The ras pathway modulator melophlin a targets dynamins

Full text of DOI:10.1002/anie.200902023

Communications
DOI: 10.1002/anie.200902023
Biological Signaling
The Ras Pathway Modulator Melophlin A Targets Dynamins**
Tanja Knoth, Karin Warburg, Catherine Katzka, Amrita Rai, Alexander Wolf,
Andreas Brockmeyer, Petra Janning, Thomas F. Reubold, Susanne Eschenburg,
Dietmar J. Manstein, Katja Hꢀbel, Markus Kaiser, and Herbert Waldmann*
The Ras/mitogen-activated protein (MAP) kinase signal
transduction pathway regulates numerous biological pro-
grams including cell growth and differentiation,[1,2] and
harbors several important anticancer-drug targets.[3] Recent
research, in particular inspired by systems biology
approaches, revealed the importance of dynamic spatiotem-
poral regulation of and interplay between the Ras network
members and their interaction with other signaling modules
for fully functional Ras signaling.[4] Because of their rapid,
conditional, and reversible mode of action, small-molecule
modulators of protein function are particularly suitable tools
for the conditional analysis of such dynamic biological
processes, and hold great promise for the study of biological
systems.[5] Therefore, the identification of novel small-mole-
cule modulators of signaling through the Ras network and the
identification of their molecular targets are of major inter-
est.[1,3,6] The naturally occurring tetramic acids melophlin A
and B (1 and 2, Scheme 1A) reverse the morphology of H-
Ras-transformed NIH3T3 fibroblasts at a concentration of
5 mgmLÀ1 (that is, IC50 = 14 mm).[7] However, the biological
targets of the melophlins and their link to the Ras network
have not been identified. Herein, we report the synthesis of a
melophlin-inspired compound collection[8] and a subsequent
chemical proteomics investigation, which revealed that melo-
[*] Dr. T. Knoth, Dipl.-Biol. K. Warburg, Dr. C. Katzka, Dr. A. Wolf,
Dipl.-Ing. (FH) A. Brockmeyer, Dr. P. Janning, Dr. K. Hꢀbel,
Prof. Dr. H. Waldmann
Figure 1. Results of the phenotypic MDCK/MDCK-F3 cell assay. MDCK
cells show a large and round phenotype and grow in a monolayer with
cell-to-cell contacts. MDCK-F3 cells are long and spindle-like without
regular cell-to-cell contacts, grow in multiple layers, and display
impaired contact inhibition as demonstrated by the loss of E-cadherin
expression on the cell surface. Cellular morphology was visualized
microscopically by staining with Celestine blue (scale bars: 19 mm).
a) MDCK-F3 cells (treated with dimethyl sulfoxide (DMSO) as negative
control) show spindle-like morphology and do not form cell-to-cell
contacts. b) MDCK-F3 cells show reversion of the phenotype after
treatment with the MEK inhibitor U0126 at 20 mm concentration.
c) MDCK-F3 cells after treatment with melophlin A (1) at 30 mm
concentration. The cells display a round morphology comparable to
the phenotype induced by treatment with U0126 and form cell-to-cell
contacts. d) Wild-type MDCK cells show a round morphology and
good cell-to-cell contacts. e) Fluorescein isothiocyanate (FITC) anti-E-
cadherin staining of MDCK-F3 cells (treated with DMSO as negative
control). Cells do not show cell-to-cell contacts, therefore E-cadherin is
not enriched at the cell interfaces. f) E-cadherin immunostaining of
MDCK-F3 cells. The arrow points to restored E-cadherin expression at
the cell/cell interfaces after treatment with 20 mm U0126. g) E-cadherin
staining of MDCK-F3 cells treated with 50 mm melophlin A (1). The
arrow points to an example for restoration of E-cadherin expression at
cell/cell interfaces. h) E-cadherin immunostaining of wild-type MDCK
cells. E-cadherin immunostaining at the interfaces shows strong cell-
to-cell contact formation.
Max-Planck-Institut fꢀr molekulare Physiologie
Abt. Chemische Biologie
Otto-Hahn-Straße 11, 44227 Dortmund (Germany)
and
Technische Universitꢁt Dortmund
Fakultꢁt Chemie, Chemische Biologie
Fax: (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Dr. T. F. Reubold, Dr. S. Eschenburg
Max-Planck-Institut fꢀr molekulare Physiologie
Abt. Strukturbiologie, Dortmund (Germany)
M.Sc. A. Rai, Prof. Dr. D. J. Manstein
Medizinische Hochschule Hannover, Zentrum Biochemie
Institut fꢀr Biophysikalische Chemie, Hannover (Germany)
Dr. M. Kaiser
Chemical Genomics Centre der Max-Planck-Gesellschaft, Dort-
mund (Germany)
[**] This research was supported by the Max-Planck-Gesellschaft,
Deutsche Forschungsgemeinschaft (MA 1081/7-2) (D.J.M.), and
the Fonds der Chemischen Industrie (H.W., D.J.M.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200902023.
