Design of Small Molecules That Compete with Nucleotide Binding to an Engineered Oncogenic KRAS Allele

Article abstract of DOI:10.1021/acs.biochem.7b01113

RAS mutations are found in 30% of all human cancers, with KRAS the most frequently mutated among the three RAS isoforms (KRAS, NRAS, and HRAS). However, directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has been difficult because of the high affinity of KRAS for GDP and GTP. We designed an engineered allele of KRAS and a covalent inhibitor that competes for GTP and GDP. This ligand-receptor combination demonstrates that the high affinity of GTP and GDP for RAS proteins can be overcome with a covalent inhibitor and a suitably engineered binding site. The covalent inhibitor irreversibly modifies the protein at the engineered nucleotide-binding site and is able to compete with GDP and GTP. This provides a new tool for studying KRAS function and suggests strategies for targeting the nucleotide-binding site of oncogenic RAS proteins.

Full text of DOI:10.1021/acs.biochem.7b01113

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Article
Design of small molecules that compete with nucleotide
binding to an engineered oncogenic KRAS allele
Yan Zhang, Mare-Helene Larraufie, Leila Musavi, Hemanth Akkiraju, Lewis M. Brown, and Brent R. Stockwell
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01113 • Publication Date (Web): 09 Jan 2018
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Biochemistry
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Design of small molecules that compete with nucleotide binding to an engineered
oncogenic KRAS allele
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Yan Zhang1, MarieꢀHélène Larraufie2, Leila Musavi2, Hemanth Akkiraju2, 3, Lewis M.
Brown2, 3 and Brent Stockwell1, 2
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Authors and Affiliations
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1Department of Chemistry, Department of Biological Sciences, Columbia University,
Northwest Corner Building, 12th Floor, MC 4846, 550 West 120th Street, New York, NY
10027.
3Quantitative Proteomics and Metabolomics Center, Columbia University, New York, NY
10027, USA
*Corresponding author: Brent R. Stockwell
Columbia University, Northwest Corner Building, 12th Floor, MC 4846, 550 West 120th
Street, New York, NY 10027.
Email: bstockwell@columbia.edu; Tel: 212ꢀ854ꢀ2948; Fax: 212ꢀ854ꢀ2951
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Abstract
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RAS mutations are found in 30% of all human cancers, with KRAS the most frequently
mutated among the three RAS isoforms (KRAS, NRAS, HRAS). However, directly
targeting oncogenic KRAS with small molecules in the nucleotideꢀbinding site has been
difficult due to the high affinity of KRAS for GDP and GTP. We designed an engineered
allele of KRAS, and a covalent inhibitor that competes for GTP and GDP. This ligandꢀ
receptor combination demonstrates that the high affinity of GTP/GDP for RAS proteins
can be overcome with a covalent inhibitor and a suitably engineered binding site. The
covalent inhibitor irreversibly modifies the protein at the engineered nucleotide binding
site and is able to compete with GDP and GTP. This provides a new tool for studying
KRAS function and suggests strategies for targeting the nucleotideꢀbinding site of
oncogenic RAS proteins.
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INTRODUCTION
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The RAS superfamily of small GTPases consists of more than 150 GTPꢀbinding
proteins that play key roles in the regulation of cell growth and survival1. There is
speculation that several members of this family, including RAS, RAC1, RHOA and
RHEB proteins, could serve as effective targets in multiple diseases. RAS mutations are
found in 30% of human cancers, with KRAS being the most frequently mutated (86%)
among the three RAS isoforms (KRAS, NRAS, HRAS)2. It has been postulated that
KRAS is essential for tumor maintenance, but not for normal adult physiology, thus
rendering inhibition of KRAS a “Holy Grail” in cancer therapy. Recently, the discoveries
of a panꢀRAS inhibitor3 and KRASG12C covalent inhibitors4ꢀ5 showed the feasibility of
directly targeting RAS protein in some cases. However, pharmacological and genetic
validation of the therapeutic index associated with targeting RAS and other small
GTPases requires further investigation.
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The KRAS protein functions as molecular switch, cycling between a GTPꢀbound
active state and a GDPꢀbound inactive state. Oncogenic mutations in KRAS impair its
intrinsic and GAPꢀmediated GTPase function, resulting in the accumulation of KRASꢀ
GTP that constitutively activates KRAS downstream signaling. Pharmacological
validation of KRAS and other small GTPases as therapeutic targets is difficult because
of their picomolar affinity to GTP/GDP. Thus, creating a potent and selective small
molecule inhibitor for these proteins is challenging.
