Lead discovery of type II BRAF V600E inhibitors targeting the structurally validated DFG-Out conformation based upon selected fragments

Article abstract of DOI:10.3390/molecules21070879

The success of the first approved kinase inhibitor imatinib has spurred great interest in the development of type II inhibitors targeting the inactive DFG-out conformation, wherein the Phe of the DFG motif at the start of the activation loop points into t

Full text of DOI:10.3390/molecules21070879

molecules
Article
Lead Discovery of Type II BRAF V600E Inhibitors
Targeting the Structurally Validated DFG-Out
Conformation Based upon Selected Fragments
Qingwen Zhang 1,*, Xuejin Zhang 2 and Qidong You 3
1
Division of Medicinal Chemistry, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Road, Pudong,
Shanghai 201203, China
State Key Laboratory of Lead Compound Research, WuXi AppTec, 288 Fute Zhong Road,
2
Waigaoqiao Pilot Free Trade Zone, Pudong, Shanghai 200131, China; cherryjin1031@gmail.com
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
3
24 Tongjia Xiang, Gulou, Nanjing 210009, Jiangsu, China; youqd@163.com
*
Correspondence: chembiomed@163.com; Tel.: +86-21-2057-2000
Academic Editors: Raymond S. Norton and Martin J. Scanlon
Received: 29 March 2016; Accepted: 28 June 2016; Published: 16 July 2016
Abstract: The success of the first approved kinase inhibitor imatinib has spurred great interest in
the development of type II inhibitors targeting the inactive DFG-out conformation, wherein the Phe
of the DFG motif at the start of the activation loop points into the ATP binding site. Nevertheless,
kinase inhibitors launched so far are heavily biased toward type I inhibitors targeting the active
DFG-in conformation, wherein the Phe of the DFG motif flips by approximately 180˝ relative to the
inactive conformation, resulting in Phe and Asp swapping their positions. Data recently obtained
with structurally validated type II inhibitors supported the conclusion that type II inhibitors are
more selective than type I inhibitors. In our type II BRAF V600E inhibitor lead discovery effort, we
identified phenylaminopyrimidine (PAP) and unsymmetrically disubstituted urea as two fragments
that are frequently presented in FDA-approved protein kinase inhibitors. We therefore defined
PAP and unsymmetrically disubstituted urea as privileged fragments for kinase drug discovery.
A pharmacophore for type II inhibitors, 4-phenylaminopyrimidine urea (4-PAPU), was assembled
based upon these privileged fragments. Lead compound SI-046 with BRAF V600E inhibitory
activity comparable to the template compound sorafenib was in turn obtained through preliminary
structure–activity relationship (SAR) study. Molecular docking suggested that SI-046 is a bona fide
type II kinase inhibitor binding to the structurally validated “classical DFG-out” conformation of
BRAF V600E. Our privileged fragments-based approach was shown to efficiently deliver a bona fide
type II kinase inhibitor lead. In essence, the theme of this article is to showcase the strategy and
rationale of our approach.
Keywords: fragment-based lead discovery; type II kinase Inhibitor; privileged fragments; classical
DFG-out conformation; BRAF V600E
1. Introduction
The protein kinase complement of the human genome, the human kinome, encodes about
518 protein kinases which play pivotal roles in virtually all aspects of cellular processes [
Dysregulation of protein kinase function has been implicated in many human diseases such as cancer,
and inflammatory and autoimmune diseases [ ]. So far, the US FDA has approved 30 small molecule
1,2].
3
protein kinase inhibitors, mainly for cancer indications [4].
Protein kinase inhibitors have been generally categorized into four classes—type I, type II, type III,
and type IV—based upon their binding mode [
5–
8
]. Type I inhibitors bind to the active DFG-in
Molecules 2016, 21, 879; doi:10.3390/molecules21070879
www.mdpi.com/journal/molecules

