Cyplecksins are covalent inhibitors of the pleckstrin homology domain of cytohesin

Article abstract of DOI:10.1002/anie.201302207

The covalent grip: A new class of 5-bromobarbiturates (Cyplecksins; see structure) act by a covalent mechanism to inhibit the biological function of the pleckstrin homology domain of cytohesins, small guanine nucleotide exchange factors for the Ras-like ARF-GTPases. In cells, Cyplecksins interfere with the phosphoinositol-dependent membrane recruitment of cytohesins. Cyplecksins may be useful in validating cytohesins as potential drug targets. Copyright

Full text of DOI:10.1002/anie.201302207

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Communications
Inhibitors
The covalent grip: A new class of 5-
bromobarbiturates (Cyplecksins; see
structure) act by a covalent mechanism
to inhibit the biological function of the
pleckstrin homology domain of cytohe-
sins, small guanine nucleotide exchange
factors for the Ras-like ARF-GTPases. In
cells, Cyplecksins interfere with the
phosphoinositol-dependent membrane
recruitment of cytohesins. Cyplecksins
may be useful in validating cytohesins as
potential drug targets.
M. Hussein, M. Bettio, A. Schmitz,
J. S. Hannam, J. Theis, G. Mayer, S. Dosa,
M. Gꢀtschow,
M. Famulok*
&&&& — &&&&
Cyplecksins Are Covalent Inhibitors of the
Pleckstrin Homology Domain of
Cytohesin
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DOI: 10.1002/anie.201302207
Inhibitors
Cyplecksins Are Covalent Inhibitors of the Pleckstrin Homology
Domain of Cytohesin**
Mohamed Hussein, Martina Bettio, Anton Schmitz, Jeffrey S. Hannam, Julian Theis,
Gꢀnter Mayer, Stefan Dosa, Michael Gꢀtschow, and Michael Famulok*
Dedicated to the Bayer company on the occasion of its 150th anniversary
Cytohesins are cytoplasmic multidomain proteins that act as
guanine nucleotide exchange factors (GEFs) for small Ras-
like GTPases called ADP-ribosylation factors (Arfs).[1] Their
Sec7 domain catalyzes the exchange of guanosine-5’-diphos-
phate (GDP) for guanosine-5’-triphosphate (GTP), which
activates Arf proteins like Arf1 and Arf6. Mammalian cells
contain four highly homologous cytohesins (cytohesins 1–4)
that are implicated in cellular processes such as b2-integrin-
mediated cell adhesion and actin dynamics, including Arf-
mediated functions, namely membrane trafficking, vesicle
transport, endocytosis, and more.[2] Moreover, cytohesin-2
(ARNO) is a cytoplasmic activator of receptor tyrosine
kinase signaling by the insulin receptor (IR)[3] and epidermal
growth factor receptor (EGFR),[4] respectively. ARNO and
Arf6 also contribute to the disruptive effects of interleukin-1b
(IL-1b) on endothelial stability by binding to the adaptor
protein MYD88.[5] These discoveries were greatly aided by
the availability of SecinH3, an inhibitor of the cytohesin Sec7
domain.[3b,6]
C-terminal to their Sec7 domain, cytohesins contain
another functional domain called the pleckstrin homology
(PH) domain (Figure 1a). Through their PH domain cytohe-
sins are recruited to the inner leaflet of the plasma membrane
and intracellular membranes by binding either to the
phosphatidylinositol phosphates PIP2 or PIP3, or to activated
Arf6-GTP.[2] Cytohesin Sec7-dependent integrin activation in
cell adhesion requires the presence of the PH domain, and the
PH domain exerts a certain level of inhibition on the Sec7
domain that is relieved upon membrane recruitment and
interaction with PIPs.[7] These findings indicate that the
functions of the cytohesin Sec7 and PH domains may be
tightly interconnected. Inspired by the usefulness of SecinH3
Figure 1. Aptamer displacement assay based on RiboGreen fluores-
cence capture. a) Domain structure of cytohesin-2. CC: Coiled coil;
PBR: polybasic region. b) The immobilized cytohesin is incubated with
the aptamer and small molecules. Nonbinding molecules are removed
by a buffer wash. Remaining bound aptamer is detected by RiboGreen
fluorescence (bottom right); reduced fluorescence is obtained upon
aptamer displacement by the small molecule (top right). c) Represen-
tative primary screening plate with a hit compound (black dot; no. 11).
