Development of highly potent inhibitors of the ras-targeting human acyl protein thioesterases based on substrate similarity design

Article abstract of DOI:10.1002/anie.201102965

A matter of common sense: A common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two

Full text of DOI:10.1002/anie.201102965

Communications
DOI: 10.1002/anie.201102965
Enzyme Inhibition
Development of Highly Potent Inhibitors of the Ras-Targeting Human
Acyl Protein Thioesterases Based on Substrate Similarity Design
Christian Hedberg, Frank J. Dekker, Marion Rusch, Steffen Renner, Stefan Wetzel,
Nachiket Vartak, Claas Gerding-Reimers, Robin S. Bon, Philippe I. H. Bastiaens, and
Herbert Waldmann*
Signal-transducing Ras guanine nucleotide binding proteins
(GTPases) play important roles in decisive cellular processes,
such as growth, division, and differentiation.[1,2] Mutations in
the S-farnesylated and S-palmitoylated H- and N-Ras iso-
forms result in oncogenic activation, which frequently occurs
in melanoma, leukemia, and cancers of the bladder, liver, and
kidney.[3]
S-Palmitoylation is required to maintain proper Ras
localization in cells,[4,5] and a dynamic de-/repalmitoylation
cycle determines the trafficking and the spatiotemporal
signaling profile of H- and N-Ras, thus regulating the
amplitude, duration, and coupling to different signaling
pathways and effectors.[6] The dynamic nature of the cycle is
established by palmitoyl transferases and thioesterases, and
recently, acyl protein thioesterase 1 (APT1) has been proven
to be a major H- and N-Ras depalmitoylating enzyme in
cells.[7,8]
global Ras signaling.[7] However, it remains unclear if APT1 is
the only Ras-depalmitoylating enzyme in cells and whether
palmostatin B targets additional proteins relevant to the Ras
acylation cycle, in particular the closely related isoenzyme
APT2.[9]
Herein we describe the substrate-based design of novel b-
lactone inhibitors of Ras depalmitoylation which led to the
development of the highly potent APT inhibitor palmosta-
tin M (30, Table 1). In the following manuscript,[10] we detail
the development of activity-based probes based on palmos-
tatin M and B as well as their use in chemical proteomics
experiments, which have revealed both APT1 and APT2 as
the targets relevant to the dynamic Ras acylation cycle.
The APT1 inhibitor palmostatin B was identified by
employing the chemical structure based principles of protein
structure similarity clustering (PSSC)[11,12] and biology-ori-
ented synthesis,[13–15] namely the structural similarity among
ligand-sensing cores of proteins and of related classes of
Chemical interference with the dynamic Ras de/reacyla-
tion cycle by using the b-lactone APT1 inhibitor palmosta-
tin B (Figure 1) disturbs the precise steady-state localization
of H- and N-Ras, thereby causing unspecific entropy-driven
redistribution to endomembranes, and down-regulation of
potential inhibitors. However, APT1 is
a
cytosolic
enzyme[16–19] with very broad substrate scope. It not only
depalmitoylates the C terminus of small GTPases, in both d-
and l-amino acid forms,[8] but also other G proteins,[20,21]
ghrelin (deoctanoylation),[22] viral glycoproteins,[23] and lyso-
phospholipids.[18] This very unusual and exceptionally wide
substrate tolerance inspired us to alternatively employ the
structural characteristics of the different substrates as guiding
arguments for the design of a more potent family of inhibitors.
The activity of palmostatin B is mainly determined by the
stereochemistry of its electrophilic b-lactone core and the
aliphatic substitution at its a position. Ideation based on
substrate similarity indicates that selective recognition will be
enhanced by the introduction of functionalities that enable
hydrogen bonding and electrostatic interactions to the
b position (Figure 1).
