A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2

Article abstract of DOI:10.1002/anie.201610957

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (K_D<10 nm), interference with Ras signaling and growth inhibition require 5–20 μm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.

Full text of DOI:10.1002/anie.201610957

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International Edition: DOI: 10.1002/anie.201610957
German Edition: DOI: 10.1002/ange.201610957
Medicinal Chemistry
A PDE6d-KRas Inhibitor Chemotype with up to Seven H-Bonds and
Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2
Pablo Martꢀn-Gago+, Eyad K. Fansa+, Christian H. Klein+, Sandip Murarka+, Petra Janning,
Marc Schꢁrmann, Malte Metz , Shehab Ismail, Carsten Schultz-Fademrecht,
Matthias Baumann, Philippe I. H. Bastiaens,* Alfred Wittinghofer,* and Herbert Waldmann*
Abstract: Small-molecule inhibition of the interaction between
the KRas oncoprotein and the chaperone PDE6d impairs
KRas spatial organization and signaling in cells. However,
despite potent binding in vitro (KD < 10 nm), interference with
Ras signaling and growth inhibition require 5–20 mm com-
pound concentrations. We demonstrate that these findings can
be explained by fast release of high-affinity inhibitors from
PDE6d by the release factor Arl2. This limitation is overcome
by novel highly selective inhibitors that bind to PDE6d with up
to 7 hydrogen bonds, resulting in picomolar affinity. Their
release by Arl2 is greatly decreased, and representative
compounds selectively inhibit growth of KRas mutated and
-dependent cells with the highest activity recorded yet. Our
findings indicate that very potent inhibitors of the KRas-
PDE6d interaction may impair the growth of tumors driven by
oncogenic KRas.
interaction impairs KRas localization and oncogenic signaling
and blocks proliferation of human pancreatic ductal adeno-
carcinoma (hPDAC) cells harboring oncogenic KRas.[27,28]
The KRas-PDE6d inhibitors Deltarasin (1) and Deltazi-
none 1 (2) bind PDE6d with low nanomolar affinity in vitro
(Figure 1A, B; Deltazinone 1: KD = 8 Æ 4 nm), but show
cellular activity only at micromolar concentrations (for
example, 5–20 mm Deltazinone 1).[28]
The release factor Arl2 stabilizes PDE6d and causes
a discharge of the medium affinity KRas cargo (KD =
453 nm).[26] We now demonstrate that Arl2 also induces
release of the high affinity inhibitors Deltarasin and Delta-
zinone 1, which explains why micromolar concentrations are
required to effectively reduce cell growth. We describe
a novel PDE6d inhibitor chemotype with picomolar affinity,
which prevents efficient release by Arl2.
Release of high-affinity cargo from PDE6d by Arl2 was
analyzed by means of fluorescence polarization measure-
ments. These experiments revealed that in the presence of
200-fold excess of unlabeled compound to prevent re-binding,
fluorescently labeled inhibitors 1L* and 2L* (Figure 1A,B)
are rapidly released by Arl 2 with koff = 38 Æ 1 and 19 Æ
1 sÀ1 10À3, respectively (Figure 2C). These results and the
fact that the concentration of Arl2 is also in the micromolar
range (Wittinghofer et al., unpublished results) explains why
ligand concentrations much higher than expected from the
determined KD values are needed to consistently establish
PDE6d inhibition in cells and to reduce cell growth.
Screening of our in-house compound library (ca. 200000
compounds) employing the Alfa Screen technology[27] for
compounds with higher potency identified bis-sulfonamides
(for example 3; KD = 13 Æ 4 nm; Figure 1C) as novel potent
ligands of the prenyl binding pocket of PDE6d.
R
as proteins are mutated in about 20–30% of human
cancers, with the KRas isoform most frequently mutated.[1–4]
However, direct targeting of KRas[4–10] by small molecules has
not yet given rise to clinical candidates.[11–22]
KRas signaling and spatial organization critically depend
on recognition of its farnesylated C-terminal cysteine methyl
ester.[23–26] Small-molecule inhibition of the KRas-PDE6d
[*] C. H. Klein,[+] Prof. Dr. P. I. H. Bastiaens
Department of Systemic Cell Biology
Max Planck Institute of Molecular Physiology
44227 Dortmund (Germany)
E-mail: philippe.bastiaens@mpi-dortmund.mpg.de
Dr. P. Martꢀn-Gago,[+] Dr. S. Murarka,[+] Dr. P. Janning,
Dr. M. Schꢁrmann, M. Metz , Prof. Dr. H. Waldmann
Department of Chemical Biology
Max Planck Institute of Molecular Physiology
44227 Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Dr. E. K. Fansa,[+] Dr. S. Ismail, Prof. Dr. A. Wittinghofer
Structural Biology Group
Max Planck Institute for Molecular Physiology
44227 Dortmund (Germany)
By analogy to Deltazinone 1,[28] compound 3 displays
three H-bonds to R61, Q78, and Y149 (Figure 3A) and two
additional interactions with the aromatic rings of W32 and
W90. An additional piperidin-4-ylmethyl H-bond donor (for
syntheses, see the Supporting Information) occupied more
space and formed a new H-bond with the carbonyl group of
C56 to yield ligand 4 (KD = 8 Æ 2 nm, Figure 1C and 3B).