Generating engineered, inhibitable alleles of proteins to validate targets has proved
successful for kinases and other proteins6ꢀ15. The essence of this strategy resides in the
design of engineered alleles of the protein of interest that are sensitized to smallꢀ
molecule inhibition, while being functionally indistinguishable from wildꢀtype counterparts
(Fig. 1A). Treatment with a complementary smallꢀmolecule probe can then provide
selective, rapid, and doseꢀdependent inactivation of the protein of interest. More
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recently, Shah and coꢀworkers reported a strategy for the selective inhibition and
activation of an engineered HꢀRas mutant by unnatural GDP and GTP analogues16.
However, future applications of that study were limited by the use of GDP analogues as
molecular probes, which are inherently nonꢀcellꢀpermeable and not selective. Moreover,
that study did not address the feasibility of designing drugꢀlike small molecule that target
engineered small GTPases.
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We aimed to address this issue by testing whether we could design a cellꢀ
permeable small molecule probe that would allow us to test the effects of
pharmacological inhibition of suitably engineered small GTPases. We focused on the
design of small molecules capable of inhibiting an engineered mutant allele of oncogenic
KRAS (termed KRAS*). We were concerned that mutation of key conserved residues in
KRAS might alter its natural nucleotide selectivity and important proteinꢀprotein
interactions, making the mutant activated in a GEFꢀindependent way and lose the switch
function16ꢀ18, as first suggested for RAS (N116I)19. Shah and coꢀworkers have reported,
however, that HꢀRAS (L19A, N116A) mutations remain fully functional as the wildꢀtype
enzyme16. We also demonstrated that the engineered inhibitable KRAS* allele we
designed has an effective “on” and “off” switch mechanism and fully functions in cells.
We report herein the computational design of a mutant engineered oncogenic allele of
KRAS bearing an enlarged nucleotide binding site and a cysteine residue that can serve
as a site for covalent targeting. We found that this mutant KRAS* was functionally
indistinguishable from KRASG12V, but could be selectively inhibited by a computationally
designed covalent inhibitor.
RESULTS
Rational design of KRAS allele
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Small GTPases in the RAS family have a conserved binding site and share similar
nucleotideꢀbinding pockets20(Fig. 1B). Shah and coꢀworkers demonstrated that mutation
of L19 and N116, located on the backside of the HRAS GTPꢀbinding site, to smaller
alanine residues allowed access to a buried hydrophobic cavity. The crystal structure of
KRASG12V in the GDPꢀbound conformation (4TQ921) was used as a template to model
the influence of point mutations in the nucleotideꢀbinding site. Prime and BioLuminate
(Schrödinger) were used to calculate the change in structure and stability of the protein
after introducing mutations (Table S1). Residues 19 and 116 were mutated in silico and
all residues within 10 Å of either mutation were minimized. A new hydrophobic cavity 3 Å
wide and 10 Å long appeared in the nucleotideꢀbinding site (Fig. 1C). We next wished to
introduce a cysteine residue in the hydrophobic pocket that could then be used as a
handle for covalent inhibition3. Indeed, we envisioned that the use of covalent inhibitors
might be key to be able to cope efficiently with the high cellular concentration of GTP (~1
mM)22. For that purpose, a cysteine residue (C114) that could be targeted by
electrophilic moieties was installed in the backside of the engineered hydrophobic
pocket. The stability of the engineered protein (KRAS*G12VL19AN116AV114C, termed KRAS*)
was predicted to have decreased energy of 52.96 kcal/mol. The predicted structure of
the engineered protein showed that C114 was orientated to perform a nucleophilic attack
on electrophilic moieties in the pocket (Fig. 1C). Importantly, the three mutated residues
in KRAS* are in a conserved site among the GTPases, so they could potentially be
translated to other members of the family.
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Figure 1
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Function of engineered KRAS allele
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To verify that the engineered mutant allele functions well as a “switch”, KRAS* was
loaded with GDP or GTP and incubated with RAF1 RBD (RAS binding domain) protein
attached to glutathione Sepharose beads. The unbound KRAS* was removed during the
washing step, while the bound KRAS* was quantified by immunobloting with antiꢀRAS
antibody. As expected, RAF 1 RBD specifically bound to GTPꢀloaded KRAS* but not
GDPꢀloaded KRAS* (Fig. 2A). To prove that the KRAS* is not nucleotideꢀfree analog of
RAS as suggested for HRAS(N116I), we tested the binding of GDP and GTP by KRAS*
and KRASG12V protein (Fig. 2B and 2C). Both KRAS* and KRASG12V were nucleotide
exchanged to BODIPYꢀGTP, a fluorescent analog of GTP which have high polarization
value when binds with protein and low polarization value in unbound form. BODIPYꢀGTP
was displaced by GDP or GTP in both KRAS* and KRASG12V, indicating KRAS* retains
nucleotide selectivity. Compared with KRASG12V, the faster nucleotide exchange rate of
BODIPYꢀGTP to GTP or GDP might come from the low binding affinity between KRAS*
and BODIPYꢀGTP.
