Rigidification Dramatically Improves Inhibitor Selectivity for RAF Kinases

Article abstract of DOI:10.1021/acsmedchemlett.9b00194

One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAF^V600E-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.

Full text of DOI:10.1021/acsmedchemlett.9b00194

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Letter
Rigidification Dramatically Improves Inhibitor Selectivity for RAF Kinases
Amir Assadieskandar, Caiqun Yu, Pierre Maisonneuve, Igor Kurinov, Frank Sicheri, and Chao Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00194 • Publication Date (Web): 04 Jun 2019
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Rigidification Dramatically Improves Inhibitor Selectivity for RAF
Kinases
§
,∥
Amir Assadieskandar,†,# Caiqun Yu,†,# Pierre Maisonneuve,§,# Igor Kurinov,⊥ Frank Sicheri, and
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†
Chao Zhang*, ,‡
†Loker Hydrocarbon Research Institute & Department of Chemistry, University of Southern California, Los Angeles, CA
90089, USA
‡USC Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA
§Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5
∥Departments of Molecular Genetics and Biochemistry, University of Toronto, Toronto, Ontario, Canada
⊥Department of Chemistry and Chemical Biology, Cornell University, NE-CAT, Argonne, IL 60439, USA
KEYWORDS: rigidification, selectivity, kinase inhibitors, BRAF
ABSTRACT: One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has
been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate
sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive
RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based
inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further
application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF
kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation
of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the
optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAFV600E-harboring cell lines consistent with its ki-
nome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor
selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.
The RAF family kinases, consisting of ARAF, BRAF and
CRAF, are essential players in the RAS-RAF-MEK-ERK sig-
naling pathway (also known as the mitogen-activated protein
kinase pathway). They perform a central role in the regulation
of cell proliferation, differentiation, and survival.1 The RAF
family kinases are activated by the heterotypic or homotypic di-
merization of their kinase domains. The dysregulation of the di-
merization and activation of RAF kinases is a potent driver of
cancer and other diseases in human.2 The most common onco-
genic mutation V600E, present in 8% of all human cancers,3
constitutively activates BRAF to drive the proliferation of a va-
riety of cancers including melanoma, colorectal cancer and thy-
roid cancer.4 In particular, nearly half of human melanomas are
driven by the BRAFV600E mutant.5
driven melanomas leading to their approval by FDA for use in
the clinic (Figure 1).10
CH3
O
S
HN
F
O
O
H3
C
O
Cl
O
N
H
N
Cl
NH
F
N
O
N
CF3
N
H
H
Sorafenib
Vemurafenib
H2N
N
F
S
N
O
N
S
O
F
H
N
F
H3C
H3C CH3
Dabrafenib
Small-molecule RAF inhibitors have long been pursued as po-
tential anti-cancer drugs. For example, sorafenib was initially
developed as a RAF inhibitor over two decades ago (Figure 1)6,
7 was later shown to inhibit a variety of receptor tyrosine kinases
in addition to RAF kinases.8 This multi-targeted characteristic
of sorafenib is thought to underlie its clinical efficacy against
kidney and liver cancers but its lack of specificity (and other
liabilities) has limited its broad utility against other cancers such
as melanomas.9 RAF inhibitors developed more recently,
namely the sulfonamides vemurafenib and dabrafenib, display
Figure 1. Chemical structures of exemplary RAF inhibitors.
In contrast to sorafenib, which targets the dimeric active state
of BRAF that is generally similar to other protein kinases,
vemurafenib and dabrafenib exhibits higher selectivity by tar-
geting the monomeric inactive conformation of RAF defined by
a laterally displaced helix αC.11, 12 This exploited feature is not
a generally shared characteristic of kinases in their inactive
states.13, 14 Despite their great initial efficacy, resistance to
vemurafenib and dabrafenib develops rapidly in melanoma
greatly improved specificity and efficacy against BRAFV600E
-
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patients. Because sulfonamide inhibitors share a highly related
binding mode, resistance to one inhibitor would be predicted to
engender resistance to all related sulfonamide chemotypes.