Positive controls (dark gray dots; nos. 87–90) lacked cytohesin-1 coat-
ing; negative controls (light gray dots; nos. 84–86) did not contain any
compound. d) Chemical structures of the most active hit compounds,
derivatives of 5-bromopyrimidine-2,4,6-trione.
as a chemical biology tool for elucidating previously unknown
functions of cytohesin Sec7 domains, we sought to identify
a small-molecule inhibitor for cytohesin PH domains. Here
we report the discovery of a class of cytohesin PH domain
inhibitors called Cyplecksins (Cytohesin pleckstrin homology
domain inhibitors) that act by a covalent mechanism.
[*] Dipl.-Chem. M. Hussein, M. Sc. M. Bettio, Dr. A. Schmitz,
Dr. J. S. Hannam, Dr. J. Theis, Prof. G. Mayer, Prof. M. Famulok
Life and Medical Sciences (LIMES) Institute, Universitꢀt Bonn
Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany)
E-mail: m.famulok@uni-bonn.de
Homepage: http://www.famuloklab.de
To develop an aptamer displacement assay for HTS, we
first selected an RNA aptamer that bound the PH domain of
cytohesin-1. After seven selection rounds, the enriched RNA
library was cloned and sequenced, and clone 6.10 was
identified as a cytohesin PH domain binder (Figure S1a in
the Supporting Information (SI)). Clone 6.10 bound PH
domains of cytohesins 1, 2, and 3 with Kd values between 0.3
and 0.7 mm, whereas no binding to the Sec7 domain and to
related PH domains could be detected (Figure S1b,c (SI)).
Dr. S. Dosa, Prof. M. Gꢁtschow
Pharmazeutisches Institut-Pharmazeutische Chemie I
University of Bonn (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 645 and SFB 704). We thank M. Lemmon for plasmids, V.
Fieberg and P. A. Ottersbach for technical assistance, and T. Quast
for help with microscopy.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302207.
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The aptamer also inhibited the binding of cytohesins to PIP3-
doped liposomes in a concentration-dependent manner (Fig-
ure S1d (SI)). Binding and inhibition required the presence of
the triphosphate group at the 5’-end of the aptamer.
The aptamer displacement screen was established as
illustrated in Figure 1b. 96-well screening plates were
coated with cytohesin-1 PH, and incubated with aptamer
6.10 in the presence of compound. Small molecules that
compete with 6.10 for binding lead to a reduced level of
aptamer complexation to the immobilized protein, whereas
noncompeting compounds do not (Figure 1b; middle). The
plates are washed with buffer to remove nonbound aptamer,
and then incubated with RiboGreen, a dye that exhibits an
increase in fluorescence that is directly proportional to the
amount of nucleic acid. Low fluorescence indicates a com-
pound that has displaced the bound aptamer from its target
protein (for possible false-positive scenarios see Figure S2
(SI)). A chemical library of roughly 12000 diversity-based
druglike small molecules was screened to identify potential
cytohesin PH domain inhibitors. The assay quality parameter
Z’ was 0.69, which is compatible with HTS conditions
(Figure 1c and Figure S3 (SI)). The screening revealed the
series of substituted 5-bromopyrimidine-2,4,6-triones 1–3 that
competed with 6.10 for cytohesin PH domain binding (Fig-
ure 1d).