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To further verify KRAS* function in cells, we used RASꢀless MEFs. K-Raslox(H-Ras-/-;N-
Ras-/-;K-Raslox/lox;RERTnert/ert) mouse embryonic fibroblasts (MEFs) were stably
transfected with KRAS* or KRASG12V alleles23. The MEFs were generated to carry null
HRAS and NRAS alleles along with a floxed KRAS locus and a knockedꢀin inducible Cre
recombinase. After transfection, 4ꢀhydroxytamoxifen was used to induce of Cre
recombinase, resulting in the complete elimination of endogenous KRAS gene after two
weeks (termed KRAS* MEFs and KRASG12V MEFs) (Fig. S1). The ability of KRAS* to
activate RAF/MEK/ERK signaling was examined by measuring the abundance of
phosphorylated ERK (pERK) and phosphorylated AKT (pAKT) in the transfected cell
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lines. Following EGF treatment, pERK and pAKT levels were increased in the
transfected cell lines (Fig. 2D), demonstrating that the engineered mutations did not
prevent KRAS* from activating effector proteins. The EGFꢀdependent signal in KRAS*
MEFs also revealed that its binding to GTP was not GEFꢀindependent as suggested for
RAS (D119N)18. KRAS mRNA levels were measured in the transfected cell lines by
qPCR. We found that the KRAS* mRNA was more abundant than the KRASG12V mRNA
(Fig. 2E). Compared with the similar protein level of KRAS in KRAS* MEFs and
KRASG12V MEFs (Fig. 2A), that might because KRAS* is less stable than KRASG12V (Fig.
S1), which providing an advantage for rapidly inducing and removing this engineered
protein from cells.
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Figure 2
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Design of small molecule ligands
A GDPꢀbound KRASG12V crystal structure (PDB: 4TQ9) was used to design compounds
that covalently lock KRAS* in the GDPꢀbound state, inactivating its signaling function.
KRASG12V was mutated in silico to KRAS*, and the engineered structure was refined by
Schrödinger Protein Preparation Wizard for docking studies. A fragmentꢀbased design
strategy was applied in the search for a covalent inhibitor24 and two series of covalent
inhibitors were designed and synthesized. 177,911 fragments (SI Software) were
screened using the Schrödinger Glide program and the topꢀranked fragments were used
as scaffolds for further design. The first series of inhibitors (Fig. 3A, S2) harbor a
carboxylic acid group, which is predicted to interact with the magnesium ion and multiple
residues in the GTPꢀbinding pocket, thus giving a high predicted binding affinity (as good
as ꢀ12 for Glidescore). These inhibitors had high predicted binding affinity to the KRAS*
protein (Fig. S3), but low cellular activity (Table S3), making it difficult to study the KRAS
therapeutic index in cells. Compound 7 had two carboxylic acid groups, thus likely could
not penetrate cell membranes (Table S2). Esterification one carboxylic acid groups can
make compounds more cellꢀmembraneꢀpermeable; the measured cellular accumulations
of compound 6 and 8 were 3.9ꢀfold and 15.6ꢀfold (Table S2), respectively. The low
cellular activity might thus derive from their inability to block KRASꢀeffector interactions
in cells.
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We then found that the conserved aspartate residue D119 is a key residue determining
the specificity for GTP over other nucleotides through hydrogen bonding. We reasoned
that the discovery of a compound that can form hydrogen bonds with D119 is thus
important for a successful design. Hꢀbond constraints to D119 were applied in fragment
screens for a second inhibitor design series. Topꢀranked fragments without carboxylic
acid groups were tested in a fluorescence polarization assay to measure binding affinity
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to KRAS*. We found that an indazole fragment G, with a docking score of ꢀ7.1, (Fig. 3B,
S2) could compete with GTP binding for KRAS in vitro in the 100 micromolar range (Fig.
S4), and was chosen as a scaffold for further design. As a next step, the hydrophobic
pocket of the engineered allele was targeted by attaching a lipophilic group (Fig. 3B) to
the appropriate site on fragment G. This produced compound G*, which had a more
favorable docking score of ꢀ9.1, compared to ꢀ7.1 for compound G (note that the Glide
docking score is a log scale, so this predicted a 100ꢀfold improvement in affinity). Based
on the structure of compound G, a number of covalent inhibitors with electrophile
warheads at different positions were designed and evaluated using Schrödinger’s
Covalent Docking program25. This showed that having a 2ꢀchloroacetamide electrophile
at the metaꢀposition (compound YZ0468) had the bestꢀpredicted affinity score in silico
(docking structure showed in Fig. 3A). On the basis of this indazole scaffold, a
customized compound library employing amide coupling, reductive aminations, and
cross coupling reactions was created (Fig. 3C) and evaluated using Schrödinger’s Glide
docking and Covalent Docking program. The topꢀscoring compounds were synthesized
and tested in the fluorescence polarization assay to further assess inhibition activity.