Thus having inhibitors of RAF that are selective and yet target
alternate conformations of the kinase domain would have value
because they might overcome certain vemurafenib-resistant
mutations.
Page 2 of 8
yield intermediates 4a-e and 4i-j.20 Compound 4f was made by
coupling 3-(trifluoromethyl)phenylboronic acid with 5-bromo-
naphthalen-1-amine in the presence of cupric acetate and tri-
ethylamine in DCM.21 To prepare the quinazoline-containing
intermediate 4g, a milder condition involving isopropanol as
solvent at room temperature was used.18 The oxygen-linked in-
termediate 4h was synthesized by refluxing 3-(trifluorome-
thyl)phenol, 5-bromo-1-chloroisoquinoline and K2CO3 in ace-
tonitrile.22
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We recently developed a rigidification strategy to improve in-
hibitor selectivity for the protein kinase BRAF. The first imple-
mentation of this strategy to a class of pyrazolopyrimidine-
based (non-sulfonamide) kinase inhibitors led to a moderate in-
crease of selectivity.15 Herein, we report that a further imple-
mentation of the strategy to non-sulfonamide pyrazolo[3,4-
d]pyrimidine-based inhibitors afforded a dramatic increase in
selectivity for RAF kinases. Structural analysis of an optimized
inhibitor confirmed our design hypothesis by revealing a bind-
ing mode that engages a dimer active-like conformation of the
BRAF kinase domain. Interestingly, the optimized molecule in-
hibited the proliferation of cancer cells driven by BRAFV600E but
also the proliferation of a cancer cell line NRASQ61R, that is nor-
mally insensitive to sulfonamides.
The precursors 5a-c were prepared using reported procedures
with slight modifications.23 3-Iodo-1H-pyrazolo[3,4-d]pyrim-
idin-4-amine was first treated with alkyl halides to install alkyl
groups at the 1 position (Scheme S2). The resulting intermedi-
ates underwent Sonogashira coupling reactions with trime-
thylsilyl acetylene and then deprotection to produce 5a-c. In the
final step, coupling of the alkyne intermediates 5a-c with the
aryl halides 4a-j afforded the final compounds 2a-l (Scheme
1).15
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We previously developed 3-alkynyl-pyrazolo[3,4-d]pyrim-
idines, exemplified as compound 1 (Figure 2), as inhibitors of
Bcr-Abl.16 Although shown to be a potent inhibitor of Bcr-Abl,
compound 1 inhibited many additional kinases at similar con-
centrations. Since our previous docking studies predicted that
the benzamide groups in compound 1 was largely situated in
one plane,16 we hypothesized that changing this group into an
isoquinoline ring (Figure 2) would introduce rigidity into the
inhibitor, resulting in preferential inhibition of conformation-
ally more flexible kinases within the kinome.15
Scheme 1. Reagents and conditions: (a) aniline (3-(trifluorome-
thyl)aniline for 4a, 4-chloroaniline for 4b, 4-chloro-3-(trifluo-
romethyl)aniline for 4c, 3-(4-methyl-1H-imidazol-1-yl)-5-(tri-
fluoromethyl)aniline for 4d, 4-methyl-3-(trifluoromethyl)ani-
line for 4e), n-butanol, reflux; (b) 3-(trifluoromethyl)phenyl-
boronic acid, Cu(OAc)2, TEA, DCM; (c) 3-(trifluorome-
thyl)aniline, isopropanol, r.t.; (d) 3-(trifluoromethyl)phenol,
K2CO3, CH3CN, reflux; (e) aniline (3-(trifluoromethyl)aniline
for 4h and 4-chloro-aniline for 4j), n-butanol, reflux; (f)
(PPh3)2PdCl2, CuI, DIPEA, DMF, 80 °C, N2 (for 4a-g );
(PPh3)4Pd, CuI, TEA, DMF, r.t., N2 (for 4i,j).
Figure 2. Cyclization of the amidobenzene group in a previ-
ously described Bcr-Abl inhibitor 1 yields compounds 2.