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Scheme 1. A) Structures of melophlin A (1) and B (2). B) Synthesis of the tetramic acid collection and design of an affinity pull-down reagent
based on structure–activity correlation. Conditions: a) N,N’-diisopropylcarbodiimide, CH2Cl2, DMF, 08C, 0.5 h; then Wang resin, DMAP, DMF, RT,
8 h; Fmoc=9-fluorenylmethoxycarbonyl, DMF=dimethylformamide, DMAP=4-dimethylaminopyridine; b) two times DMF/piperidine 4:1, RT,
15 min; c) R2CHO, THF, NaB(OAc)3H, NaOAc, RT, 12 h; d) acyl Meldrum’s acid 6, toluene, 808C, 3 h; e) KOH (0.1m in methanol), dioxane/
CH2Cl2, RT, 3 h. Overall yield 5–83%. C) Structure–activity relationship deduced from the Ras reporter gene assay for compounds active up to a
concentration of 30 mm, which are not toxic in this concentration range. D) Chemical structures of the biotinylated affinity probes 9 (active and
nontoxic analogue of melophlin A) and 10 (inactive and nontoxic negative control probe).
phlin A unexpectedly targets dynamins in cells and thereby
modulates signal transduction through the Ras network.
To strengthen the link between melophlin A and Ras
signaling, we synthesized the natural product (see below) and
subjected it to a PathDetect reporter gene assay (Stratagene),
which quantifies Ras/MAP kinase signaling, and a WST-1
proliferation assay (see the Supporting Information).[9] Melo-
phlin A inhibited the reporter signal with an IC50 value of
(18.1 Æ 1.2) mm and was not cytotoxic to the employed cell line
up to a concentration of 30 mm. In addition, melophlin A was
investigated in a phenotype-based assay using epithelial
Madine–Darby canine kidney (MDCK) cells and MDCK-F3
cells, which are MDCK cells transfected with the constitu-
tively active H-RasG12Voncogene (Figure 1).[10] Since the H-
Ras oncogene is the effector of the typical visible morpho-
logical differences between these cell lines, quantification of
the phenotypic reversion of H-Ras-transformed cells can be
used to measure the potency of small-molecule inhibitors of
the activated H-Ras pathway.[6,11]
Treatment of MDCK-F3 cells with melophlin A at the
noncytotoxic concentration of 30 mm (see the Supporting
Information and Table 1) caused reversal to the phenotype
that is characteristic for untransformed MDCK cells
(Figure 1). A comparable phenotypic reversal was induced
by the Ras pathway (MEK) inhibitor U0126[12] (Figure 1). To
correlate the activity of melophlin A with its chemical
structure, we synthesized a focused collection of 60 melophlin
analogues on the solid phase by following established
methods[13] (Scheme 1B; for a list of all synthesized ana-
logues, see the Supporting Information). To this end, Wang
resin was loaded by means of Steglich esterification with
various Fmoc-protected amino acids containing aliphatic,
benzylic, polar, acidic, or basic side-chain residues. After
removal of the Fmoc protecting group, the liberated amino
group was subjected to reductive amination with different
aliphatic, benzylic, or alkenyl aldehydes. Treatment of the
resulting secondary amines 5 with acyl Meldrumꢀs acid 6
embodying alkyl, cycloalkyl, alkenyl, and aromatic residues
gave the intermediate b-ketoamides 7 which, upon treatment
with 0.1m KOH, were cyclized with simultaneous release of
the desired tetramic acid potassium salts from the polymeric
carrier. If required, additional amino acid side-chain protect-
ing groups were removed before melophlin A-derived tetra-
mic acids were purified to homogeneity by reversed-phase
HPLC (RP-HPLC) and finally obtained in 5–83% overall
yield. These compounds were then subjected to the assays
described above. Noncytotoxic compounds that induced both
phenotype reversal in MDCK-F3 cells and more than 30%
inhibition of the signal in the reporter gene assay at 30 mm
concentration were considered as hits. These criteria were
met by only three compounds including melophlin A (Table 1
and Supporting Information).