Figure 2. Binding behavior of Cyplecksins 1–3, and their analogues 4
We next performed microscale thermophoresis measure-
ments to quantify the binding of Cyplecksins 1–3 and the
related derivatives 4 and 5 (Figure 2a), which lack the 5-
bromo substituent, to the Alexa647-labeled cytohesin-1 PH
domain (Figure 2b). Increasing concentrations of 1–3 led to
sigmoidal binding curves corresponding to Kd values around
2 mm. In contrast, neither 4 nor 5 showed any binding to the
cytohesin-1 PH domain. To test the specificity of 1–3 for other
PH domains we carried out the same binding test using the
Alexa647-labeled DH-PH domain of the “T-lymphoma
invasive and metastasis inducing protein 1” (Tiam1). No
concentration-dependent change in thermophoresis was
detected, indicating that the PH domain of the Tiam1
fragment is not recognized by 1–3 (Figure 2c). We then
investigated whether Cyplecksins 1–3 compete with PIP2
binding to the cytohesin-1 PH domain in vitro (Figure 2d).
We used PIP2 labeled with tetramethylrhodamine (TMR) and
measured fluorescence polarization (FP) in the presence of
increasing concentrations of 1–5. The complex between
TMR-PIP2 and the cytohesin-1 PH domain leads to higher
levels of FP than the free TMR-PIP2. Indeed, increasing
Cyplecksin concentrations led to inhibition of TMR-PIP2
binding to the cytohesin-1 PH domain with IC50 values
below 10 mm. Derivatives 4 and 5 again were inactive in this
assay. The same experiment was done using the PH domains
of GEP100, another Arf6-GEF (Figure 2e), and of DAGK,
ARHGAP25, IRS1, Pleckstrin, and full-length Akt2 (Fig-
ure S4 (SI)). Cyplecksins 1 and 2 either did not interfere at all
with the binding of TMR-PIP2 or TMR-PIP3 to these proteins,
or exhibited only weak inhibition with IC50 values at least
above 100 mm. In the latter cases the precise IC50 values could
not be determined because compound concentrations beyond
the solubility limit would have been required. Only Cypleck-
sin 3 showed a somewhat less specific inhibitory profile.
and 5. a) Chemical structures of nonbinding Cyplecksin analogues 4
and 5. b),c) Cyplecksins 1–3 bind specifically to cytohesin-1 PH but
not to Tiam1-DH-PH. 100 nm Alexa647-labeled Cyth1-PH (b) and
Tiam1-DH-PH domain (c) were incubated with compounds (0.1–
20 mm), followed by microscale thermophoresis (MST) measurements.
For color and symbols legend see box insert in (c). d),e) Cyplecksins
specifically inhibit the binding of cytohesin to PIP2. 250 nm Cyth1-PH
(d) and 500 nm GEP100-PH (e) were incubated with compounds (1–
100 mm) and 30 nm tetramethylrhodamine(TMR)-conjugated PIP2. PIP2
binding was quantified by fluorescence polarization. mP=millipolari-
zation.
Cyplecksins 1–3, but not 4 and 5, also inhibited the binding of
PIP3 to cytohesin-1 PH, and to nearly full-length cytohesins-
1 and -2 that lacked only the short polybasic region (Figure S5
(SI)). Taken together, these data indicate that Cyplecksins
1 and 2 exhibit a high degree of specificity for the PH domain
of cytohesins.
The lack of inhibitory activity of the Cyplecksin analogues
4 and 5 suggests that the bromine substituent in 1–3 is crucial
for binding and inhibition. In aqueous solution, Cyplecksins
slowly hydrolyze within several hours into mixtures of
derivatives including those that result from ring opening
(data not shown). In the presence of amines, however,
a substitution of the bromine for the amine occurs in related
5-methyl- or 5-phenyl-substituted pyrimidine-2,4,6-triones.[8]
This suggests that upon binding of Cyplecksins to their
binding site in the cytohesin PH domain, a substitution
reaction may occur, in which either the C5 in 1–3 is attacked
by a nucleophile (lysine or cysteine) to displace the Br atom,
or one of the carbonyl C atoms in the heterocycle is attacked
in a ring-opening reaction.[9] In any case, both mechanisms
should lead to a covalent attachment of Cyplecksins to the
cytohesin PH domains.