To further improve kinase selectivity, a new series of alkyne-
linked naphtyl, isoquinoline, or quinazoline pyrazolopyrim-
idines were designed (Figure 2) and synthesized (Scheme 1).
The precursor compounds 3a-d were either commercially avail-
able or synthesized based on previous literature methods. 3a
was synthesized via N-oxidation of 5-bromoisoquinoline with
mCPBA followed by chlorination with phosphorus oxychlo-
ride.17 3c was prepared by cyclization of 2-amino-3-bromoben-
zoic acid in formamide followed by chlorination with phospho-
rus oxychloride.18 A precursor of 3d, 5-iodo-6-methylisoquino-
line, was made by a sequence of transformations including ni-
tration, reduction and diazotization-iodination of 6-methyli-
soquinoline,19 and then converted into 3d using similar methods
to those used for preparing 3a (Scheme S1).
We chose 2a as a representative of the panel of synthesized
compounds (Table 1) because of its closest structural similarity
to the parent compound 1, and evaluated its kinome inhibition
profile against 245 protein kinases in comparison with 1 (Table
S1). Consistent with our previous reported profiling results
against a smaller panel of kinases,24 the selectivity of 1 was ra-
ther poor, inhibiting 42 kinases in the panel by over 85% activ-
ity at 1 µM. In contrast, compound 2a showed drastically im-
proved selectivity, inhibiting only BRAF and CRAF, by over
85% activity (Figure 3). Hence a rigidification strategy imple-
mented via ring closure here greatly improved the specificity
characteristics of the promiscuous inhibitor 1.
The intermediates 4, except 4f, were synthesized from com-
pounds 3 using nucleophilic aromatic substitution reactions. 3a
and 3d, the isoquinoline-containing precursors, were reacted
with different anilines in n-butanol under reflux condition to
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on the proliferation of A375 cells harboring the BRAFV600E mu-
tant (Table 1).26 Notably, 2l, the most potent inhibitor of
BRAFV600E of the panel in vitro, also displayed the highest po-
tency at inhibiting the proliferation of A375 cells, with an IC50
of 417 nM, only 3-fold worse than vemurafenib (IC50 = 127
nM). The diminished potency of 2l relative to vemurafenib in
cells was not unexpected considering that as an FDA-approved
drug vemurafenib has been greatly optimized for permeability
and bioavailability. In conclusion, the optimized compound, 2l,
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was effective at suppressing the proliferation of BRAFV600E
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driven cells, in agreement with its potent inhibition of
BRAFV600E in vitro.
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We next compared the effects of 2l with vemurafenib on down-
stream signaling in A375 cells. In these cells, expression of the
active mutant BRAFV600E leads to constitutively elevated levels
of phospho-ERK (pERK), which provides a useful marker for
effects on pathway signaling. In agreement with a previous re-
port25, vemurafenib repressed pERK levels with an IC50 of 80
nM, whereas 2l inhibited phospho-ERK levels with an IC50 of
420 nM (Figure 4). These results were consistent with the rel-
ative potency of 2l in the A375 cell proliferation assay (Table
1). In sum, our data showed we could use a rigification strategy
to design a highly specific inhibitor that was both potent in vitro
and in A375 cells.
Figure 3. Kinome inhibition profiles of compounds 1 and
2a illustrated in a phylogenetic tree format. The inhibitors were
screened against 245 kinases at 1 µM. The kinases that were
>85% inhibited by 1, 2a, 2l are colored blue, red, and orange,
respectively, on the kinome trees.