For activity in the phenotypic screen, variation of the
substituent at C5 is tolerated. Increasing the size of the
nitrogen substituent beyond an ethyl group and even mod-
erate shortening of the tetramic acid side chain at C3
drastically reduce the phenotype reversion (Scheme 1C; see
also the Supporting Information). Based on these results,
compound 9 (Scheme 1D), which incorporates a glutamic
acid side chain and an alkyl chain of the same length as in the
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Table 1: Results of the phenotype and reporter gene assay and determination of cytotoxicity. Data for
melophlin A (1) are shown for comparison.
Both biotinylated probes were
immobilized on magnetic strepta-
vidin-coated beads and incubated
with HeLa cell lysates. Bound
proteins were released by heating,
separated by SDS-PAGE, and
visualized by Coomassie staining.
The proteins were identified by in-
gel tryptic digest, separation of the
resulting peptides by nano-RP-
HPLC, and online mass spectrom-
etry (MS) followed by MS/MS
analysis (see the Supporting Infor-
mation). Only proteins that were
identified in the affinity chroma-
tography experiment with active
probe 9 but not with the inactive
compound 10, and that were iden-
tified in at least three out of four
pull-down experiments, were con-
sidered potential targets.
Compound
Phenotype
ED50MDCK-
F3 [mm][a]
Cytotoxicity
LD25MDCK-
F3 [mm][b]
Reporter gene
assay
Cytotoxicity
LD25HLR [mm][d]
IC50HLR [mm][c]
30
20
50
30
18.1Æ1.2
16.8Æ3.4
30
30
20
30
50
30Æ2.5
30
30
no effect
no effect
By means of this method,
dynamin II, dynamin I-like pro-
tein, and p120 catenin were iden-
tified as potential targets with
relevance to Ras signaling. The
sequence coverages for the pro-
teins were approximately 6% for
p120 catenin, 13% for dynamin II,
and 8% for dynamin I-like pro-
tein. The identities of dynamin II
and p120 catenin were confirmed
by Western blotting with specific
antibodies, which also revealed
that the proteins were not bound
by the control probe 10 (Figure 2).
p120 Catenin has been linked
no effect
50
no effect
30
no effect
no effect
50
30
36.5Æ5
30
30
23.8Æ0.8
to the Ras pathway only indirectly
through its putative role as binding
partner of E-cadherins, regulating
their expression level, and an asso-
ciated change of the endothelial to
the mesenchymal cellular pheno-
type, but little is known about the
relevance of this finding.[14] How-
ever, the observed binding of
melophlin A to dynamins is partic-
ularly interesting. Dynamins are
GTPases (GTP = guanosine-5’-tri-
9
10
no effect
no effect
30
50
25.7Æ2.9
30
30
no effect
[a] Concentration at which 50% of the MDCK-F3 cells show the reversed phenotype. [b] Concentration at
which the number of living MDCK-F3 cells is reduced by 25%. All compounds were initially tested at
50 mm concentration. If no toxicity was observed at the highest tested concentration, this concentration
is given as LD25. [c] IC50 value for inhibition of luciferase activity in a reporter gene assay (PathDetect
from Stratagene) in HLR cells. All compounds were initially tested at 30 mm concentration.
[d] Concentration at which the number of living HLR cells was reduced by 25%. All compounds were
initially tested at 30 mm concentration. If no cytotoxicity was observed at the highest concentration
tested, this is given as LD25.
guiding natural product, was synthesized and employed in
affinity pull-down experiments from cell lysate to identify
potential target protein(s) of melophlin A. Compound 10
(Scheme 1D), with a much shorter alkyl chain, was designed
as inactive control molecule (for the synthesis of 9 and 10, see
the Supporting Information). Affinity pull-down reagent 9
was active in the reporter gene assay [IC50 = (25.7 Æ 2.9) mm]
and was noncytotoxic up to 30 mm concentration, whereas
control compound 10 did not display any activity in the
reporter gene assay.
phosphate) involved in many processes including budding of
transport vesicles, division of organelles, and cytokinesis,[15–19]
and they hold a prominent role in endocytosis.[15,16] Endocy-
tosis is required for full activation of MAP kinase (ERK) by
growth factor receptors.[17] Dominant negative interfering
dynamin mutants inhibit endocytosis and EGF- and insulin-
stimulated ERK activation without interfering with Ras, Raf-
1, and MAP kinase kinase (MEK) activity.[16,18] This finding
led to the suggestion that after activation at the plasma
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Figure 2. Identification of proteins isolated in the affinity pull-down
experiment with the melophlin A-derived probe 9 and Western blot of
resin-bound proteins. Proteins that were resolved by SDS-PAGE after
affinity pull down were transferred to a nitrocellulose membrane and
analyzed with specific antibodies. Lane 1 shows the control pull down
with 10, and lane 2 shows the pull down with active probe 9. The cell
lysates were analyzed with antibodies against the proteins p120
catenin and dynamin II. The results demonstrate that dynamin II and
p120 catenin do not bind to negative control probe 10 but to active
probe 9.