[*] Dr. C. Hedberg, Dipl.-Chem. M. Rusch, Dr. C. Gerding-Reimers,
Prof. Dr. H. Waldmann
Max-Planck-Institut fꢀr molekulare Physiologie
Abt. Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
and
Technische Universitꢁt Dortmund, Fakultꢁt Chemie
Lehrbereich Chem. Biologie
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Dr. F. J. Dekker
Univ. of Groningen, University Centre for Pharmacy
A. Deusinglaan 1, 9713 AV Groningen (The Netherlands)
We compared two known native APT1 substrates, lyso-
phospholipid 1 and the H-Ras C terminus 2, and identified a
common recognition motif that consists of a negatively
charged group at a distance of five to six bonds (red) from
the (thio-)ester functionality (green) and a positively charged
tail group ten to twelve bonds away (blue) (Figure 1a). We
found that the phosphatidyl choline moiety 3 (Figure 1b)
fulfilled the design requirements to provide the required
electrostatic interactions. However, since the zwitterionic
nature of the phosphatidyl choline moiety might limit
application in cells, it was replaced by a sulfonyl-1,3-propyl-
Dr. S. Renner, Dr. S. Wetzel
Novartis Pharma AG, Novartis Institute of Biomedical Research
4056 Basel (Switzerland)
Dr. R. S. Bon
School of Chemistry, University of Leeds and
Biomedical and Health Research Centre
LS2 9JT Leeds (UK)
Dr. N. Vartak, Prof. Dr. P. I. H. Bastiaens
Max-Planck-Institut fꢀr molekulare Physiologie
Abt. Systemische Zellbiologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
ene-N,N-dimethylamino functionality
fused fragment 4 through a variable spacer to a trans-b-
4 (Figure 1b). We
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201102965.
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designed
a small focused
library of three series of
inhibitors, denoted A–C,
which would define the struc-
ture–activity
relationship
(Figure 1d). The design was
further verified in silico by
modeling the binding mode
through electrostatic interac-
tions in the active site of the
APT1 apo-crystal struc-
ture.[25] In silico, the polar
residues Q114 and L25 make
strong interactions to the
opened b-lactone, in which
the carbonyl group is cova-
lently bound to the nucleo-
philic S114. Notably, the
polar head group of the inhib-
itor makes contact with resi-
dues T28 and H203, thus
creating an electrostatic sta-
bilizing interaction (Fig-
ure 1e).
The synthesis of the inhib-
itor commenced with the
dicyclohexylboron
triflate
mediated anti-aldol reac-
tion[26] between the O-dode-
cylated ephedrine auxiliaries
6 and 7 with aldehydes 8–11
(Scheme 1, and see the Sup-
porting Information). The
whole synthesis sequence
was carried out with both
enantiomers of the ephedrine
auxiliary, thus resulting in two
series, denoted (S,S) and
(R,R), of reaction products
as single enantiomers. The
aldol reaction products were
purified to diastereomeric
homogeneity by column chro-
matography to yield com-
pounds 12–19. A subsequent
S oxidation of 12–19 with per-
acetic acid yielded the corre-
sponding sulfones, which
were not isolated. Some over-
Figure 1. Inhibitor development based on substrate similarity.