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conformation, wherein the Asp of the DFG motif at the start of the activation loop points into the ATP
binding site. Type I inhibitors are ATP-competitive and represent the majority of currently approved
kinase inhibitors. Type II inhibitors bind to the inactive DFG-out conformation, wherein the Asp of
the DFG motif flips by approximately 180˝ relative to the active conformation, resulting in Asp and
Phe swapping their positions. Type II inhibitors occupy a characteristic hydrophobic pocket adjacent
to the ATP binding pocket, which is only accessible in the DFG-out conformation. Type II inhibitors
can be either ATP-competitive or not, depending on whether they extend past the gatekeeper into the
adenine pocket and form hydrogen bonds with the hinge residues. Type III and Type IV inhibitors
are not ATP-competitive. Type III inhibitors bind to an allosteric pocket opposite the ATP binding
pocket and is also known to induce conformational changes in the activation loop, forcing the αC
helix to adopt an inactive conformation. They do not form any hydrogen bonding interaction with the
hinge residues. Type IV inhibitors bind to any allosteric sites distant from the ATP binding pocket and
induce conformational changes that render the kinase inactive.
The serendipitous discovery of imatinib binding to the ABL DFG-out conformation has spurred
great interest in the development of type II inhibitors targeting the inactive kinase conformation [9–11].
One underlying view was that type II inhibitors are intrinsically more selective than type I inhibitors
based on the observation that the residues surrounding the hydrophobic pocket exposed in the
DFG-out conformation are not as highly conserved as those in the ATP binding pocket [
it was originally thought that many kinases are unable to adopt the inactive DFG-out conformation [
However, this view was challenged by Zhao et al. who demonstrated that 220 kinases can be targeted
with a small library of 36 type II inhibitors [ ]. Nevertheless, Vijayan et al. had recently obtained
4]. In addition,
4
].
7
profiling results for structurally validated type II inhibitors identified through conformational analysis
and reached the conclusion that type II inhibitors are statistically significantly (p < 10´4) more selective
than type I inhibitors [6].
It has been established that the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK)
signaling pathway is essential to cellular growth and survival [12]. Constitutive activation resulting
from mutations in this pathway impacts approximately one-third of human cancers [13]. BRAF (V-RAF
murine sarcoma viral oncogene homologue B1) is a serine/threonine kinase that functions in this
pathway as a downstream effector of RAS. Its mutant BRAF V600E has proven to be a highly
tractable target in this cascade for cancer therapy [14]. FDA has already approved four BRAF
V600E inhibitors, namely, vemurafenib (Zelboraf, 2011) [15], dabrafenib (Tafinlar, 2013) [16], sorafenib
(Nexavar, 2005) [17], and regorafenib (Stivarga, 2012) [18]. Vemurafenib and dabrafenib are type I
inhibitors, while sorafenib and regorafenib are type II inhibitors.
Although there are still controversies regarding the relative merits of type I and type II kinase
inhibitors, the fact is that launched products are heavily biased toward type I inhibitors. This is
the reason why we choose to target DFG-out conformation in our effort to discover lead for BRAF
V600E inhibition. We believe that type II inhibitors research is still a vibrantly developing and highly
rewarding field for kinase drug discovery [19,20].
2. Results and Discussion
Phenylaminopyrimidine (PAP), 4-anilinoquinazoline, and unsymmetrically disubstituted urea
are identified as fragments that are frequently presented in 30 FDA-approved small molecule protein
kinase inhibitors. PAP is presented in five (17%) launched protein kinase inhibitors (imatinib,
nilotinib, pazopanib, ceritinib, and osimertinib) (Figure 1), 4-anilinoquinazoline is presented in
five launched products (17%) (gefitinib, erlotinib, lapatinib, vandetanib, and afatinib) (Figure 2),
while unsymmetrically disubstituted urea is presented in three launched products (10%) (sorafenib,
regorafenib, and lenvatinib) (Figure 3). It is noteworthy that 4-anilinoquinazoline contains PAP in its
skeleton, with PAP presented in 34% of approved protein kinase inhibitors. We therefore defined PAP
and unsymmetrically disubstituted urea as privileged fragments for kinase drug discovery.