Since the kinome profiling experiment showed that compound
2a possessed an improved specificity profile for RAF kinases,
we next sought to explore the activity characteristics of the full
compound series 2a-l against BRAF, with the goal of identify-
ing a specific lead with greatest potency. To this end we per-
formed dose-response analyses against BRAFV600E activity in
vitro (Table 1). Vemurafenib, employed as a control, displayed
an IC50 value of 21 nM, consistent with the value reported pre-
viously.25 Compounds 2a, 2c, 2i, and 2j inhibited BRAFV600E
with IC50 values ranging between 21 nM to 140 nM while com-
pounds 2b, 2d, 2e, 2f, 2g, 2h and 2k inhibit BRAFV600E with
IC50 values greater than 300 nM (Figure S1). The SAR analysis
indicated that a para-chloro substituent on the anilino moiety
(as in 2c) confers a 4-fold increase in affinity relative to a meta-
trifluoromethyl substituent (as in 2a). Furthermore, a methyl
group at position 6 of the isoquinoline ring confers a 7-fold in-
crease in affinity (comparing 2a with 2i) while an ethyl group
at position 1 of the pyrazolopyrimidine relative to an iso-propyl
group confers a 3-fold increase in affinity to BRAF (comparing
2a and 2j). Gratifyingly, when the favored substituents at each
position were combined, the resulting compound 2l exhibited
the highest potency in the series with an IC50 of 8 nM against
BRAFV600E. This corresponded to an 18-fold increase in affinity
relative to 2a. Notably, the potency of 2l was over two-fold
more potent than vemurafenib in the same assay.
2l
VEM
µM
pERK
ERK
Figure 4. Effects of vemurafenib and compound 2l on down-
stream signaling of BRAFV600E in A375 cells. Vemurafenib and
2l inhibited the phosphorylation of ERK with IC50 values of 80
nM and 420 nM, respectively.
To determine the binding mode of 2l, we solved its co-structure
in complex with the BRAF kinase domain to 3.05Å resolution
(Rwork/Rfree= 23.5/27.5; Table S2). Unbiased electron density in
the ATP binding pocket of each kinase domain allowed unam-
biguous modeling of 2l in BRAF (Figure 5).
The kinase domain of RAF adopted the characteristic side-to-
side dimer configuration reflective of a kinase active-like state
(Figure S2A). This configuration, defined by a productive in-
ward conformation of helix αC is also notably preferred by the
poorly selective, non-sulfonamide inhibitor sorafenib and con-
trasts with the monomeric inactive-like conformation of the
BRAF kinase domain engaged by sulfonamide inhibitors,
which display a laterally displaced helix αC (Figure 5B). 2l is
stabilized in the ATP binding pocket by a mixture of hydrogen
bonds and hydrophobic interactions (Figure 5C; D).
We next profiled 2l at 1 µM against 245 kinases as performed
for 1 and 2a, and observed a slight reduction of selectivity
against RAFs with just four kinases, specifically BRAF, CRAF,
BRK and FMS, inhibited by more than 85% (Figure 3; Table
S1). Importantly, this result demonstrated that the specificity
characteristics arising from our original rigidification strategy
were largely maintained following lead optimization.
Having shown that a few rigidified inhibitors were both specific
and potent at inhibiting BRAF in vitro, we next investigated the
effect of the five most potent inhibitors (2a, 2c, 2i, 2j, and 2l)
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Table 1: Inhibitory activity of compounds 2a-l against BRAF V600E and in vitro and in cells.
1
2
3
4
5
6
7
8
9
IC50(nM)
Compounds
Aryl
R1
R2
X1
X2
X3
BRAF
A375b
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V600Ea
(BRAFV600E
)
2a
2b
2c
2d
i-Pr
i-Pr
i-Pr
i-Pr
H
H
H
H
NH
NH
NH
NH
N
CH
N
CH
CH
CH
CH
140±28
>300
>10,000
Ndc
>10,000
Ndc
29±10
>300
N
2e
i-Pr
H
NH
N
N
>300
Ndc
2f
2g
i-Pr
i-Pr
i-Pr
i-Pr
Et
H
H
NH
NH
O
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
>300
>300
>300
21±6
41±6
>300
8±1
Ndc
Ndc
2h
2i
H
Ndc
CH3
H
NH
NH
NH
NH
869±79
>10,000
Ndc
2j
2k
2l
n-Pr
Et
H
CH3
417±39
127±3
Vem
21±7
aIC50 values were determined by following the in vitro kinase assay protocols. The data represent the mean values of two inde-
pendent experiments.
