Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation

Article abstract of DOI:10.1002/anie.201201358

Looking for fragments: A fragment-based screen using NMR spectroscopy was applied to discover ligands that bind to the GTPase K-Ras and modulate the activity of the nucleotide exchange factor Sos. Structural data on how these fragment-derived hits bind to the guanosine diphosphate-K-Ras complex (see picture) provides a starting point for the future discovery of drugs that target K-Ras activation and signaling. Copyright

Full text of DOI:10.1002/anie.201201358

Angewandte
Chemie
DOI: 10.1002/anie.201201358
Fragment-Based Screening
Discovery of Small Molecules that Bind to K-Ras and Inhibit Sos-
Mediated Activation**
Qi Sun, Jason P. Burke, Jason Phan, Michael C. Burns, Edward T. Olejniczak, Alex G. Waterson,
Taekyu Lee, Olivia W. Rossanese, and Stephen W. Fesik*
K-Ras is a small GTPase that functions as a molecular switch
cycling between inactive (guanosine diphosphate [GDP]-
bound) and active (guanosine triphosphate [GTP]-bound)
states. The conversion of K-Ras–GDP to K-Ras–GTP is the
rate-limiting step in the activation of K-Ras and is catalyzed
by guanine nucleotide exchange factors, such as the son of
sevenless (Sos). Mutations in K-Ras fix the protein in the
active state and endow cells with capabilities that represent
the hallmarks of cancer.[1] These include the ability to
proliferate, evade apoptosis, reprogram cell metabolism,
induce angiogenesis, activate invasion and metastasis, and
escape immune destruction.[2] Indeed, aberrant K-Ras signal-
ing plays a role in 30% of all human cancers, with the highest
incidence of activating mutations found in pancreatic (70–
90%), colon (30–50%), and lung (20–30%) carcinomas.[3]
Downregulation of activated Ras reverses the transformed
phenotype of cells and results in the dramatic regression of
tumors in murine xenograft models.[4] Thus, K-Ras inhibition
represents an attractive therapeutic strategy for many can-
cers. However, Ras activation and signaling is accomplished
primarily through protein–protein interactions. Such protein
interfaces typically lack well-defined binding pockets and
have been difficult to target with small molecules.[5]
Herein we report on the discovery of novel small
molecules that bind directly to K-Ras between switch I and
switch II and inhibit Sos-catalyzed K-Ras activation. To
identify compounds that bind directly to K-Ras, we conducted
a fragment screen[6] using uniformly 15N-labeled GDP-bound
K-Ras (G12D). An NMR-spectroscopy-based screen of
11000 fragments yielded approximately 140 fragments that
bind to GDP-bound K-Ras (G12D) (hit rate = 1.3%) as
determined by changes of 1H and 15N chemical shifts in 1H/15N
HSQC spectra of uniformly 15N-labeled K-Ras (G12D).
Representative examples of some of the hits that were
identified in the fragment-based screen (1, 2, 3) as well as
analogues that were synthesized to increase water solubility
and binding affinity (4, 5, 6) are depicted in Scheme 1. These
compounds bind to K-Ras (G12D) with affinities of 1.3–2 mm.
[*] Q. Sun,[+] Dr. J. P. Burke,[+] Prof. J. Phan, M. C. Burns,
Prof. E. T. Olejniczak, Prof. T. Lee, Prof. O. W. Rossanese,
Prof. S. W. Fesik
Scheme 1. Multiple chemotypes were identified in the fragment-based
screen, including indoles (1), phenols (2), and sulfonamides (3).
Analogues of these compounds (4, 5, 6) were synthesized to increase
their water solubility and binding affinity.
Department of Biochemistry
Vanderbilt University School of Medicine
2215 Garland Ave., 607 Light Hall, Nashville, TN 37232 (USA)
E-mail: stephen.fesik@vanderbilt.edu
Prof. A. G. Waterson
Department of Pharmacology
Vanderbilt University School of Medicine
2200 Pierce Ave., 442 RRB, Nashville, TN 37232 (USA)
The fragment hits identified in the screen were found not only
to bind the G12D mutant of K-Ras but also bind to wild-type
K-Ras, K-Ras (G12V), and H-Ras (data not shown), thus
indicating that these compounds bind to a site that is
conserved among Ras isoforms and different K-Ras mutants.
To determine how the fragment hits and analogues bind to
K-Ras, we obtained their cocrystal structures. Initial attempts
to cocrystallize K-Ras (G12D) with these compounds failed
to produce suitable crystals. Owing to the limited number of
space groups available to this mutant form of the protein, we
performed crystallization screens of both wild-type and G12V
mutant K-Ras. Both proteins crystallized across a broad range
of conditions in multiple space groups and yielded high-
resolution cocrystal structures. In all 20 cocrystal structures
obtained thus far, the compounds occupy a hydrophobic
pocket located between the a2 helix of switch II (60–74) and
the central b sheet of the protein. Figure 1a depicts a high-
[+] These authors contributed equally to this work.