Binding is further enhanced by a sulfur–p interaction to
M117.
E-mail: alfred.wittinghofer@mpi-dortmund.mpg.de
Prof. Dr. P. I. H. Bastiaens, Prof. Dr. H. Waldmann
TU Dortmund, Faculty of Chemistry and Chemical Biology
44227 Dortmund (Germany)
To establish an additional H-bond interaction with the
E88 side-chain, which plays a major role in KRas binding to
Dr. C. Schultz-Fademrecht, Dr. M. Baumann
Lead Discovery Center GmbH
44227 Dortmund (Germany)
PDE6d,[29]
a (2-(methylamino)pyrimidin-4-yl)methyl sub-
[+] These authors contributed equally to this work.
stituent was introduced to give inhibitor 5 (Deltasonamide 1,
Figure 1C). Deltasonamide 1 displays two new water-medi-
ated H-bonds to the aromatic pyrimidine nitrogen atoms
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201610957.
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Figure 1. Chemical structures and binding affinities (KD, determined by competitive fluorescence polarization assay of three independent
experiments, see the Supporting Information for details) of Deltarasin and labeled Deltarasin (A, 1 and 1L*), Deltazinone 1 and labeled
Deltazinone 1 (B, 2 and 2L*), the newly developed bis-sulfonamides (C, 3–8), and a fluorescently labeled bis-sulfonamide derivative (C, 8L*). For
kinetic solubility and schematic binding modes, see the Supporting Information, Figure S1.
Figure 2. Arl2-mediated displacement of the different chemotypes and CETSA melting curves. A),B) Release by Arl2 under equilibrium conditions
of 1L*, 2L*, and 8L*. 0.2 mm fluorescently labeled inhibitor was placed in a cuvette, followed by the addition of 0.5 mm PDE6d resulting in the
increase of the polarization signal owing to complex formation. The formed complex was titrated with Arl2 (1 and 5 mm) as indicated.
C),D) Kinetics of release by Arl2 under competition with an unlabeled inhibitor. A mixture of 0.5 mm Arl2 and 40 mm 5 was added to a preformed
complex of 0.2 mm of the fluorescently labeled inhibitor and 0.5 mm PDE6d. The Arl2- mediated release was monitored by the decrease of the
polarization. Dissociation rate constants (koff) were obtained by single exponential fitting of the data. E),F) Representative melting curves for
PDE6d (E) and RPL31 (F) as observed in a thermal protein profiling experiment. Melting curves for the DMSO controls are shown in dark blue
and melting curves after treatment with inhibitor 8 are shown in red.
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methyl group yielded inhibitor 8 (Figure 1C and 3F), which
binds to PDE6d with a total of 7 H-bonds, including a H-bond
with E88, and 3 aromatic–p interactions (KD = 358 Æ 36 pm).
Measurement of apparent permeability (Papp) from the
apical (A) to the basolateral (B) side of Caco-2 cells (see the
Supporting Information for details) revealed that both
Deltasonamide 1 and 2 migrate slowly but show enhanced
apparent permeability from the B to A side suggesting
a potential interaction with efflux transporters. However, this
effect is only moderate.
Assessment of general cytotoxicity employing human
peripheral blood mononuclear cells (hPBMCs) revealed that,
in contrast to Deltarasin,[28] Deltasonamides 1 and 2 do not
impair hPMBC proliferation up to 30 mm concentration.
Labeling of 8 at the carboxylic acid that points towards the
exit of the prenyl binding tunnel (Supporting Information,
Figure S2) yielded fluorescent analogue 8L* (Figure 1C).