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Synthesis of covalent KRAS* inhibitors
A set of promising covalent small molecule inhibitors was thus synthesized (Table S3).
As an example, the synthetic route developed for compound YZ0711 is illustrated in Fig.
3D. The indazole scaffold was synthesized by heating 2ꢀfluoroꢀ5ꢀnitrobenzonitrile and
hydrazine monohydrate in refluxing ethanol followed by the selective BOCꢀprotection of
the Nꢀ1 position of the indazole and subsequent palladiumꢀcatalyzed hydrogenation26.
The resulting diaminoindazole could be acylated selectively at the Cꢀ5 amine using an
acid chloride. In the next step, the Cꢀ3 amine was acylated in pyridine to yield compound
4. Catalytic hydrogenation of the nitro group in compound 4 followed by acylation of the
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resulting aniline with chloroacetyl chloride and selective deprotection of the BOCꢀgroup
provided the desired product.
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Figure 3
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Biophysical measurements
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We developed an in vitro fluorescence polarization assay to test the ability of small
molecules to displace BODIPYꢀGTP, a fluorescent analog of GTP. In this assay, test
compounds that were able to competitively replace BODIPYꢀGTP bound to KRAS*
resulted in a decrease in the polarization of BODIPYꢀGTP (Fig. 4A).
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The designed covalent inhibitors and nonꢀcovalent inhibitors were able to compete with
fluorescent GTP binding for the engineered mutant, but not for KRASG12V (Fig. 4, Fig.
S5), thus satisfying the selectivity criterion. In addition, inhibitor binding was catalyzed by
EDTA but not SOS1, a guanine nucleotide exchange factor that promotes guanine
nucleotide exchange (Fig. S5). Compared with compounds bearing acrylamide and vinyl
sulfonate warheads, compounds bearing a chloroacetyl warhead had a better ability to
bind to KRAS* (Fig. 4B). This might result from the relatively small pocket in KRAS*.
Indeed, an extra chloro group on the benzyl moiety of YZ0571ꢀ1 decreased potency,
consistent with a small available space in the binding site. In addition, a nonꢀcovalent
analog (YZ0571ꢀ4) was not able to displace BODIPYꢀGTP. Based on this result, a series
of covalent inhibitors incorporating chloroacetyl warheads were synthesized. Compound
YZ0714 and YZ0719 had comparable binding affinity to YZ0571ꢀ1. Compound YZ0719
has low activity in a cell viability assay, which might result from its low cell membrane
permeability (Table S2). Compound YZ0711 had the best selectivity at inhibiting KRAS
signaling among these electrophiles (data not shown) and was selected for further
analysis.
WaterMap (Schrödinger), which calculates thermodynamics associated with water
hydration sites in protein active sites27ꢀ28, showed that compound YZ0711 displaced
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nearly all highꢀenergy hydration sites in the nucleotide binding site of KRAS* (Fig. 4D,
Fig. S6). This result indicated possible favorable binding between compound YZ0711
and KRAS* protein.
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Figure 4
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Mass-spectrometry analysis of covalent binding
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To determine whether compound YZ0711 could covalently bind to KRAS*, purified
KRAS* (20 ꢁM) was incubated with 200 ꢁM YZ0711 and 1 mM EDTA overnight at 4°C.
The protein was then labeled with iodoacetamide and digested with trypsin. The
resulting peptides were analyzed by nano LC/MS. LC/MS analysis revealed a
modification of peptide (103ꢀ117) with YZ0711 at cysteine (C114) or lysine (K117)
residues (Fig. 5A). A peptide (103ꢀ117) lacking cysteine but containing lysine in
KRASG12V was not modified with YZ0711 (Fig. 5B), indicating that YZ0711 modification
was exclusive to C114 in KRAS*. C114 modification with YZ0711 was estimated to be
94% in KRAS* (Table MS1).
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Cell-based assay
The KRAS* MEFs and KRASG12V MEFs described above were used in cellular assays.
We investigated whether the test compounds were able to penetrate cell membranes
and covalently label KRAS*. Using LCꢀMS, we found accumulation of compound
YZ0711 in KRAS* MEFs was 1.4 fold compared to the compound concentration in the
medium (Table S2). DesthiobiotinꢀGTP, which can covalently modify conserved lysine
residues in the nucleotideꢀbinding site, was used to pull down KRAS in cell lysates.
KRAS* MEFs treated with 50 ꢁM YZ0711 has a decreased KRAS enrichment (Fig. 6A),
suggesting that YZ0711 is able to penetrate the cell membrane and target the GTP/GDP
binding site.