bGrowth inhibition was determined using MTT assay. A375 cells were plated in 96-well plates at 1×103 cells per well and incu-
bated with different concentrations of the inhibitor for 96 h. The data represent the mean values of two independent experi-
ments, each carried out in triplicates.
cNd: not determined
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Specifically, the exocyclic amino group (N4) and the N5 atom
group of 2l occupies the hydrophobic pocket conventionally oc-
cupied by the phenyl ring of Phe595, resulting in an outward
shift of the DFG loop that is characteristic of a type-II inhibitor
binding mode (Figure 5C). The crystal structure of the
BRAF:2l complex also nicely explain the structure-activity re-
lationship for the compound 2 series observed above. For ex-
ample, the substitution of isoquinoline nitrogen by a carbon (as
in 2b) is unfavorable as it is expected to impair the hydrogen
bond with the carboxyl backbone of Asp594 based on our crys-
tal structure.
1
2
3
4
5
6
7
8
of the pyrazolo[3,4-d]pyrimidine ring form hydrogen bonds
with the carboxyl and the amide backbone of Gln530 and
Cys532 from the hinge region, respectively. The pyrazolopy-
rimidine core is stabilized by hydrophobic interactions with the
side chain of Phe583, Phe595, Trp531, and Ala481. The ethyl
group at 1 position is poised to form hydrophobic interactions
with the side chain of Ile463. The isoquinoline is stabilized by
hydrophobic interactions with the Thr529, Ala481, Ile527,
Lys483 and Leu514 as well as a hydrogen bond with the car-
boxyl backbone of Asp594 (part of the DFG loop). The aryl
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Figure 5. The X-ray crystal structures of BRAF protein in complex with 2l. (A) Unbiased Fo−Fc electron density map contoured at
2.0σ in green showing 2l binding in the ATP pocket of the kinase domain of BRAF. (B) Comparison of the BRAF:2l complex
structure with (on left panel) the BRAF:sorafenib complex structure (PDB:1UWH) illustrating the dimeric ON-state conformation of
the RAF kinase and with (right panel) the BRAF:PLX4720 complex structure (PDB:4WO5) illustrating the monomeric OFF-state
conformation of the RAF kinase. Lower panel represents a close-up view of the inward and outward conformations of the αC helix.
(C) Stick representation of 2l bound to BRAF kinase domain. BRAF residues interacting with 2l are represented in stick. Yellow dash
lines represent hydrogen bonds. (D) Flat schematic representation of the binding mode of 2l with BRAF. Black sticks represent
hydrophobic interaction and yellow dash lines represent hydrogen bonds.
The binding mode of 2l revealed by our structure analysis im-
plied that, similar to other inhibitors that preferentially bind to
the dimer state of RAF, that it might also promote kinase do-
main dimerization. To test this hypothesis further, we per-
formed analytical ultracentrifugation analyses with recombi-
nant BRAF kinase in the presence or absence of 2a (Figure
S2B). We observed that at the concentration tested, recombi-
nant BRAF kinase sedimented predominantly as a monomer in
absence of inhibitor while in the presence of saturating concen-
tration of 2a, BRAF sedimented mostly as a dimer. These re-
sults showed that 2a promotes BRAF kinase dimerization. As
suggested by our crystal structure showing 2l bound to the di-
mer state of BRAF and based on the similar structure of
compounds 2a and 2l, we predict that 2l also promotes BRAF
kinase domain dimerization.
To further evaluate the potency and selectivity of 2l at inhibiting
BRAF, it was screened against a collection of 57 distinct cancer
cell lines at the National Cancer Institute according to the re-
ported protocol (http://dtp.nci.nih.gov) (Table S3). Im-
portantly, among the 57 cell lines, 10 harbor the BRAFV600E mu-
tation. The profiling data showed that 2l at 10 µM inhibited the
proliferation of the diverse cell lines to various degrees. Notably
3 out of the 5 most sensitive cell lines (MALME-3M, MDA-
MB-435 and SK-MEL-5) corresponded to melanoma cells har-
boring the BRAFV600E mutation (Figure 6). Further studies con-
firmed that 2l inhibited the proliferation of these three cell lines
in a dose-dependent manner with Total Growth Inhibition (TGI)
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values of 7.1, 4.6, and 6.6 µM respectively. Interestingly, one
Corresponding Author
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2
3
4
5
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7
of the most sensitive cell lines in the panel, SK-MEL-2, is
known to harbor a mutant NRAS (Q61R). As it has been estab-
lished that elevated RAS activity drive RAF dimerization in
cells, these data indicate that 2l might inhibit RAF dimers under
certain cellular settings.