membrane, MEK needs to traffic through the cytosol by
means of the endocytotic pathway to fully activate ERK1/2.[19]
Decreased MAP kinase phosphorylation and activity
upon inhibition of endocytosis by means of a dominant
negative dynamin mutant has been shown in HeLa cells, that
is, the model cell line investigated here.[18] To determine
whether by analogy treatment of HeLa cells with melo-
phlin A also leads to reduced MAP kinase phosphorylation,
activation of MAP kinase (ERK1/ERK2) was investigated.
As shown in Figure 3a, treatment of HeLa cells with 10 mm
melophlin A led to a decrease of ERK1/2 phosphorylation by
70% (for quantification, see Figure 6 in the Supporting
Information).
Figure 3. a) Representative Western blot showing reduced ERK phosphor-
ylation in HeLa cells upon treatment with melophlin A. HeLa cells were
treated with DMSO, melophlin A (10 mm), and the known MEK inhibitor
U0126 (30 mm) overnight. After stimulation with EGF for 5 min the cells
were lysed, and the lysate was separated by SDS-PAGE and subjected to
immunoblotting for ERK. pERK1/2 as well as tubulin (as loading control)
Direct interaction of melophlin A with recombinantly
produced human dynamin I-like protein, dynamin I, and
dynamin II was monitored by surface plasmon resonance
experiments with the biotinylated probe 9 immobilized on a
Series S sensor chip SA. For full-length dynamin II, a
dissociation constant Kd = (13.8 Æ 0.12) mm [kon = (1.42 ꢁ
were detected with specific antibodies. Lane 1 shows the pERK signal after
treatment of cells with DMSO. The signal is strongly reduced in lane 3,
which shows reduction of ERK phosphorylation after treatment with
U0126 (30 mm). In lane 4 only a weak pERK signal is visible, and shows
reduction of ERK phosphorylation after incubation with melophlin A
(10 mm). b) Binding of dynamin II to melophlin A determined by means of
surface plasmon resonance. Melophlin A probe 9 was immobilized on the
surface of a Series S sensor chip SA and binding to dynamin II was
determined at a concentration of 3 mm. The net binding curve is shown.
Melophlin A analogue 9 binds dynamin II with Kd =(13.8Æ0.12) mm.
c) Domain structure of dynamins. Domain boundaries are denoted by
residue numbers. DNM1L (dynamin I-like protein) contains an extra
domain of unknown function between the middle domain and GED.
d) Effect of melophlin A on the GTPase activity of full-length dynamin II
(residues 1–866) at a concentration of 0.5 mm (left panel) and effect of
melophlin A on the GTPase activity of full-length dynamin II (residues 1–
866) stimulated with GST-Grb2 at a concentration of 0.5 mm (right panel).
Measurements were done in triplicate; error bars are standard deviations.
103 Æ 1.54 ꢁ 102) mÀ1 sÀ1,
k
off = (1.95 ꢁ 10À2 Æ 9.2 ꢁ 10À3) sÀ1]
was determined (Figure 3b). Because of complex binding
kinetics, the Kd values for dynamin I and dynamin I-like
protein were calculated by direct measurement of the
response at equilibrium for several concentrations.[20] The
obtained data were analyzed by a sigmoidal equilibrium
model and the Kd values were calculated to be Kd = (0.4 Æ
0.099) mm for dynamin I (1–760) and Kd = (0.6 Æ 0.042) mm for
dynamin I-like protein (see also Figure 7 in the Supporting
Information).
The dynamins embody a GTPase domain,[21] a middle
domain, and a GTPase effector (GE) domain (Figure 3c). For
dynamins I and II this basic set of domains is supplemented by
two targeting domains, the pleckstrin homology (PH) domain
and the proline-rich (PR) domain.[15] The truncated dynamin I
(1–760) form employed here lacks the PR and the dynamin I-
like protein used in our experiments does not possess the two
targeting domains. Still, both proteins interact with melo-
phlin A, which suggests that PH and PR are not crucial for the
interaction.
assay, which revealed that melophlin A does not inhibit the
GTPase activity of dynamin II in the presence or absence of
Grb2. Rather, melophlin A slightly stimulates GTPase activ-
ity (Figure 3d).