oxidation on the amine func-
tionality occurred (N-oxide
lactone core 5, which addresses the stereochemical preference
of APT1[7] and serves as a covalent modifier of the nucleo-
philic residue in the active site of the enzyme. Variation of the
spacer length enables identification of the optimal distance
between fragments 4 and 5. A lipophilic tail mimicking the
palmitate moiety was introduced on the opposite side of the
b-lactone core to create affinity to the lipid-binding pocket of
the enzyme[24] (Figure 1c). Based on these criteria, we
formation); however, brief treatment with H2 over Pd/C in
MeOH reduced the N-oxides quantitatively back to the
amines. Removal of the auxiliary from the crude sulfones by
hydrogenolysis over Pearlmans catalyst (Pd(OH)2/C) at
slightly elevated temperature (408C) provided the free b-
hydroxy acids 20–27 without epimerization. A subsequent b-
lactonization with phenylsulfonyl chloride/pyridine con-
cluded the synthesis of sublibrary A (28–35). Series A (28–
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Communications
Table 1: IC50 values for the inhibition of APT1 by the focused b-lactone
libraries A–C.[a]
Compound (S,S) IC50 APT1 [nm]
Compound (R,R) IC50 APT1 [nm]
28: n=2
29: n=3
30: n=4
31: n=5
6.75Æ0.53
3.98Æ0.13
2.13Æ0.31
2.96Æ0.48
32: n=2
33: n=3
34: n=4
35: n=5
42.20Æ2.27
37.27Æ1.91
20.46Æ3.04
28.18Æ4.48
36: n=3
37: n=4
38: n=5
2.65Æ0.15
1.67Æ0.28
1.30Æ0.07
39: n=3
40: n=4
41: n=5
38.32Æ2.33
23.61Æ0.58
17.38Æ1.65
50: n=2
51: n=4
52: n=5
5.93Æ0.79
5.61Æ0.74
6.36Æ0.88
53: n=2
54: n=4
55: n=5
22.06Æ3.02
34.15Æ4.84
29.83Æ3.18
[a] All IC50 values were determined with DiFMUO as substrate, and
released DiFMU was observed by the increase in the fluorescence
intensity at 510 nm. Data were determined by at least three independent
measurements. Z’ factors were in all cases higher than 0.65. Inhibition
curves are given in the Supporting Information.
35) was quarternized at the dimethylamino functionality by
treatment with an excess of methyl iodide in acetonitrile to
yield sublibrary B (36–41). To validate the design principle, a
third sublibrary (C), not carrying the polar sulfone moiety,
was prepared. The auxiliary was removed from intermediates
12–19 under basic conditions, thereby yielding b-hydroxy
acids 42–49, which were subjected to subsequent b-lactoniza-
tion (50–55).
Scheme 1. Synthesis of a b-lactone library. Reagents and conditions:
a) (CyHex)2BOTf (2.2 equiv), Et3N (1.5 equiv), CH2Cl2, À788C (2 h);
b) aminoaldehyde (1.1 equiv), CH2Cl2, À788C (1 h) then 08C (1 h), 60–
80% over 2 steps; c) 3 equiv AcO2H, MeOH, RT (3 h); d) 20 mol%
Pd(OH)2/C, H2, MeOH, 408C, 24 h, 55–85% over 2 steps; e) PhSO2Cl
(2 equiv), pyridine, 08C, overnight, 50–80%; f) MeI (4 equiv), MeCN,
RT, 3 h, quantitative; g) LiOH (3 equiv), dioxane/MeOH/H2O (8:1:1),
RT, 48 h, 65–73%; h) PhSO2Cl (2 equiv), pyridine, 08C, overnight, 60–
82%. CyHex=cyclohexyl, OTf=trifluoromethanesulfonyl.
To identify and characterize APT1 inhibitors, focused b-
lactone libraries (A–C) were screened for inhibitory activity
of recombinant hAPT1 by employing 7-octoyl-6,8-difluor-
oumbelliferone (DiFMUO) as the substrate. The released 6,8-
difluoroumbelliferone (DiFMU) was detected by the increase
in fluorescence intensity at 510 nm. IC50 values were deter-
mined to estimate the residual enzyme activity after preincu-
bation with inhibitors. We identified the (S,S)-b-lactones in
sublibraries A (28–31, Table 1) and B (36–38, Table 1) as
highly potent inhibitors, and the postulated importance of the
spacer length between the b-lactone core and the polar head
group was verified. Investigation of the APT1 inhibitory
activity also revealed a crucial role of the sulfone moiety; thus
replacement by a sulfide (series C) led to a decrease in
activity. The S,S compounds with a spacer length of 4 or 5
carbon atoms from series A and B proved to be most potent,
with IC50 values of about 2.5 nm. In comparison, palmosta-
tin B displays an IC50 value of 5.4 nm under identical assay
conditions. Investigation of the enzyme kinetics upon treat-
ment of APT1 with the most potent compound (30) from
sublibrary A revealed an apparent competitive mode of
inhibition. This finding indicates a similar binding mechanism
as observed for palmostatin B, in which the enzyme is quickly
inactivated by covalent acylation by the inhibitor, followed by
a relatively slow reactivation by hydrolysis of the acyl–
enzyme intermediate. We verified the importance of the b-
lactone core (see the following manuscript for details).