Notably, in contrast to Deltarasin and Deltazinone 1, 8L*
could not be directly displaced from PDE6d by 5 and even
25 equiv of Arl2 (Figure 2Avs. 2B) Acceleration of release by
2.5 equiv of Arl2 in the presence of 200-fold excess of non-
fluorescent inhibitor was 45- and 25-fold slower than the
release of 1L* and 2L*, respectively (Figure 2C vs. 2D). Thus,
increase of the number of inhibitor-PDE6d interactions leads
to a higher stabilization of the complex and higher resistance
to displacement by Arl2.
PDE6d engagement by inhibitor 8 was confirmed by
means of a cellular thermal shift assay (CETSA)[31–33] in which
the thermal stabilization of the protein of interest upon ligand
binding is determined. Mass spectrometric analysis of Jurkat
cell lysate treated with 1 mm compound 8 identified proteins
showing a characteristic shift in melting temperature by at
least 28C, or for which the differences in the signal intensities
for at least two characteristic peptides were at least 10% (see
the Supporting Information and Figure 2E,F) Ligand 8 only
engaged PDE6d and ribosomal protein L31 (RPL31). Liter-
ature analysis did not reveal a link between RPL31, Ras
proteins or PDE6d. Thus, compound 8 is a very selective
inhibitor of PDE6d.
Disruption of the interaction between mCherry-PDE6d
and farnesylated Ras proteins (mCitrine-RheB) in living cells
was measured in MDCK cells by FLIM-FRET.[27] The
homogenous fluorescence patterns of both proteins in the
absence of inhibitor indicate solubilization of mCitrine-RheB
by mCherry-PDE6d (Figure 4A,B), which was also reflected
in the high molar fraction a of interacting mCitrine-RheB and
mCherry-PDE6d as derived from global analysis of FLIM
data.[34] Treatment with increasing concentrations of Delta-
sonamides resulted in a reduced interacting fraction a of
RheB-PDE6d. Fitting the dose-dependent measurements to
an equilibrium model with a clamped compound concentra-
tion[27] yielded an apparent “in-cell” KD of 85 Æ 18 nm for
Deltasonamide 1 and 61 Æ 5 nm for Deltasonamide 2. In
contrast to Deltarasin,[27] these apparent KD values surpass
the in vitro measured KD values by an order of magnitude. If
the partitioning coefficient P of the compounds between
cytoplasm and extracellular environment is independent of
administered compound concentration, the ratio of the
in vivo and in vitro KD values reflect P. This indicates that
Figure 3. Comparison among the binding modes of inhibitors 3–8.
Crystal structure of inhibitor 3 (A; PDB code: 5ML2), 4 (B; PDB:
5ML8), 5 (C, Deltasonamide 1; PDB: 5ML3), 7 (E; PDB: 5ML4), and 8
(F; PDB: 5ML6) in complex with PDE6d and best docking pose of 6
(D, Deltasonamide 2). Important H-bonding interactions of the ligand
to the protein (dotted yellow line) between PDE6d residues (gray) and
aromatic–p interactions (blue) are shown. Only key amino acids are
displayed as sticks (see also the Supporting Information).
(Figure 3C). A total of 10 non-covalent interactions stabilize
the binding of Deltasonamide 1 inside the PDE6d binding
pocket, including H-bonds to R61, Q78, E88, and Y149 side-
chains, to the carbonyl of C56, two water molecule-mediated
H-bonds to the Glu88 side-chain and the amide proton of
M118, two aromatic–p interactions to the indole side-chains
of W32 and W90, and one M117–aryl interaction. This
enhanced the in vitro potency to the picomomolar level
(KD = 203 Æ 31 pm; see the Supporting Information and Fig-
ure S3). The side-chains of E88 and W90 are in an arrange-
ment characteristic for the open RheB-bound conformation
of PDE6d.[26,29] Alternative introduction of a 4-aminocyclo-
hexane moiety resulted in an analogous binding mode, and
compound 6 (Deltasonamide 2, Figure 1C) displayed pico-
molar affinity as well (KD = 385 Æ 52 pm) (Figure 3D).
Energetically favorable replacement of a water molecule
with less than three H-bonds[30] by a carboxylate pointing
towards M118 yielded compound 7 (Figures 1C and 3E). An
additional water molecule establishes a new H-bond between
the carboxylate and the carbonyl backbone of Q116. Com-
pound 7 maintains high in vitro potency, but lacks the H-bond
to the E88 side-chain, probably because an intramolecular H-
bond between the carboxylate and the aniline proton forces
the methyl substituent out of the pocket. Removal of the
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Figure 4. Inhibition of the interaction between Ras proteins and PDE6d in cells by Deltasonamides resulting in delocalization. A),B) Left: FLIM
measurements of the mCitrine-RheB and mCherry-PDE6d interaction in dependence on Deltasonamide dose. Upper and middle rows:
fluorescence intensity of mCitrine-RheB and mCherry-PDE6d. Lower rows: molar fraction a of interacting mCitrine-RheB and mCherry-PDE6d.