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Figure 1. FDA-approved kinase inhibitors containing phenylaminopyrimidine (PAP). The PAP
fragments are colored red. INN, brand name, year FDA approved, and main target kinases are
provided for each kinase drug. Kinase abbreviations: ABL: Abelson kinase; KIT: stem cell factor
receptor; PDGFR: platelet derived growth factor receptor; VEGFR: vascular endothelial growth factor
receptor; ALK: anaplastic lymphoma kinase; EGFR: epidermal growth factor receptor.
Figure 2. FDA-approved kinase inhibitors containing 4-anilinoquinazoline. The contained PAP
fragments are colored red. INN, brand name, year FDA approved, and main target kinases are
provided for each kinase drug. Kinase abbreviations: HER2 (ERRB2): erythroblastic leukemia viral
oncogene homolog 2; RET: rearranged during transfection; ERRB4: erythroblastic leukemia viral
oncogene homolog 4.

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Figure 3. FDA-approved kinase inhibitors containing unsymmetrically disubstituted urea. The urea
fragments are colored green. INN, brand name, year FDA approved, and main target kinases
are provided for each kinase drug. Kinase abbreviations: RAF: rapidly growing fibrosarcoma;
FLT3: Fms-like tyrosine kinase 3; FGFR: fibroblast growth factor receptor; TIE2: tyrosine kinase
with immunoglobulin and epidermal growth factor homology domains 2.
Thus, we were prompted to design type II BRAF V600E inhibitors based upon privileged
fragments of PAP and unsymmetrically disubstituted urea. Sorafenib was employed as the template
type II inhibitor which traps the structurally validated classical DFG-out conformation [
6] of BRAF
V600E (PDB code 1UWJ) [21].
After changing the O linkage to NH and displacing 2-carboxamidopyridinyl with 4-pyrimidinyl,
we traveled from sorafenib to a scaffold that fuses the two privileged fragments into one
pharmacophore, which we coined 4-phenylaminopyrimidine urea (4-PAPU) (Figure 4). Importantly,
the 4-PAPU scaffold fits the generalized pharmacophore model of type II inhibitors: the 4-pyrimidinyl
hinge-binding moiety (HBM) is connected through a nitrogen atom to the central phenyl that is
expected to occupy the DFG-out pocket (BPII). The urea functionality not only links the central phenyl
and the terminal phenyl, but also serves as the hydrogen bond donor/acceptor to interact with the
conserved glutamic acid in the
the terminal phenyl occupy the lipophilic pockets created by the DFG-out flip (BPIII and BPIV) [22
α
C helix and aspartic acid in the DFG motif. Substitutes R1 and R2 on
23].
,
In our preliminary SAR campaign, a focused compound library based upon the pharmacophore
4-PAPU was synthesized and tested in a biochemical assay. It was found that the lead compound
SI-046 (IC50 298 nM) exhibits BRAF V600E inhibitory activity comparable to the template compound
sorafenib (IC50 263 nM). SI-008 (IC50 685 nM) is moderately active, while SI-098 (IC50 > 10,000 nM) is
devoid of activity (Figure 5) (Table 1). The GlideScores of selected compounds generated with Glide,
version 6.9 (Schrödinger, LLC, New York, NY, USA, 2015), which agrees with the biochemical results,
are also presented in Table 1.
Figure 4. Evolution of type II kinase inhibitor pharmacophore 4-PAPU based upon privileged fragments
of PAP (colored red) and unsymmetrically disubstituted urea (colored green). Potential hydrogen bond
interactions are depicted as dashed lines.