* E-mail: zhang.chao@usc.edu
Author Contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
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9
# These authors contributed equally to this work as first authors.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank N. Graham and A. Delfarah for help with HR-MS
data collection, National Cancer Institute for screening the com-
pounds against cancer cell lines, and the Northeastern Collabo-
rative Access Team beamlines for conducting diffraction work
(NIH P41 GM103403 and S10 RR029205). This work was sup-
ported by National Science Foundation (CHE-1455306), Cana-
dian Cancer Society Research Institute (704116), Canada Re-
search Chair Program, and Canadian Institutes for Health Re-
search (FDN143277). A.A. was supported by Stauffer Endowed
Fellowship by USC Graduate School. C.Y. was supported by
USC Dornsife Chemical Biology Training Program (T32). P.M.
was supported by a TD Bank postdoctoral fellowship.
Figure 6. Compound 2l was profiled for antiproliferative ef-
fects against diverse cancer cell lines. (A) 2l at 10 µM preferen-
tially inhibited the proliferation of a few out of a collection of
57 distinct cell lines. (B) 2l inhibited dose-dependent growth
inhibition of MALME-3M, MDA-MB-435, and SK-MEL-5
ABBREVIATIONS
cells. * indicates sensitive cell lines bearing BRAFV600E
.
RAF, rapidly accelerated fibrosarcoma; RAS, rat sarcoma;
MEK, mitogen activated protein kinase; ERK, extracellular sig-
nal-regulated kinase; TGI, total growth inhibition.
In summary, we have demonstrated that using a strategy of in-
hibitor rigidification, via ring closure, can lead to a dramatic in-
crease in selectivity towards RAF kinases. Our structural and
biochemical characterizations confirm that the rigidified inhib-
itors interact with BRAF kinase domains with a type-II binding
mode that can drive RAF dimerization in vitro. The selectivity
of our inhibitors reported here for RAF kinases is unprece-
dented for non-sulfonamide RAF inhibitors. We posit that, a ri-
gidification strategy could be readily applied to other RAF in-
hibitor scaffolds as a potential means to generate second-gener-
ation targeted therapies where first-generation inhibitors fail to
function. Furthermore, rigidification could be a general strategy
for enhancing inhibitor selectivity against other protein kinases,
which may open up therapeutic opportunities not afforded by
other approaches.
REFERENCES
1.
J.; Rapp, U. R. The ins and outs of Raf kinases. Trends Biochem Sci
1994, 19, 474-80.
2.
signalling pathways in cancer. Oncogene 2007, 26, 3279-90.
3. Bond, C. E.; Whitehall, V. L. How the BRAF V600E
Daum, G.; Eisenmann-Tappe, I.; Fries, H. W.; Troppmair,
Dhillon, A. S.; Hagan, S.; Rath, O.; Kolch, W. MAP kinase
Mutation Defines a Distinct Subgroup of Colorectal Cancer: Molecular
and Clinical Implications. Gastroenterology research and practice
2018, 2018.
4.
Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.;
Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.;
Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes,
R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.;
Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson,
B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner, D. D.;
Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W.; Leung,
S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson,
H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal,
P. A. Mutations of the BRAF gene in human cancer. Nature 2002, 417,
949-54.
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org
5.
Mann, M. B.; Black, M. A.; Jones, D. J.; Ward, J. M.; Yew,
Supplementary Figures and Tables, experimental methods, X-
ray crystallography data, synthetic procedures, and compound
characterization data
C. C.; Newberg, J. Y.; Dupuy, A. J.; Rust, A. G.; Bosenberg, M. W.;
McMahon, M.; Print, C. G.; Copeland, N. G.; Jenkins, N. A.