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In a cell growth inhibition assay (Fig. 6B and Fig. S7), KRAS* MEFs and KRASG12V
MEFs were treated with DMSO, 25 ꢁM YZ0711, or 25 ꢁM YZ0756 (a structurally similar
compound as YZ0711) for 4 days and cell numbers were counted on a ViꢀCell XR Cell
Viability Analyzer. Compound YZ0711 showed 80% inhibition of KRAS* MEF number
and 50% inhibition to KRASG12V MEFs, indicating that YZ0711 exhibits some offꢀtarget
effect, but it is more selective to KRAS*. The cellular inhibition test using BRAFV600E
-
CAAX RASless MEFs also indicated that compound YZ0711 selectively inhibit KRAS*
MEFs’ growth (Fig. S7). Analog YZ0756 exhibited 25% inhibition in KRAS* MEFs and
15% inhibition to KRASG12V MEFs, which is less potent than compound YZ0711.
Inhibition of RAS signaling pathway
To test whether compound YZ0711 can disrupt KRAS*ꢀeffector binding, RAF RBD
protein, which can selectively pull down GTP loaded KRAS*, was used. The GTPꢀloaded
KRAS* protein was incubated with DMSO or YZ0711 (40uM) for 15 min and the reaction
was stopped by adding MgCl2 (65mM), then incubated with RAF RBD protein. Minimal
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KRAS* was observed for YZ0711ꢀbound KRAS* compared with GTPꢀloaded KRAS*
(DMSO control group) (Fig. 6C). To further investigate whether compound YZ0711 can
inhibit the RASꢀRAFꢀMEKꢀERK and RASꢀPI3KꢀAKT signaling pathways in cells, the
phosphorylation levels of ERK and AKT were analyzed by western blot (Fig. 6D).
Treatment of KRAS* MEFs with YZ0711 resulted in decreased pERK and pAKT levels.
KRASG12V MEFs were more resistant to this effect. Treatment of KRAS* MEFs with
YZ0711 also resulted in decreased RASꢀbound BRAF, suggesting YZ0711 can disrupt
RAS signaling (Fig. 6E).
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DISCUSSION
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Before embarking on a small molecule drug discovery project directed against a specific
target protein, it is critical to first validate the therapeutic effectiveness of the target in a
specific disease, as well as the therapeutic index associated with target inhibition29.
“Chemical” and “genetic” methods are two main approaches used in this target validation
where small molecules or genetic tools are used to modulate the target function30.
Genetic methods including gene knockout and RNAi may have problems of generating
null mutants for essential genes and lacking alignment between RNAi and inhibitor
studies30. Small molecule approaches can overcome above difficulties and provide
additional information about druggability, toxicity, and safety of inhibiting a target.
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KRAS is an essential gene and its gene product interacts with multiple upstream and
downstream effectors. However, there is a lack of suitable chemical tools that can
directly target KRAS. We developed a scalable system for testing the in vitro
consequences of pharmacological inhibition of KRAS. An enlarged binding pocket was
engineered into GTP binding site of KRAS through mutation of two conserved residues
to alanine. The engineered KRAS is functionally distinguishable from KRASG12V in cell.
We have designed and synthesized a smallꢀmolecule probe that covalently binds to an
engineered KRAS mutant with high affinity and displaces GTP from the binding pocket.
The probe allows selective binding to the engineered protein, but not the wildꢀtype
protein. The probe is also cell membrane permeable and can selectively inhibit RAS
signaling in cells. Covalent inhibitors are known to have offꢀtarget activity by forming a
covalent bond with other proteins. We employed chloroacetyl warhead, which has low
intrinsic reactivity, to minimize this offꢀtarget effect24. The probe we designed is selective
to the engineered KRAS but also has some extent of offꢀtarget effect in cells. The better
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selectivity and higher potency could be achieved by getting coꢀcrystal structure of ligandꢀ
protein complex, and further optimization of ligand structure and activity. The
crystallization research is ongoing in our lab and we’ll report it in future after we have
progress.
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Small GTPases contribute to multiple cellular processes and different stages of cancer
development and progression31ꢀ36. They have conserved GTP/GDP binding pockets and
share similar activation/deactivation mechanisms. Guanine nucleotide exchange factors
(GEFs) catalyze the exchange of GDP by GTP, while GTPaseꢀactivating proteins
(GAPs) accelerate the intrinsic GTPase activity of small GTPases. In the active state
(GTPꢀbound state), small GTPases associate with a variety of effectors and promote
downstream signaling. The system we designed here can be expanded for the design of
allele selective small molecule inhibitors of other proteins in small GTPase family, thus
providing tools to validate small GTPases. Considering the high affinity of small
GTPases for GTP (nanoꢀmolar range) and the high concentration of GTP in cells (~0.5
mM)22, a covalent inhibitor is generally necessary to compete with nucleotide binding. To
achieve high selectivity and minimize offꢀtarget effect, a targeted covalent inhibitor need
to be designed, which requires available protein crystal structures and structureꢀandꢀ
activity studies. Besides, choosing a protein which is easy to be crystalized will be highly
beneficial for the study.
Supporting Information
Supporting Information includes five figures, three tables, LCꢀMS data, and methods file.