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Figure 5. Selected compounds from the focused library based upon the pharmacophore 4-PAPU. PAP is
colored red and unsymmetrically disubstituted urea is colored green. Molecular formula, molecular
weight, and CLogP are generated with ChemDraw.
Table 1. BRAF V600E inhibitory activity and GlideScore of selected compounds from the focused
library based upon the pharmacophore 4-PAPU.
Compound
IC50 (nM)
GlideScore (kJ/mol)
SI-046
SI-008
SI-098
298
685
>10,000
263
11.3
10.3
10.2
12.1
sorafenib
Vijayan et al. [6] labeled those conformations for which D1 < 7.2 Å and D2 > 9 Å as “classical
DFG-out” conformations. Those conformations that do not satisfy their structural definition of
“classical DFG-out” were labeled “nonclassical DFG-out,” which cannot accommodate a type II
inhibitor. D1 is the DFG Phe to Asn at HRD + 5 distance, while D2 is the DFG Phe to salt-bridge Glu
distance. We measured the distance criteria D1 (5.4 Å) and D2 (11.6 Å) and confirmed that sorafenib
binds to the classical DFG-out conformation of BRAF-V600E (PDB code 1UWJ) (Figure 6a). This agrees
with the literature [6]. Molecular modeling was performed to establish the binding mode of selected
title compounds. Docking of SI-046 in the above-mentioned classical DFG-out conformation of BRAF
V600E bound to sorafenib is depicted in Figure 6b. SI-046 was shown to be bound to the DFG-out
conformation of BRAF V600E. Its morpholino group binds in the hydrophobic pocket surrounded
by Trp530, Cys531, and Phe582 and forms an H-bond with the backbone amide NH of Cys531 in the
hinge region. The meta-disubstituted phenyl linking 2-methylpyrimidine ring through NH occupies
the hydrophobic pocket of the ATP binding site and interacts with the side chain of Lys482 through
a cation-p interaction. The side chain of Trp530 forms favorable p–p interaction (face-to-face) with
2-methylpyrimidine and p–p interaction (face-to-edge) with meta-disubstituted phenyl. Carbonyl and
two NHs of urea form H-bond interactions with the backbone of Asp593 and the side chain of Glu500,
respectively. Terminal 2-methyl-5-fluorophenyl extends into the hydrophobic pocket produced by the
flip of Phe594 and lined with Leu504, Thr507, Leu566, Ile512, lys600, and His573. However, when the
morpholino is displaced with 4-methylpiperazin-1-yl (SI-098), the interaction with the hinge region
disappears due to clashes caused by the positively charged nitrogen on the piperazine ring (Figure 6c).
Moreover, the higher absolute value of GlideScore of SI-046 compared with SI-098 indicated that
SI-046 forms more ideal interactions with the structurally validated DFG-out conformation of BRAF
V600E. This agrees well with the experimental results (Table 1).

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(a)
(b)
(c)
Figure 6.
(
a) The distance criteria D1 (5.4 Å) and D2 (11.6 Å) confirmed that sorafenib binds to the
classical DFG-out conformation of BRAF-V600E (PDB code 1UWJ). Docking of (
b
)
SI-046 and ( SI-098
c)
in the crystal structure of sorafenib bound to the classical DFG-out conformation of BRAF-V600E
(PDB code 1UWJ). Sorafenib is colored magenta. SI-046 and SI-098 are colored green. H-bonds are
depicted as orange dashed lines. Key interacting residues are illustrated in golden sticks.
In summary, our approach extracted two privileged fragments—phenylaminopyrimidine
(PAP) and unsymmetrically disubstituted urea—from FDA-approved small molecule protein kinase
inhibitors and assembled them into 4-PAPU, a pharmacophore for type II inhibitors. Lead compound
SI-046, which exhibited BRAF V600E inhibitory activity comparable to the template compound
sorafenib in the biochemical assay, was in turn identified through preliminary SAR study. Docking data
are consistent with the hypothesis that SI-046 targets the classical DFG-out conformation of the
oncogenic mutant BRAF V600E.