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To test this hypothesis, we synthesized the probe 6,
a biotinylated analogue of Cyplecksin-1 (Figure 3a). The non-
brominated variant 7 was used as a negative control. We then
incubated the cytohesin-1 PH domain, full-length cytohesin-1,
and the Sec7 domain of cytohesin-2, to which no binding of 6
should occur, with increasing concentrations of 6 and 7. After
denaturing polyacrylamide gel electrophoresis (PAGE) and
western blotting, covalently attached biotin groups in the
proteins were detected with neutravidin. Indicative of the
modification of the cytohesin-1 PH domain with the biotin-
ylated Cyplecksin-1 variant 6.
As a first step to identify the Cyplecksin-reactive nucle-
ophile in the protein, the cysteine residues of cytohesin-1 PH
were alkylated by iodoacetamide. Neither the binding of
TMR-PIP2 nor its inhibition by Cyplecksins 1–3 were affected
by the treatment, arguing against cysteine residues being the
sites of the covalent attachment of Cyplecksins (Figure S6
(SI)). To unambigously identify the binding mode and binding
site of Cyplecksins a combined mass spectroscopic/crystallo-
graphic approach would be required which, however, is
beyond the scope of this work.
To analyze whether Cyplecksins inhibit membrane target-
ing of cytohesin-2 PH, nanodisks[10] were used as a membrane
surrogate. Cytohesin-2 PH bound only to PIP2-doped nano-
disks, indicating that nanodisks are suitable to reliably detect
the PIP2-dependent membrane recruitment of cytohesins
(Figure 4a). Cyplecksins 1–3 but not the control compounds 4
and 5 inhibited the binding.
Having shown that Cyplecksins inhibit the binding of
cytohesin PH domains to PIP2/PIP3 phospholipids by a cova-
lent mechanism in vitro we next sought to test the activity of
these compounds in living cells. When HeLa cells are
stimulated with insulin, insulin receptor (IR)-dependent
signaling cascades lead to production of PIP3, which stim-
ulates the translocation of cytohesins to the inner leaflet of
the plasma membrane by means of their PH domains. To
analyze the effect of Cyplecksins on this process, we trans-
fected HeLa cells with a cytohesin-2 construct that was fused
to green fluorescent protein (GFP) (Figure 4b, top row,
Cyth2-GFP). The cells were then analyzed for membrane
recruitment of cytohesin-2 GFP by confocal fluorescence
microscopy. Without insulin stimulation almost no Cyth2-
GFP can be detected at the membrane (Figure 4b, column 1)
but after insulin stimulation, Cyt2-GFP translocates to the
membrane (white arrows, column 2) and colocalizes with
membrane proteins stained by wheat germ agglutinin (white
arrows, merge, column 2). In the presence of 50 mm Cypleck-
sins 1, 2, or 3, however, no insulin-dependent Cyth2-GFP
translocation to the plasma membrane can be detected
(columns 3–5). In contrast, the inactive Cyplecksin analogues
4 and 5 at similar concentrations have no influence on the
membrane recruitment of Cyth2-GFP (columns 6 and 7). The
analysis of Cyth2-GFP membrane recruitment in a large
number of cells revealed a statistically highly significant
inhibition by Cyplecksins 1–3 (Figure 4c). These results
clearly demonstrate that Cyplecksins 1–3 effectively inhibit
the binding of PIP3 to cytohesin PH domains also in the
context of living cells.
Figure 3. Cyplecksins bind covalently to cytohesins a) Chemical struc-
ture of biotinylated Cyplecksin-2 analogues 6 and 7. b) Increasing
concentrations of 6 and its inactive analogue 7 incubated with
cytohesin-1 PH domain (top panel), full-length cytohesin 1 (Cyth1-fl;
middle panel), and cytohesin-2 Sec7 domain (bottom panel), analyzed
by denaturing PAGE. Covalently bound biotin is detected by neutravi-
din and total protein by an anti-His5 antibody. Cytohesin-1 PH domain
and full-length cytohesin-1, but not the cytohesin-2 Sec7 domain, were
found to be biotinylated. In all experiments, compound 7 was inactive.
c) Competition of biotinylated adduct formation by 6 using Cyplecksins
1–5. Cyplecksins 1–3, but not 4 and 5, competed with 6 for binding to
the cytohesin-1 PH domain; analysis was the same as in (b).
formation of the covalent adduct, cytohesin-1 PH and full-
length protein both showed concentration-dependent biotin-
ylation with 6 but not with 7, whereas cytohesin-2 Sec7
remained unmodified (Figure 3b).