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Acknowledgements
This research was funded by NIH (R21CA177591, R35CA209896 and P01CA087497 to
BRS).
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References
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7
8
1.
of Small GTPase Activity and Function. Angewandte Chemie 2015.
2. Cox, A. D.; Fesik, S. W.; Kimmelman, A. C.; Luo, J.; Der, C. J., Drugging the
Cromm, P. M.; Spiegel, J.; Grossmann, T. N.; Waldmann, H., Direct Modulation
9
undruggable RAS: Mission Possible? Nature reviews. Drug discovery 2014, 13 (11),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
828-51.
3.
Welsch, M. E.; Kaplan, A.; Chambers, J. M.; Stokes, M. E.; Bos, P. H.; Zask, A.;
Zhang, Y.; Sanchez-Martin, M.; Badgley, M. A.; Huang, C. S.; Tran, T. H.; Akkiraju, H.;
Brown, L. M.; Nandakumar, R.; Cremers, S.; Yang, W. S.; Tong, L.; Olive, K. P.;
Ferrando, A.; Stockwell, B. R., Multivalent Small-Molecule Pan-RAS Inhibitors. Cell
2017, 168 (5), 878-889 e29.
4.
Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M., K-Ras(G12C)
inhibitors allosterically control GTP affinity and effector interactions. Nature 2013,
503 (7477), 548-51.
5.
Lim, S. M.; Westover, K. D.; Ficarro, S. B.; Harrison, R. A.; Choi, H. G.; Pacold, M.
E.; Carrasco, M.; Hunter, J.; Kim, N. D.; Xie, T.; Sim, T.; Janne, P. A.; Meyerson, M.;
Marto, J. A.; Engen, J. R.; Gray, N. S., Therapeutic targeting of oncogenic K-Ras by a
covalent catalytic site inhibitor. Angewandte Chemie 2014, 53 (1), 199-204.
6.
Bishop, A.; Buzko, O.; Heyeck-Dumas, S.; Jung, I.; Kraybill, B.; Liu, Y.; Shah, K.;
Ulrich, S.; Witucki, L.; Yang, F.; Zhang, C.; Shokat, K. M., Unnatural ligands for
engineered proteins: new tools for chemical genetics. Annual review of biophysics
and biomolecular structure 2000, 29, 577-606.
7.
of Protein Kinase Cascades. The Scientific World Journal 2002, 2, 108-110.
8. Specht, K. M.; Shokat, K. M., The emerging power of chemical genetics.
Current opinion in cell biology 2002, 14 (2), 155-9.
9. Denzel, A.; Hare, K. J.; Zhang, C.; Shokat, K.; Jenkinson, E. J.; Anderson, G.;
Alaimo, P. J.; Shogren-Knaak, M. A.; Shokat, K. M., Chemical Genetic Analysis
Hayday, A., Cutting edge: a chemical genetic system for the analysis of kinases
regulating T cell development. Journal of immunology 2003, 171 (2), 519-23.
10.
Bishop, A. C.; Zhang, X. Y.; Lone, A. M., Generation of inhibitor-sensitive
protein tyrosine phosphatases via active-site mutations. Methods 2007, 42 (3), 278-
88.
11.
Coward, P.; Wada, H. G.; Falk, M. S.; Chan, S. D.; Meng, F.; Akil, H.; Conklin, B.
R., Controlling signaling with a specifically designed Gi-coupled receptor.
Proceedings of the National Academy of Sciences of the United States of America
1998, 95 (1), 352-7.
12.
Peet, D. J.; Doyle, D. F.; Corey, D. R.; Mangelsdorf, D. J., Engineering novel
specificities for ligand-activated transcription in the nuclear hormone receptor RXR.
Chemistry & biology 1998, 5 (1), 13-21.
13.
Liberles, S. D.; Diver, S. T.; Austin, D. J.; Schreiber, S. L., Inducible gene
expression and protein translocation using nontoxic ligands identified by a
mammalian three-hybrid screen. Proceedings of the National Academy of Sciences of
the United States of America 1997, 94 (15), 7825-30.
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Biochemistry
Page 26 of 36
1
2
3
4
5
6
7
8
9
14.
Clemons, P. A.; Gladstone, B. G.; Seth, A.; Chao, E. D.; Foley, M. A.; Schreiber, S.
L., Synthesis of calcineurin-resistant derivatives of FK506 and selection of
compensatory receptors. Chemistry & biology 2002, 9 (1), 49-61.
15.
Belshaw, P. J.; Schoepfer, J. G.; Liu, K. Q.; Morrison, K. L.; Schreiber, S. L.,
Rational Design of Orthogonal Receptor-Ligand Combinations. Angewandte Chemie-
International Edition in English 1995, 34 (19), 2129-2132.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16.