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3. Materials and Methods
3.1. Chemistry
The title compounds SI-046
,
SI-008, and SI-098 were prepared via the synthetic route outlined in
Scheme 1 according to known procedures [24,25].
Scheme 1. Synthesis of title compounds SI-046, SI-008 and SI-098.
Melting points were determined on a Tianjing YRT-3 melting point apparatus (Tianda Tianfa
1
Technology Co., Ltd., Tianjin, China) and were uncorrected. H-NMR spectra were recorded on a
Varian INOVA-400 spectrometer (Varian, Inc., Palo Alto, CA, USA) at 400 MHz. Chemical shifts (δ) are
reported in ppm relative to the DMSO-d6 signal (1H 2.50 ppm). Mass spectra were taken on a Waters
Micromass Q-Tof micro instrument, and ionization was positive ion electrospray (MS-ESI) (Waters
Corporation, Milford, MA, USA). Elemental analyses were performed on a Thermo SCIENTIFIC
FLASH 2000 Organic Elemental Analyzer (Thermo Fisher Scientific Inc., New York, NY, USA).
1-(5-Fluoro-2-methylphenyl)-3-(3-(2-methyl-6-morpholinopyrimidin-4-ylamino)phenyl)urea (SI-046)
A mixture of A2-CAR-2 [24] (0.73 g, 1.5 mmol), 5-fluoro-2-methylaniline (0.22 g, 1.76 mmol),
and triethylamine (0.46 g, 4.5 mmol) in anhydrous DMF (6 mL) under nitrogen was stirred at 40 ˝C
for 9 h. The resulting reaction mixture was diluted with methylene chloride (90 mL), then washed
consecutively with aqueous 0.5 mol/L sodium hydroxide (2
over anhydrous sodium sulfate, and evaporated in vacuo. The obtained residue was subjected to
ˆ
25 mL) and water (2
ˆ
25 mL), dried
silica gel column chromatography eluting with a gradient of ethanol in ethyl acetate (0%–16%, v/v)
to deliver SI-046 as a white solid (0.23g, 35% yield): m.p. 220.5–221.5 ˝C; 1H-NMR (DMSO-d6)
δ 8.93
(s, 1H, exchangeable), 8.70 (s, 1H, exchangeable), 8.59 (s, 1H, exchangeable), 7.73 (t, J = 2.0 Hz, 1H),
7.43 (dd, J = 2.0, 12.0 Hz, 1H), 7.13–7.17 (m, 3H), 7.00 (dd, J = 2.4, 8.0 Hz,1H), 6.95–6.97 (m, 1H), 5.95
(s, 1H), 3.66–3.68 (m, 4H), 3.46–3.48 (m, 4H), 2.31 (s, 3H), 2.16 (s, 3H); MS-ESI (m/z) 437.17 [M + H]+,
873.40 [2M + H]+; elemental analysis (C23H25FN6O2
62.62, 5.86, 18.84.
¨
0.25H2O) C, H, N, calcd. 62.64, 5.83, 19.06; found
1-(3-Chloro-4-fluorophenyl)-3-(4-(2-methyl-6-morpholinopyrimidin-4-ylamino)phenyl)urea (SI-008)
This compound was prepared from A1-CAR-2 26] (0.49 g, 1.0 mmol) and 3-chloro-4-fluoro
aniline (0.16 g, 1.1 mmol) in essentially the same way as that of SI-046 to deliver SI-008 as pale
[
˝
1
yellow crystalline powders (0.32g, 70% yield): m.p. 228.6–232.6 C; H-NMR (DMSO-d6)
δ 8.77
(s, 1H, exchangeable), 8.75 (s, 1H, exchangeable), 8.54 (s, 1H, exchangeable), 7.74 (d, J = 6.8 Hz, 1H),
7.29–7.45 (m, 6H), 5.73 (s, 1H), 3.65 (m, 4H), 3.42 (m, 4H), 2.29 (s, 3H); MS-ESI (m/z) 457.14 [M + H]+,
913.43 [2M + H]+; elemental analysis (C22H22ClFN6O2) C, H, N, calcd. 57.83, 4.85, 18.39, found 58.12,
5.16, 19.00.
1-(5-Fluoro-2-methylphenyl)-3-(3-(2-methyl-6-(4-methylpiperazin-1-yl)pyrimidin-4-ylamino)phenyl)urea (SI-098)
This compound was prepared from A2-CAR-3 [24] (0.75 g, 1.5 mmol) and 5-fluoro-2-methylaniline
(0.22 g, 1.76 mmol) in essentially the same way as that of ˝SI-046 to deliver SI-098 as pale yellow
1
crystalline powders (0.53g, 79% yield): m.p. 177.8–179.1 C; H-NMR (DMSO-d6)
δ
8.88 (s, 1H,
exchangeable), 8.71 (s, 1H, exchangeable), 8.59 (s, 1H, exchangeable), 7.75 (s, 1H), 7.45 (d, J = 12.0 Hz,
1H), 7.16 (m, 3H), 7.01 (s, 1H), 6.95 (s, 1H), 5.97 (s, 1H), 3.50 (m, 4H), 2.37 (m, 4H), 2.31 (s, 3H), 2.22 (s, 3H),