[**] This work was supported by the US National Institutes of Health:
5DP1OD006933 (NIH Director’s Pioneer Award) to S.W.F., an ARRA
stimulus grant (5RC2A148375) to L. J. Marnett, and a NCI SPORE
grant in GI cancer (5P50A095103-09) to R.J. Coffey. This work was
also funded by the Lustgarten Foundation grant awarded to S.W.F.
and the American Cancer Society (Postdoctoral Fellowship,
PF1110501CDD) to J.P.B. We thank Matt Mulder (Craig Lindsley lab,
Vanderbilt University) for providing HRMS data.
Supporting information (including details of the protein purifica-
tion, the fragment screen, X-ray crystallography, nucleotide
exchange, 1H/15N-HSQC spectra of K-Ras with and without ligands
and Sos, and the synthesis of compounds) for this article is
available on the WWW under http://dx.doi.org/10.1002/anie.
201201358.
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for binding. This hypothesis is supported by
the lack of binding of analogues containing
substituents at these positions that occlude
hydrogen-bond formation.
Analysis of the ligand–protein cocrystal
structures reveal that all the compounds bind
to a pocket that is not readily observed in the
ligand-free form (Figure 2a) but in an “open”
form of the protein (Figure 2b). The pocket is
created by a conformational change (Fig-
ure 2c) in which the a2 helix moves away
from the central b sheet, and the side chain of
Tyr-71 breaks the hydrogen-bond network
present in the ligand-free form. Moreover,
the side chain of Met-67 rotates out of the way
to form a secondary binding cleft. This rota-
tion creates a new binding site for small
molecules that is not present in the “closed”
form. In subsequent X-ray structures obtained
of ligand-free K-Ras under different experi-
mental conditions as well as recently published
molecular dynamics simulations, the “open”
form has been observed, thus suggesting that
the “open” and “closed” conformations are
present in equilibrium.[7]
The secondary binding cleft is electroneg-
ative in character (Figure 2b) and contains
two acidic residues, Glu-37 and Asp-38. To
Figure 1. Ribbon and molecular surface representations of the X-ray structures of GDP–
K-Ras complexed to: a) 4 (PDB code 4EPV), b) 5 (PDB code 4EPW), c) 2 (PDB code
4EPT), and d) 6 (PDB code 4EPX).
resolution structure of 4, an analogue of the screening hit 1,
complexed to the GDP-bound form of wild-type K-Ras. The
indole of 4 binds into a hydrophobic pocket formed by Val-7,
Leu-56, and Tyr-71, as well as the aliphatic portion of the side
chains of Lys-5 and Thr-74. The imidazopyridine portion of
the molecule lies flat in an adjacent binding cleft formed on
one side by the side chain of Tyr-71. The nitrogen atom at the
1-position of the imidazopyridine is involved in a water-
mediated interaction with the side chain of Ser-39, and the
indole NH group forms a hydrogen bond with the side chain
of Asp-54. Another member of the indole series (5) uses
a thiocarbonyl instead of a methylene linker to access the
secondary binding cleft. In this case the indole moiety in 5 is
rotated towards the a2 helix and the switch II loop region and
forms a hydrogen bond with the side chain of Ser-39 instead of
Asp-54. This positions the piperidine ring closer to the helix
(Figure 1b). In addition to an indole, a phenol moiety is also
able to bind into this hydrophobic pocket, as demonstrated by
the X-ray structure of compound 2 bound to K-Ras (Fig-
ure 1c). The hydroxy group of the phenol forms a hydrogen
bond with Asp-54 while the pyrrolidine moiety forms stacking
interactions with Tyr-71. The binding mode of a member of
the sulfonamide series is shown in Figure 1d. The pyridine
nitrogen atom of 6 forms three water-mediated hydrogen
bonds to the side chains of Asp-54, Arg-41, and Ser-39, and
the ortho amino group interacts with the Asp-54 side chain
and a backbone carbonyl group. From these structures, it
appears that a hydrogen-bond donor, such as the -NH on the
indole moiety or the -OH on the phenol moiety, is important
Figure 2. Electrostatic surface representations (red negative, blue pos-
itive) of GDP–K-Ras a) in the absence of a ligand (PDB code 4EPR)
and b) in the “open” form showing the primary hydrophobic binding
pocket and the adjacent electronegative cleft. c) Schematic representa-
tion of the transition of GDP–K-Ras from the “closed” form (green) to
the “open” form (cyan).
bind to this region of the protein, we synthesized amide-
linked amino acid analogues of the indole-benzimidazole
fragment 7 containing positively charged amine groups
(Table 1). Improved binding to K-Ras was observed for
2
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Table 1: Compound binding affinity with GDP–K-Ras (G12D) and func-
tional activity in a Sos-catalyzed nucleotide exchange assay.
interacts with the carboxylic acid side chain of Asp-38 in the
secondary binding cleft.