Briefly, IC50 values were shifted by a factor of about 1000
upon hydrolysis of the b-lactone, or by converting it into the
corresponding methyl ester.[10] In addition, the initial pre-
steady-state kinetics for inactivation of the enzyme was fast
(rate constant greater than 5 ꢀ 103 molÀ1 sÀ1), and the half-life
1
(t = ) of the enzyme–inhibitor complex was in the range of 2–
2
3 min (see the Supporting Information), similar to that of
palmostatin B.
To verify the results of the biochemical assay in cells, the
three b-lactone sublibraries were subjected to a MDCK-F3
back-transformation assay,[27,28] in which HRasG12V-trans-
formed MDCK-F3 cells were subjected to chronic overnight
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incubation with a high, but noncytotoxic concentration of
each inhibitor (30 mm, see the Supporting Information).
Down-regulation of oncogenic Ras signaling in these cells
would result in a partial phenotypic reversion,[7] including a
change from a spindle-shaped to a more round-shaped cell
morphology and restoration of cell adhesion and cadherin
expression at the cell–cell contact sites. Under these con-
ditions, only compounds from sublibrary A (more potent) and
C (less potent) induced the expected reversion from the
spindlelike to the round-shaped phenotype of untransformed
MDCK cells. This behavior is comparable to treatment with
the specific MEK inhibitor U0126,[29] a known blocker of the
MAP-kinase pathway downstream of Ras. This result indi-
cates that cationic sublibrary B, which provided potent APT1
inhibitors in the biochemical assay, has no or minor activity in
cells, possibly because of poor cell permeability. Immunos-
taining for E-cadherin expression at the cell surface[27] to
quantify the degree of phenotypic reversion by the expression
level of E-cadherin revealed that 30 is distinctly more potent
than palmostatin B (Figure 2A). In light of these findings, we
chose 30 as the most potent inhibitor for further character-
ization and termed it palmostatin M.
Inhibition of APT1 leads to an interruption of dynamic
Ras trafficking between the Golgi and the plasma membrane,
since palmitoylated Ras cannot be retrapped kinetically at the
Golgi and subsequently directed to the plasma membrane
through the secretory pathway.[7] To show that palmostatin M
interrupts the dynamic Ras de/reacylation cycle, we treated
MDCK cells expressing the fluorescent protein fusion con-
structs mCitrine-N-Ras, mCitrine-H-Ras, or mCitrine-K-Ras,
Figure 3. Palmostatin M (30) interrupts the dynamic Ras cycle. A) Top
row left: Colocalization images of mCitrine-N-Ras and mCherry-H-
RasC181S,C184S, which can not be palmitoylated showing palmosta-
tin M induced mislocalization of mCitrine-N-Ras after 60 min. Similar
images of mCitrine-K-Ras (top row right) and mCherry-H-
RasC181S,C184S (see the Supporting Information) show no detectable
redistribution at 150 min. Second row left: Intensity scatter plots of
mCitrine-N-Ras and mCherry-H-RasC181S,C184S showing palmosta-
tin M induced merging of the N-Ras and H-RasC181S,C184S pixel
populations. Second row right: Intensity scatter plots show mCitrine-K-
Ras maintains its localization after treatment with palmostatin M. The
dotted diagonals represent theoretically identical populations. B) Top
row: Colocalization images of mCitrine-H-Ras and mCherry-H-
RasC181S,C184 showing minimal effect of H-Ras on the plasma
membrane after 150 min, but a complete depletion of mCitrine H-Ras
on the Golgi apparatus (arrows). Bottom Row: GalT-mCerulean is a
Golgi marker. Scale bars represent 10 mm in all cases. C) Graph
showing a comparison of the rate at which N-Ras redistributes to the
unspecific localization of mCherry-HRasC181S,C184S, as quantified by
Manders’ correlation coefficients (R), upon treatment with palmosta-
tin M (30; black) versus treatment with 38 (red). Trend lines are
indicated.