Inhibitor concentrations are indicated above each image in nm. Right: fit of averaged dose-response of five cells to a binding model (see
methods) yielded an “in-cell” KD of 85Æ18 nm (s.e.m.) for Deltasonamide 1 and 61Æ5 nm for Deltasonamide 2. C) Time series of mCitrine-KRas
redistribution upon administration of 5 mm Deltasonamide 2 in MiaPaCa-2. Upper row: fluorescence intensity of mCitrine-KRas, lower row:
intensity profiles of corresponding ROI in the images. Right: KRas mean intensity Æ s.d. at the plasma membrane (black) and inside the cell (red)
over time (N=8).
only about 0.2–0.6% of the extracellular Deltasonamide
concentration is available in the cytoplasm to inhibit PDE6d.
In general, this finding is consistent with the Caco-2 perme-
ability data described above and calculated logP/logD values
(see the Supporting Information for details). However, in
contrast to these data, the apparent KD values monitor target
engagement in cells, as opposed to general membrane
permeability.
The new inhibitors also led to a loss of KRas plasma
membrane (PM) localization as apparent from MiaPaCa-2
cells ectopically expressing mCitrine-KRas treated with 5 mm
Deltasonamide 2 (Figure 4C). Analysis of variance showed
significance for loss of PM localization between 0 min and
30 min onwards in agreement with the effective rate of KRas
PM dissociation.[24]
Analysis of anti-proliferative activity by means of impe-
dance-based real-time cell analyzer (RTCA) measurements
revealed that treatment of the oncogenic KRas-dependent
Panc-Tu-I, and MiaPaCa-2[17,18] cells with either Deltasona-
mide 1 or 2 resulted in a strongly reduced proliferation, even
at submicromolar concentrations (Figure 5). This was most
pronounced in MiaPaCa-2 cells treated with Deltasona-
mide 2, with about 50% reduction of growth rate at 750 nm
(Figure 5B). Growth of Panc Tu-I cells was reduced by ca.
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Panc-1 cells and their weakest anti-proliferative effect on
KRas wild type BxPC-3 cells.
In conclusion, release of even high-affinity inhibitors by
Arl2 activity explains why high inhibitor concentrations are
needed to effectively reduce cell growth by PDE6d inhibition.
Highly potent selective inhibitors with up to seven H-bonds
and picomolar affinity reduce release by Arl2 and show the
highest activity recorded yet in proliferation assays employing
KRas mutated and -dependent cell lines. These results suggest
that very potent inhibitors of the KRas- PDE6d interaction
may become accessible that impair the growth of tumors
driven by oncogenic KRas.
Acknowledgements
This research was supported by the Deutsche Forschungsge-
meinschaft (Sonderforschungsbereich SFB 642 to A.W.,
H.W., and P.I.H.B.), the Deutsche Krebshilfe (Grant 110995
to P.I.H.B.), and the European Research Council under the
European Unionꢀs Seventh Framework Program (FP7/2007–
2013; ERC Grant 322637 to P.I.H.B., ERC Grant 268309 to
H.W. and ERC Grant 268782 to A.W.). We are grateful to
Andreas Brockmeyer for assistance in the cellular thermal
shift assay.
Conflict of interest
The authors declare no conflict of interest.
Keywords: drug design · medicinal chemistry · PDEd ·
Ras protein · structure–activity relationships
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Manuscript received: November 14, 2016
Final Article published: && &&, &&&&
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Communications
Medicinal Chemistry
Small-molecule inhibition of the interac-
tion between KRas oncoprotein and the
chaperone PDE6d impairs KRas spatial
organization and signaling in cells. Del-
tasonamides are highly selective inhibi-
tors that bind to PDE6d with up to 7
hydrogen bonds, resulting in picomolar
affinity.
P. Martꢁn-Gago, E. K. Fansa, C. H. Klein,
S. Murarka, P. Janning, M. Schꢂrmann,
M. Metz , S. Ismail,
C. Schultz-Fademrecht, M. Baumann,
P. I. H. Bastiaens,* A. Wittinghofer,*
H. Waldmann*
&&& — &&&
A PDE6d-KRas Inhibitor Chemotype with
up to Seven H-Bonds and Picomolar
Affinity that Prevents Efficient Inhibitor
Release by Arl2
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