Sugimoto, Y. Ijiri, A. Takeda, Y. Nishiyama, C. Sato, S. Muraoka, A.
Tamura, T. Osoda, K. Tsuda, T. Miyakawa, H. Fukunishi, J. Shimada, T.
Kumasaka, M. Yamamoto, T. Kataoka, Proc. Natl. Acad. Sci. U.S.A.
2013, 110, 8182-8187.
[35] J. Sun, Y. Qian, A. D. Hamilton, S. M. Sebti, Oncogene 1998, 16, 1467-
1473.
[36] E. T. Efuet, K. Keyomarsi, Cancer Res. 2006, 66, 1040-1051.
[37] T. Watanabe, T. Seki, T. Fukano, A. Sakae-Sawano, S. Karasawa, M.
Kubota, H. Kurokawa, K. Inoue, J. Akatsuka, A. Miyawaki, Sci. Rep.
2017, 7, 46380.
[5]
[6]
J. M. Ostrem, U. Peters, M. L. Sos, J. A. Wells, K. M. Shokat, Nature
2013, 503, 548-551.
S. M. Lim, K. D. Westover, S. B. Ficarro, R. A. Harrison, H. G. Choi, M.
E. Pacold, M. Carrasco, J. Hunter, N. D. Kim, T. Zie, T. Sim, P. A. Janne,
M. Meyerson, J. A. Marto, J. R. Engen, N. S. Gray, Angew. Chem. Int.
Ed. 2014, 53, 199-204.
[38] M. P. Patricelli, M. R. Janes, L-S. Li, R. Hansen, U. Peters, L. V. Kessler,
Y. Chen, J. M. Kucharski, J. Feng, T. Ely, J. H. Chen, S. J. Firdaus, A.
Babbar, P. Ren, Y. Liu, Cancer Discov. 2016, 6, 316-329.
[39] P. J. Casey, P. A. Solski, C. J. Der, J. E. Buss, Proc. Natl. Acad. Sci. U.
S. A. 1989, 86, 8323-8327.
[7]
H. Waldmann, I-M. Karaguni, M. Carpintero, E. Gourzoulidou, C.
Herrmann, C. Brockmann, H. Oschkinat, O. Müller, Angew. Chem. Int.
Ed. 2004, 43, 454-458.
[40] J. F. Hancock, A. I. Magee, J. E. Childs, C. J. Marshall, Cell 1989, 57,
1167-1177.
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
[41] I. Mohammed, S. E. Hampton, L. Ashall, E. R. Hildebrandt, R. A. Kutlik,
S. P. Manandhar, B. J. Floyd, H. E. Smith, J. K. Dozier, M. D. Distefano,
W. K. Schmidt, T. M. Dore, Bioorg. Med. Chem. 2016, 24, 160-178.
[42] J. D. Majmudar, H. B. Hodges-Loaiza, K. Hahne, J. L. Donelson, J. Song,
L. Shrestha, M. L. Harrison, C. A. Hrycyna, R. A. Gibbs, Bioorg. Med.
Chem. 2012, 20, 283-295.
[43]
W. R. Judd, P. M. Slattum, K. C. Hoang, L. Bhoite, L. Valppu, G. Alberts,
B. Brown, B. Roth, K. Ostanin, L. Huang, D. Wettstein, B. Richards, J.
A. Willardsen, J. Med. Chem. 2011, 54, 5031-5047.
[44] B. Papke, S. Murarka, H. A. Vogel, P. Martin-Gago, M. Kovacevic, D. C.
Truxius, E. K. Fansa, S. Ismail, G. Zimmermann, K. Heinelt, C. Schultz-
Fademrecht, A. A. Saabi, M. Baumann, P. Nussbaumer, A. Wittinghofer,
H. Waldmann, P. I. H. Bastiaens, Nat. Commun. 2016, 7, 11360.
This article is protected by copyright. All rights reserved.

10.1002/chem.201903129
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Cell-permeable bivalent dual
Mai Tsubamoto, Toan Khanh Le,
inhibitors for FTase and GGTase I
were designed aiming to disrupt
protein-protein interactions with K-
Ras. Cell-based evaluation
Minghua Li, Taku Watanabe, Chiemi
Matsumi, Prakash Parvatkar, Hiroshi
Fujii, Nobuo Kato, Jiazhi Sun, Junko
Ohkanda*
demonstrated that the compound
inhibits K-Ras processing without
apparent interference with Rap-1A
processing. The compound was also
shown to disrupt localization of K-
Ras to the plasma membrane and
impairs interaction with c-Raf, a
downstream effector.
Page No. – Page No.
A Guanidyl-based Bivalent
Peptidomimetic Inhibits K-Ras
Prenylation and Association with c-
Raf
This article is protected by copyright. All rights reserved.