as well as the mutant mCherry-H-RasC181S,C184S, which can
not be palmitoylated, and the Golgi marker GalT mCerulean
with inhibitor (1 mm) for up to 3 h. N-Ras and H-Ras are
typically enriched on the Golgi and at the plasma membrane,
albeit to different extents on each organelle. K-Ras is
enriched on the plasma membrane through a palmitoyla-
tion-independent mechanism, while the solely prenylated H-
RasC181S,C184S, which can not be palmitoylated is distrib-
uted nonspecifically in all cellular membranes.[8] We moni-
tored the influence of the inhibitor on the redistribution of N-,
H-, and K-Ras through their typical localization patterns
relative to nonspecific distribution, as indicated by H-
RasC181S,C184S, through confocal imaging of live cells, and
quantified mislocalization through intensity correlation anal-
ysis (for details see the Supporting Information).[30] Treatment
Figure 2. Characterization of palmostatin M (30) in cells. A) E-cadherin
immunostaining of HRasG12V-transformed MDCK-F3 cells showing
restoration of E-cadherin expression at the cell–cell interfaces (arrow-
heads) after treatment with 10 mm palmostatin M. B) DMSO control.
C) HRasG12V-transformed MDCK-F3 cells treated with palmostatin B
at 50 mm show a similar, but weaker effect on the phenotype. D) Treat-
ment of HRasG12V-transformed MDCK-F3 cells with the known MEK
inhibitor U0126 as a positive control. MDCK cells (non-HRasG12V
transformed) treated with DMSO and palmostatin B (50 mm) are
controls for the reversed phenotype (see the Supporting Information).
Scale bars represent 20 mm.
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with palmostatin M caused efficient mislocalization of N-Ras,
with complete unspecific distribution reached one hour after
treatment with the inhibitor. H-Ras enrichment, on the other
hand, was not clearly affected at the plasma membrane on this
timescale, but H-Ras was depleted from the Golgi apparatus
because of the directional influence of the secretory path-
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localization, which does not require the de/reacylation
cycle[32] was completely unaffected by palmostatin M, thus
indicating that palmostatin M exerts a specific effect on the
acylation cycle. The results of the redistribution assays are in
accordance with the phenotypic assay. Compound 38, which
showed poor phenotypic effects on transformed cells,
although highly potent in the biochemical assay, was also
comparably inefficient in the redistribution assay relative to
palmostatin M. Complete unspecific distribution of N-Ras
upon treatment with 38 at 1 mm required more than three
hours, compared to less than one hour for palmostatin M
(Figure 3C).
We have described the development of the APT1
inhibitor palmostatin M based on the principle of substrate
similarity and its characterization in vitro and in cells.
Palmostatin M perturbs the acylation cycle as well as the H-
and N-Ras signaling activity at the level of depalmitoylation,
thereby leading to decreased MAP-kinase signaling and
partial endothelial to mesenchymal phenotypic reversion of
H-Ras-transformed MDCK-F3 cells.
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Received: April 29, 2011
Revised: August 8, 2011
Published online: September 9, 2011
Keywords: acyl protein thioesterase · inhibitors · ras protein ·
.
signal transduction
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