Vincent, F.; Cook, S. P.; Johnson, E. O.; Emmert, D.; Shah, K., Engineering
unnatural nucleotide specificity to probe G protein signaling. Chemistry & biology
2007, 14 (9), 1007-18.
17.
Fletcher, T. M.; Cathers, B. E.; Ravikumar, K. S.; Mamiya, B. M.; Kerwin, S. M.,
Inhibition of human telomerase by 7-deaza-2'-deoxyguanosine nucleoside
triphosphate analogs: potent inhibition by 6-thio-7-deaza-2'-deoxyguanosine 5'-
triphosphate. Bioorg Chem 2001, 29 (1), 36-55.
18.
Cool, R. H.; Schmidt, G.; Lenzen, C. U.; Prinz, H.; Vogt, D.; Wittinghofer, A., The
Ras mutant D119N is both dominant negative and activated. Molecular and cellular
biology 1999, 19 (9), 6297-305.
19.
p21ras by a novel mechanism. Science 1986, 233 (4764), 649-52.
20. Wennerberg, K.; Rossman, K. L.; Der, C. J., The Ras superfamily at a glance.
Journal of cell science 2005, 118 (5), 843-846.
21. Hunter, J. C.; Manandhar, A.; Carrasco, M. A.; Gurbani, D.; Gondi, S.; Westover,
K. D., Biochemical and Structural Analysis of Common Cancer-Associated KRAS
Mutations. Mol Cancer Res 2015, 13 (9), 1325-35.
22.
Walter, M.; Clark, S. G.; Levinson, A. D., The oncogenic activation of human
Ostrem, J. M.; Shokat, K. M., Direct small-molecule inhibitors of KRAS: from
structural insights to mechanism-based design. Nature reviews. Drug discovery
2016, 15 (11), 771-785.
23.
Drosten, M.; Dhawahir, A.; Sum, E. Y.; Urosevic, J.; Lechuga, C. G.; Esteban, L.
M.; Castellano, E.; Guerra, C.; Santos, E.; Barbacid, M., Genetic analysis of Ras
signalling pathways in cell proliferation, migration and survival. The EMBO journal
2010, 29 (6), 1091-104.
24.
drugs. Nature reviews. Drug discovery 2011, 10 (4), 307-17.
25. Zhu, K.; Borrelli, K. W.; Greenwood, J. R.; Day, T.; Abel, R.; Farid, R. S.; Harder,
E., Docking covalent inhibitors: a parameter free approach to pose prediction and
scoring. Journal of chemical information and modeling 2014, 54 (7), 1932-40.
Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A., The resurgence of covalent
26.
Krishnamurty, R.; Brock, A. M.; Maly, D. J., Protein kinase affinity reagents
based on a 5-aminoindazole scaffold. Bioorganic & medicinal chemistry letters 2011,
21 (1), 550-4.
27.
Abel, R.; Young, T.; Farid, R.; Berne, B. J.; Friesner, R. A., Role of the active-site
solvent in the thermodynamics of factor Xa ligand binding. Journal of the American
Chemical Society 2008, 130 (9), 2817-31.
28.
Young, T.; Abel, R.; Kim, B.; Berne, B. J.; Friesner, R. A., Motifs for molecular
recognition exploiting hydrophobic enclosure in protein-ligand binding. Proceedings
of the National Academy of Sciences of the United States of America 2007, 104 (3),
808-13.
29.
Carney, S., Target validation. Drug discovery today 2012, 17 Suppl, S1-2.
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6
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8
9
30.
Weiss, W. A.; Taylor, S. S.; Shokat, K. M., Recognizing and exploiting
differences between RNAi and small-molecule inhibitors. Nature Chemical Biology
2007, 3 (12), 739-744.
31.
Nishikimi, A.; Uruno, T.; Duan, X.; Cao, Q.; Okamura, Y.; Saitoh, T.; Saito, N.;
Sakaoka, S.; Du, Y.; Suenaga, A.; Kukimoto-Niino, M.; Miyano, K.; Gotoh, K.; Okabe, T.;
Sanematsu, F.; Tanaka, Y.; Sumimoto, H.; Honma, T.; Yokoyama, S.; Nagano, T.;
Kohda, D.; Kanai, M.; Fukui, Y., Blockade of inflammatory responses by a small-
molecule inhibitor of the Rac activator DOCK2. Chem. Biol. 2012, 19 (4), 488-97.
10
11
12
13
14
15
16
17
18
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41
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45
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49
50
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52
53
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60
32.
Montalvo-Ortiz, B. L.; Castillo-Pichardo, L.; Hernandez, E.; Humphries-Bickley,
T.; De la Mota-Peynado, A.; Cubano, L. A.; Vlaar, C. P.; Dharmawardhane, S.,
Characterization of EHop-016, novel small molecule inhibitor of Rac GTPase. JBC
2012, 287 (16), 13228-38.
33.