To further substantiate this result, we tested whether the
biotinylation of the cytohesin-1 PH domain with 6 was
competed by non-biotinylated Cyplecksins 1–3. At concen-
trations of 50 mm or higher, Cyplecksins 1–3 resulted in
a marked reduction of the biotinylation by 6, and at 5 mm
a reduction was already detectable. Neither 4 nor 5 were
able to compete with 6 for cytohesin-1 PH domain binding,
not even at 100 mm concentrations (Figure 3c). Altogether,
these results can only be explained by a specific covalent
In conclusion, our study demonstrates that the method-
ology of aptamer displacement screening is feasible for
identifying small-molecule inhibitors of the cythohesin PH
domain. Aptamer-directed screening assays have previously
led to useful druglike inhibitors.[3b,6,11] Our screening assay,
based on RiboGreen detection, revealed a series of substi-
tuted 5-bromopyrimidine-2,4,6-triones 1–3, termed Cypleck-
sins, which inhibit phospholipid binding to PH domains of
cytohesins. Within the subset of PH domains of other proteins
tested here, Cyplecksins exhibited high selectivity for cyto-
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Figure 4. Cyplecksins inhibit the binding of cytohesin-2 PH to PIP2-containing nanodiscs and
insulin-induced translocation of cytohesin-2-GFP to the plasma membrane in HeLa cells. a) Cytohe-
sin-2 PH tagged with a streptavidin-binding peptide was incubated with 100 mm Cyplecksin 1–3 or
the inactive analogues 4 and 5, and subsequently with PIP2-containing nanodisks. After incubation,
cytohesin-2 PH was enriched by pull-down on StrepTactin beads. Cytohesin-2 PH and the co-
enriched nanodisk scaffold protein MSP1D1 were visualized by Coomassie staining after PAGE. T:
total protein; PD: pull down. b) HeLa cells were transfected with cytohesin-2-GFP and treated with
DMSO and 50 mm Cyplecksins 1–3 or the inactive analogues 4 and 5. Membrane recruitment was
detected by confocal fluorescence microscopy. Arrows point to membrane-recruited cytohesin-2-GFP
(Cyth2-GFP, top row) that colocalizes (bottom row) with stained membrane (row 3). For enlarged
micrographs, see Figures S7–S9 (SI). c) Statistical analysis of membrane translocation of Cyth2-GFP
in HeLa cells (n=3, counted cells >150) in percent. ***p<0.001. Data are represented as mean
ÆSEM.
hesin PH domains, but did not discriminate within the
conserved three-dimensional architecture without similarities
in their primary sequence. Besides the common structural
elements, PH domains contain variable loop regions that
connect antiparallel b-sheets within a b-sandwich motif, and
often contribute to ligand specificity to some extent.[14]
Recently it was shown that certain PH domains possess the
specificity required for discriminating between various inosi-
tol pentakisphosphate isomers, while others could not.[15]
These structural differences may allow for recognition of
cytohesin family. In this respect, Cyplecksins behave similar
to SecinH3, a Sec7 domain-specific cytohesin inhibitor that
targets cytohesins 1–3 but not other Sec7 domains. Whether
Cyplecksins are cytohesin-specific over the entire human
proteome, which contains more than 250 proteins with PH
domains,[12] remains to be investigated.
PH domains are widely distributed within the proteomes
of higher organisms.[13] They share a common and highly
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specific PH domains by a given inhibitor despite the overall
similarity of this domain. Another study reported the
identification of PITenins, a class of (thio)urea derivatives
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A remarkable feature of the Cyplecksins described here is
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Cyplecksins represent the first example of covalent inhibitors
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Keywords: aptamers · Cyplecksins · cytohesin ·
drug development · inhibitors
.
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