Transposon mutagenesis identifies genetic drivers of Braf(V600E)
melanoma. Nat Genet 2015, 47, 486-95.
Accession codes. Protein Data Bank code for BRAF-2l com-
plex structure is 6NSQ.
6.
Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas,
J.; Smith, R. A.; Schwartz, B.; Simantov, R.; Kelley, S. Discovery and
development of sorafenib: a multikinase inhibitor for treating cancer.
Nature reviews Drug discovery 2006, 5, 835.
AUTHOR INFORMATION
6
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
7.
Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-
drug resistance of Bcr-Abl Kinase. Journal of medicinal chemistry
2015, 58, 9228-9237.
Duvaz, D.; Good, V. M.; Project, C. G.; Jones, C. M.; Marshall, C. J.;
Springer, C. J. Mechanism of activation of the RAF-ERK signaling
pathway by oncogenic mutations of B-RAF. Cell 2004, 116, 855-867.
1
2
17.
Kitade, M.; Yamashita, S.; Ohkubo, S. Bicyclic compound
or salt thereof. U.S. Patent 8,912,181, December 16, 2014. In.
3
4
5
6
7
8
9
8.
Wilhelm, S. M.; Adnane, L.; Newell, P.; Villanueva, A.;
18.
Kung, A.; Chen, Y.-C.; Schimpl, M.; Ni, F.; Zhu, J.; Turner,
Llovet, J. M.; Lynch, M. Preclinical overview of sorafenib, a
multikinase inhibitor that targets both Raf and VEGF and PDGF
receptor tyrosine kinase signaling. Molecular cancer therapeutics
2008, 7, 3129-3140.
M.; Molina, H.; Overman, R.; Zhang, C. Development of specific,
irreversible inhibitors for a receptor tyrosine kinase EphB3. Journal of
the American Chemical Society 2016, 138, 10554-10560.
19.
Smith, A. L.; DeMorin, F. F.; Paras, N. A.; Huang, Q.;
9.
Mousa, A. B. Sorafenib in the treatment of advanced
Petkus, J. K.; Doherty, E. M.; Nixey, T.; Kim, J. L.; Whittington, D.
A.; Epstein, L. F. Selective inhibitors of the mutant B-Raf pathway:
discovery of a potent and orally bioavailable aminoisoquinoline.
Journal of medicinal chemistry 2009, 52, 6189-6192.
hepatocellular carcinoma. Saudi journal of gastroenterology: official
journal of the Saudi Gastroenterology Association 2008, 14, 40.
10.
Van Allen, E. M.; Wagle, N.; Sucker, A.; Treacy, D. J.;
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
Johannessen, C. M.; Goetz, E. M.; Place, C. S.; Taylor-Weiner, A.;
Whittaker, S.; Kryukov, G. V.; Hodis, E.; Rosenberg, M.; McKenna,
A.; Cibulskis, K.; Farlow, D.; Zimmer, L.; Hillen, U.; Gutzmer, R.;
Goldinger, S. M.; Ugurel, S.; Gogas, H. J.; Egberts, F.; Berking, C.;
Trefzer, U.; Loquai, C.; Weide, B.; Hassel, J. C.; Gabriel, S. B.; Carter,
S. L.; Getz, G.; Garraway, L. A.; Schadendorf, D.; Dermatologic
Cooperative Oncology Group of, G. The genetic landscape of clinical
resistance to RAF inhibition in metastatic melanoma. Cancer Discov
2014, 4, 94-109.
20.
Chao, Q.; Deng, L.; Shih, H.; Leoni, L. M.; Genini, D.;
Carson, D. A.; Cottam, H. B. Substituted isoquinolines and
quinazolines as potential antiinflammatory agents. Synthesis and
biological evaluation of inhibitors of tumor necrosis factor α. Journal
of medicinal chemistry 1999, 42, 3860-3873.
21.