Shang, X.; Marchioni, F.; Sipes, N.; Evelyn, C. R.; Jerabek-Willemsen, M.; Duhr,
S.; Seibel, W.; Wortman, M.; Zheng, Y., Rational Design of Small Molecule Inhibitors
Targeting RhoA Subfamily Rho GTPases. Chem. Biol. 2012, 19 (6), 699-710.
34.
Nassar, N.; Cancelas, J.; Zheng, J.; Williams, D. A.; Zheng, Y., Structure-function
based design of small molecule inhibitors targeting Rho family GTPases. Current
topics in medicinal chemistry 2006, 6 (11), 1109-16.
35.
Vigil, D.; Cherfils, J.; Rossman, K. L.; Der, C. J., Ras superfamily GEFs and GAPs:
validated and tractable targets for cancer therapy? Nature reviews. Cancer 2010, 10
(12), 842-57.
36.
2008, 582 (14), 2093-101.
Vega, F. M.; Ridley, A. J., Rho GTPases in cancer cell biology. FEBS letters
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Figure 1. Design of engineered KRAS allele (A) The engineered KRAS* harbors an
enlarged pocket and a cysteine residue, which enables the design of a covalent
molecule selectively target KRAS* over KRASG12V. (B) Sequence alignment of RAS and
small GTPases superfamily (conserved residues are in BOLD). (C) Representation of
KRAS G12V (PDB code: 4TQ9) and engineered KRAS* (structure was minimized using
Schrödinger Prime).
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Figure 2. Establishment of engineered KRAS knock in cell lines (A) RAFꢀRBD can
selectively pull down GTPꢀloaded KRAS* but not GDPꢀloaded KRAS*. (B) Amount of
exchanged GDP and GTP in KRASG12V as a function of time. (C) Amount of exchanged
GDP and GTP in KRAS* as a function of time. (D) Western blot indicated the
overexpression of RAS in KRASG12V MEFs and KRAS* MEFs. RAS downstream
signaling was activated upon EGF treatment. The endogenous KRAS MEFs are MEFs
transfected with empty pBabe vector but without hydroxygamoxifen selection. The
transfected pBabe puro KRAS G12V vector has a FLAG tag (Nꢀterminal on insert)
causing it to migrate slower through the gel compared with the endogenous KRAS and
KRAS* (KRAS* w/o FLAG tag was inserted into the pBabe vector). (E) qPCR shows
over expression of KRAS in the transfected cell lines.
Figure 3. Design and synthesis of covalent inhibitors (A) Design of a first series of
small molecules and representation of the proposed binding mode of compound 6 in
KRAS*. The benzyl moiety is designed to target the engineered hydrophobic pocket.
Electrophile warheads are designed to covalently react with C114. Covalent docking of
compound 6 in KRAS* showed favorable formation of covalent bond between C114 and
the vinyl sulfone moiety (The docking pose is shown in the figure). The ligand is
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predicted to form hydrogen bond interaction with A146, C114, S17, Y32 residues; and
Piꢀcation interaction with K117, and K147 residues (docking was conducted by
Schrödinger Covalent Docking). (B) Design of a second series of small molecule and
representation of the proposed binding mode of compound YZ0468 in KRAS*. Covalent
docking of compound YZ0468 in KRAS* showed favorable formation of covalent bond
between C114 and the chloroacetyl moiety (The docking pose is shown in the figure).
The ligand is predicted to form hydrogen bond interaction with D30, D119, T144, S145
residues; Piꢀcation interaction with K117; and PiꢀPi interaction with F28 (C)
Combinatorial library design (created by MOE) (libraries of carboxylic acids, aldehydes,
acid chlorides, and amines are collected from SigmaꢀAldrich). (D) Synthesis route of
compound YZ0711
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Figure 4. Fluorescent polarization assay shows the competitive displacement of
GDP in KRAS* protein (A) FluoroꢀGTP binds in GTPꢀbinding site to yield a high
fluorescence polarization (FP) value. With the displacement of small molecule inhibitors,
fluoroꢀGTP shows a small FP value. (B) The structure and activity of selected covalent
inhibitors. Covalent inhibitors selectively occupy GTP binding pocket of KRAS*, but not
KRASG12V protein. Compound YZ0571ꢀ1, bearing a 2ꢀchloroacetyl chloride moiety, has
optimal activity in the fluorescence polarization assay and cell viability assays. (C) The
structure and activity of selected covalent inhibitors. Covalent inhibitors selectively
displace GDP from KRAS* protein, but not KRASG12V protein. (D) Compound YZ011 is
covalently docked in KRAS* (The docking pose is shown in figure). WaterMap shows the
thermodynamicꢀproperties of hydration site around the protein active site. Stable
hydration sites (ΔG < 3 kcal/mol) are shown in green, significantly unstable
hydration sites (∆G > 5 kcal/mol) are shown in red, and moderately unstable sites
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