To determine whether melophlin A acts as a GTPase
inhibitor, the GTPase activity of dynamin II was monitored
by using an enzyme-coupled continuous regenerative GTPase
To determine whether melophlin A interferes with endo-
cytosis, internalization of the transferrin/transferrin receptor
complex by means of receptor-mediated endocytosis was
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Figure 4. Inhibition of dynamin-dependent internalization of transferrin
by melophlin. a) Alexa 546–transferrin was allowed to bind to the
surface receptor of HeLa cells by incubation at 258C (time point 0 min
(1)). Upon gentle heating to 378C, the bound transferrin was internal-
ized. HeLa cells were incubated overnight at 378C in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum (FCS) to confluency of about 50%. Medium was removed and
cells were incubated for 3 h at 378C in DMEM without serum
containing melophlin A (3; uptake of transferrin–Alexa is reduced in
these cells; most of the transferrin is still localized at the cell surface
(left arrow); only a little transferrin is internalized (right arrow)),
dynasore (4, reduced transferrin uptake; transferrin is not internalized
but still binds to the cell-surface receptors (arrow)), or 0.8% DMSO
(2, the arrows point to transferrin taken up into the cells) as solvent
control, followed by incubation for 2 min at room temperature in the
same medium containing 25 mgmLÀ1 transferrin–Alexa 546 (Invitro-
gen). Cells were washed three times with the same medium and
incubated at 378C for 0 and 5 min, respectively, in the presence of
inhibitors. Transferrin attached to the cell surface was removed by
washing. Cells were fixed, mounted on cover slips, and imaged.
b) Reversibility of the inhibition of transferrin internalization by melo-
phlin A. The cells were incubated for 3 h at 378C with 25 mm
melophlin A (image 7) or 0.8% DMSO (image 5) in DMEM without
serum. The cells were washed three times with the medium without
inhibitor and incubated for 12 h at 378C with DMEM with 10% FCS.
The positive control was incubated for the last 3 h in DMEM without
serum in the presence of 25 mm melophlin A (image 6). Transferrin
internalization was visualized as described above. Image 5 shows
internalization of transferrin in cells treated with DMSO. In cells
pretreated with melophlin A (image 6) internalization is inhibited. After
wash-out of the compound, the cells were allowed to recover.
Recovered cells (image 7) show full restoration of transferrin–Alexa
internalization, thus revealing that the effect is reversible.
because of inhibition of receptor-mediated endocytosis.
Melophlin A binds to dynamin II and dynamin I-like protein,
that is, the mitochondrial homologue of dynamin in HeLa
cells. Dynamin I is not expressed in these cells and conse-
quently could not be identified in the affinity pull-down
experiments. However, as shown in the surface plasmon
resonance experiment, melophlin A binds dynamin I as well.
Our results identify melophlin A as a new modulator of
dynamin activity that inhibits dynaminꢀs functions by means
of a yet unidentified mechanism, which differs from the mode
of action of dynasore, the only other known dynamin inhibitor
so far. We provide experimental proof that the previously
described connection between dynamin-mediated endocyto-
sis and Ras/MAP kinase signaling can be antagonized by
small molecules. These insights will open up new opportu-
nities for the study of the Ras/MAP kinase network and may
provide new avenues for medicinal chemistry research.
monitored, which requires normal dynamin function.[22] As a
positive control we used dynasore, the only known inhibitor,
which interferes with endocytosis by noncompetitive inhib-
ition of the GTPase activity of dynamin.[23] As shown in
Figure 4, the uptake of transferrin was reduced to 30% after
incubation with 20 mm melophlin A for three hours. Uptake
inhibition is dose-dependent (see Figure 8 in the Supporting
Information) and can be reversed by wash-out of melophlin A
and 12 hours of recovery without the compound. Note that at
concentrations greater than 20 mm the reduction of ERK
phosphorylation and the inhibition of transferrin endocytosis
decrease. This may be a result of aggregation of melophlin A
in an aqueous environment at greater than or equal to 20 mm,
as indicated by dynamic light scattering experiments (see the
Supporting Information) and the observed increase in
GTPase stimulation by melophlin A at such concentrations
(see above). These findings suggest that melophlin A should
preferably be employed at concentrations up to about 20 mm
in biological experiments.
Received: April 15, 2009
Revised: July 21, 2009
Published online: August 31, 2009
Keywords: biological activity · natural products · proteomics ·
.
solid-phase synthesis · structure–activity relationships
In the light of these results, the observed phenotypic
effects induced by treatment with melophlin A are most likely
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