Chan, D. M.; Monaco, K. L.; Wang, R.-P.; Winters, M. P.
New N-and O-arylations with phenylboronic acids and cupric acetate.
Tetrahedron Letters 1998, 39, 2933-2936.
22.
Gharat, L. A.; Banerjee, A.; Khairatkar-Joshi, N.; Kattige,
11.
Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho,
V. G. Bicyclic compounds as mPGES-1 inhibitors. U.S. Patent
9,006,257, April 14, 2015. In.
23.
M.; Zhong, L.; Li, G.-B.; Zhang, R.-J.; Lin, W.-T.; Jiao, Y. Design,
synthesis, and structure–activity relationship studies of 3-
(Phenylethynyl)-1 H-pyrazolo [3, 4-d] pyrimidin-4-amine derivatives
as a new class of Src inhibitors with potent activities in models of triple
negative breast cancer. Journal of medicinal chemistry 2015, 58, 3957-
3974.
H.; Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G. Clinical efficacy of
a RAF inhibitor needs broad target blockade in BRAF-mutant
melanoma. Nature 2010, 467, 596.
Zhang, C.-H.; Zheng, M.-W.; Li, Y.-P.; Lin, X.-D.; Huang,
12.
Zhang, C.; Spevak, W.; Zhang, Y.; Burton, E. A.; Ma, Y.;
Habets, G.; Zhang, J.; Lin, J.; Ewing, T.; Matusow, B. RAF inhibitors
that evade paradoxical MAPK pathway activation. Nature 2015, 526,
583.
13.
Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.;
Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K. Discovery of a selective
inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.
Proceedings of the National Academy of Sciences 2008, 105, 3041-
3046.
24.
Liu, X.; Kung, A.; Malinoski, B.; Prakash, G. K.; Zhang, C.
Development of Alkyne-Containing Pyrazolopyrimidines To
Overcome Drug Resistance of Bcr-Abl Kinase. J Med Chem 2015, 58,
9228-37.
14.
Rheault, T. R.; Stellwagen, J. C.; Adjabeng, G. M.;
25.
Zhang, C.; Spevak, W.; Zhang, Y.; Burton, E. A.; Ma, Y.;
Hornberger, K. R.; Petrov, K. G.; Waterson, A. G.; Dickerson, S. H.;
Mook Jr, R. A.; Laquerre, S. G.; King, A. J. Discovery of dabrafenib:
A selective inhibitor of Raf kinases with antitumor activity against B-
Raf-driven tumors. ACS medicinal chemistry letters 2013, 4, 358-362.
Habets, G.; Zhang, J.; Lin, J.; Ewing, T.; Matusow, B.; Tsang, G.;
Marimuthu, A.; Cho, H.; Wu, G.; Wang, W.; Fong, D.; Nguyen, H.;
Shi, S.; Womack, P.; Nespi, M.; Shellooe, R.; Carias, H.; Powell, B.;
Light, E.; Sanftner, L.; Walters, J.; Tsai, J.; West, B. L.; Visor, G.;
Rezaei, H.; Lin, P. S.; Nolop, K.; Ibrahim, P. N.; Hirth, P.; Bollag, G.
RAF inhibitors that evade paradoxical MAPK pathway activation.
Nature 2015, 526, 583-6.
15.
Assadieskandar, A.; Yu, C.; Maisonneuve, P.; Liu, X.; Chen,
Y.-C.; Prakash, G. S.; Kurinov, I.; Sicheri, F.; Zhang, C. Effects of
rigidity on the selectivity of protein kinase inhibitors. European journal
of medicinal chemistry 2018, 146, 519-528.
26.
Poulikakos, P. I.; Zhang, C.; Bollag, G.; Shokat, K. M.;
16.
Liu, X.; Kung, A.; Malinoski, B.; Prakash, G. S.; Zhang, C.
Rosen, N. RAF inhibitors transactivate RAF dimers and ERK
signalling in cells with wild-type BRAF. Nature 2010, 464, 427-30.
Development of alkyne-containing pyrazolopyrimidines to overcome
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