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2.17 (s, 3H); MS-ESI (m/z) 450.06 [M + H]+, 899.14 [2M + H]+; elemental analysis (C24H28FN7O) C, H,
N, calcd. 64.12, 6.28, 21.81, found 63.72, 6.28, 21.55.
3.2. Biochemical Assays
A LanthaScreen kinase assay (Invitrogen) was used to measure the potency of title compounds
against BRAF V600E. Shanghai ChemPartner Co., Ltd. (Pudong, Shanghai 201203, China) generated
the kinase inhibitory data.
3.3. Docking
All of the modeling calculations in this work were performed using Glide, version 6.9 (Schrödinger,
LLC, 2015) [27–29]. Before docking was carried out, the OPLS3 (Schrödinger, LLC, 2013) force
field [30,31] was used to model the ligand and protein, and their charges were assigned using the
Prime, version 4.2 (Schrödinger, LLC, 2015). The X-ray cocrystal structure of BRAF V600E (PDB code
1UWJ) was downloaded from RCSB Protein Data Bank. Water molecules and small inorganic ions
were removed. Default parameters in Glide 6.9 were used in this study. The ligand poses that Glide
generated passed through a series of hierarchical filters that evaluated the ligand’s interaction with the
target. The initial filters tested the spatial fit of the ligand to the defined active site. Poses that passed
these initial screens entered the final stage of energy minimization.
The Schrödinger’s proprietary GlideScore multi-ligand scoring function was used to score
the poses:
GScore “ 0.05 ˆ vdW ` 0.15 ˆ Coul ` Lipo ` Hbond ` Metal ` Rewards ` RotB ` Site (1)
where GScore stands for GlideScore, vdW stands for Van der Waals energy, Coul stands for Coulomb
energy, Lipo stands for Lipophilic term, HBond stands for Hydrogen-bonding term, Metal stands for
Metal-binding term, Rewards stands for various features such as buried polar groups, hydrophobic
enclosure, correlated hydrogen bonds, amide twists, and so on, RotB stands for Penalty for freezing
rotatable bonds, and Site stands for Polar interactions in the active site.
The best poses were selected for representation. This score is intended to be more suitable
for comparing the binding affinities of different ligands than is the “raw” Coulomb-van der Waals
interaction energy. GlideScore shows exceptionally high accuracy in ranking the binding modes of
the ligands.
4. Conclusions
Our privileged fragments-based approach was shown to efficiently deliver a bona fide type
II kinase inhibitor lead targeting the structurally validated DFG-out conformation. Furthermore,
the theme of this article is to showcase the strategy and rationale of our approach. Additional work
will be required to enhance the potency and assess the selectivity of these molecules to help answer
the question of whether type II inhibitors are more selective than type I. Finally, novel privileged
fragments and assembling strategies should be innovatively pursued to explore diversified and
emerging kinase targets.
Acknowledgments: We are grateful to the Basic Research Key Program of Shanghai Municipal (No. 09JC1413200)
and the National Science and Technology Major Project ‘Key New Drug Research and Development’
(No. 2009ZX09301-007) for financial support. Qingwen Zhang is indebted to the organizers of HLTH:
Fragment-based Lead Discovery (#145) at Pacifichem 2015.
Author Contributions: Qingwen Zhang and Qidong You conceived and designed the experiments;
Qingwen Zhang and Xuejin Zhang performed the experiments and analyzed the data; Qingwen Zhang wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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Abbreviations
The following abbreviations are used in this manuscript:
ATP
DFG
FDA
HRD
INN
PDB
SAR
adenosine triphosphate
Asp-Phe-Gly
Food and Drug Administration
His-Arg-Asp
International Nonproprietary Names
Protein Data Bank
structure-activity relationship
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