To examine the functional consequences of binding to K-
Ras, compounds were tested for their ability to inhibit Sos-
mediated nucleotide exchange. In this assay, unlabeled GDP
is exchanged for the fluorescently labeled nucleotide
BODIPY-GTP; this exchange is catalyzed by Sos and results
in an increase in fluorescence.[8] As shown in Table 1, the
extended analogues with binding affinities below 500 mm
inhibited the nucleotide exchange process at a concentration
of 1 mm. For example, the analogue containing an isoleucine
(12) inhibited nucleotide exchange at 78% (see the Support-
ing Information). The inhibition of Sos-mediated nucleotide
exchange that we observe can be rationalized from a model
that was prepared by overlaying the X-ray structure of the K-
Ras/13 complex (Figure 3b) onto a reported structure of H-
Ras complexed with Sos.[9] The amino acid of 13 clashes with
the aH helix of Sos. The model predicts that Sos would not be
able to bind to K-Ras when complexed to a small molecule
that extends into this space (e.g., 8–13). This model is
supported by NMR spectroscopy experiments in which the
1H/15N HSQC spectrum of 15N-labeled K-Ras (G12D) when
complexed to unlabeled Sos dramatically changes upon the
addition of compound 13. This new spectrum resembles that
of the K-Ras/13 complex without Sos (see the Supporting
Information). Furthermore, in the absence of K-Ras, com-
pound 13 does not bind to Sos, and an analogue of 13 with the
indole moiety N-methylated does not bind to K-Ras and does
not inhibit Sos-mediated nucleotide exchange.
Compound
R
KD [mm][a]
Inhibition [%][b]
7
8
9
10
11
12
13
-H
ꢀ1300
420
no inhibition
27Æ9
-Gly
-Ala
-b-Ala
-Val
-Ile
350
340
240
190
51Æ4
32Æ10
73Æ12
78Æ8
-Pro
340
58Æ8
[a] KD values were measured from the changes in chemical shifts
observed in HSQC spectra of uniformly 15N-labeled protein as a function
of added compound. [b] The inhibition in % of Sos-catalyzed nucleotide
exchange observed using a 1 mm concentration of compound.
several of these analogues. The best compound in this series
contains an isoleucine (12) and binds to K-Ras with an affinity
of 190 mm, an improvement of roughly 10-fold over the
unsubstituted analogue 7. We were able to obtain a high-
resolution X-ray structure of one of these analogues (13)
complexed to GDP-bound K-Ras (G12V) (Figure 3a). As
designed, the indole is located in the primary binding pocket,
and the positively charged amino group of the amino acid
In conclusion, by using a fragment-based screen, we have
identified small molecules that bind to K-Ras in a hydro-
phobic pocket that is occupied by Tyr-71 in the apo-Ras
crystal structure. Using structure-based design, we obtained
analogues of the fragment hits with improved binding affinity
as well as functional activity in a Sos-catalyzed nucleotide
exchange assay. These compounds bind to K-Ras and block
binding to Sos, thereby causing the inhibition of Sos-mediated
nucleotide exchange. These molecules represent a starting
point for obtaining probe molecules that may be useful in
elucidating new insights into Ras signaling and for discovering
K-Ras inhibitors for the treatment of cancer.
Received: February 17, 2012
Published online: && &&, &&&&
Keywords: fragment-based screening · GTPases ·
.
K-Ras inhibitors · ligand design · NMR spectroscopy
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Figure 3. a) Ribbon and molecular surface representations of GDP-
bound K-Ras complexed to 13 (PDB code 4EPY). b) K-Ras/13 X-ray
structure overlaid with a reported[9] H-Ras–Sos complex crystal struc-
ture (PDB code 1BKD).
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4
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Communications
Fragment-Based Screening
Looking for fragments: A fragment-based
screen using NMR spectroscopy was
applied to discover ligands that bind to
the GTPase K-Ras and modulate the
activity of the nucleotide exchange factor
Sos. Structural data on how these frag-
ment-derived hits bind to the guanosine
diphosphate–K-Ras complex (see picture)
provides a starting point for the future
discovery of drugs that target K-Ras
activation and signaling.
Q. Sun, J. P. Burke, J. Phan, M. C. Burns,
E. T. Olejniczak, A. G. Waterson, T. Lee,
O. W. Rossanese,
S. W. Fesik*
&&&& — &&&&
Discovery of Small Molecules that Bind to
K-Ras and Inhibit Sos-Mediated
Activation
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!