Discovery of Potent and Noncovalent Reversible EGFR Kinase Inhibitors of EGFRL858R/T790M/C797S

Article abstract of DOI:10.1021/acsmedchemlett.8b00564

In this paper, we describe the discovery and optimization of a series of noncovalent reversible epidermal growth factor receptor inhibitors of EGFR^{L858R/T790M/C797S}. One of the most promising compounds, 25g, inhibited the enzymatic activity of EGFR^{L858R/T790M/C797S} with an IC₅₀ value of 2.2 nM. Cell proliferation assays showed that 25g effectively and selectively inhibited the growth of EGFR^{L858R/T790M/C797S}-dependent cells. This series of compounds, which occupy both the ATP binding site and the allosteric site of the EGFR kinase, may serve as a basis for the development of fourth-generation EGFR inhibitors for L858R/T790M/C797S mutants.

Full text of DOI:10.1021/acsmedchemlett.8b00564

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of Potent and Noncovalent Reversible EGFR Kinase
Inhibitors of EGFRL858R/T790M/C797S
Qiannan Li,†,§ Tao Zhang,‡,§ Shiliang Li,†,§ Linjiang Tong,‡ Junyu Li,† Zhicheng Su,† Fang Feng,‡
Deheng Sun,† Yi Tong,† Xia Wang,† Zhenjiang Zhao,† Lili Zhu,† Jian Ding,‡ Honglin Li,*,† Hua Xie,*,‡
and Yufang Xu*,†
†Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China
‡Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: In this paper, we describe the discovery and
optimization of a series of noncovalent reversible epidermal
growth factor receptor inhibitors of EGFRL858R/T790M/C797S
.
One of the most promising compounds, 25g, inhibited the
enzymatic activity of EGFRL858R/T790M/C797S with an IC50 value
of 2.2 nM. Cell proliferation assays showed that 25g
effectively and selectively inhibited the growth of
EGFRL858R/T790M/C797S-dependent cells. This series of com-
pounds, which occupy both the ATP binding site and the
allosteric site of the EGFR kinase, may serve as a basis for the
development of fourth-generation EGFR inhibitors for
L858R/T790M/C797S mutants.
KEYWORDS: EGFR, mutant, inhibitor, C797S
activating EGFR mutations.14 The third-generation EGFR
inhibitors selectively and irreversibly target EGFRT790M and
other activated EGFR mutations. AZD9291 (4, osimertinib,
Figure S1, Supporting Information),15,16 the only FDA
approved third generation inhibitor,17 has good potency
against the EGFRT790M mutant and minimal toxicities, with
excellent selectivity for wild-type EGFR. Nevertheless, a
clinical study showed that 20−30% of patients treated with
AZD9291 developed the tertiary point mutation C797S,18
which prevents irreversible inhibitors from covalently binding
to Cys797. Loss of the covalent interaction results in a marked
decrease in inhibition, which then leads to the development of
resistance.
Jia et al. reported on compound EAI001 (6, Figure S1,
Supporting Information) as the first non-ATP competitive
EGFRL858R/T790M/C797S inhibitor.19 After structural optimiza-
tion, a more potent compound 1 (EAI045, Figure 1) was
obtained. EAI045 binds to the allosteric site created by the
outward displacement of the αC-helix of the EGFR kinase,
located next to the ATP binding pocket.19 The discovery of
this allosteric site laid the theoretical foundation for our
research.
INTRODUCTION
■
Epidermal growth factor receptor (EGFR), a member of the
HER family, is an essential transmembrane glycoprotein in cell
signaling pathways that regulate cell proliferation, differ-
entiation, and apoptosis.1 The overexpression of EGFR has
been observed in many types of solid tumors.2−4 Nonsmall cell
lung cancer (NSCLC) is one of the most malignant cancer
types worldwide.5 With the drug development, various small
molecular EGFR inhibitors (Figure S1, Supporting Informa-
tion) have been developed as therapeutic agents for NSCLC.
The first generation of reversible EGFR inhibitors, gefitinib
and erlotinib, delivered significant therapeutic effects for
NSCLC patients with activating EGFR mutations.6−8 The
L858R point mutation and exon 19 deletion are the most
common activating mutations, with enhanced sensitivity to
inhibitors.9 However, after 12 months of clinical treatment, the
T790M mutation appeared in 50%−60% of drug-resistant
patients.10 The presence of T790M increases the affinity of the
receptor for ATP, thereby reducing the ability of EGFR
inhibitors to effectively compete with ATP.11 Then, the
second- and third-generation EGFR irreversible inhibitors were
developed12 that had increasing cellular potency against
T790M mutants, mainly by covalently binding to Cys797
13
.
However, because the aniline moiety of the second-generation
EGFR inhibitors may not interact as effectively with the side
chain of Met790, the T790M activity is lower against the
Received: November 21, 2018
Accepted: May 21, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00564
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Letter
position of 2 with an amide bond as the linker. The resultant
compounds, 18a−18i (Figure 2C), had no inhibitory activity
against EGFRL858R/T790M/C797S (Table S1, Supporting Informa-
tion). Referring to the structure of EAI045, oxoisoindolin-2-
phenylacetamide was introduced into the R1 position,
synthesizing compound 25a. The kinase assay showed that
25a has a nanomolar level bioactivity (IC50 = 9.3 nM) against
EGFRL858R/T790M/C797S. We surmised that the substituted group
at the R1 position of 25a has a “Y-shaped” configuration,22
making it more likely to embed in the allosteric site. To further
explore the structure−activity relationship and acquire
compounds with higher potency, we selected compound 25a
as the new lead compound.
Figure 1. Structures of compound 1 (EAI045) and compound 2.
We found that compound 2 (2, Figure 1), a known EGFR
inhibitor (vandetinib),20 exhibited modest potency (IC50
=
After a docking simulation, we found that the interactions
between 25a and EGFR include three parts (Figure 2C and
Figure 3): (1) the quinazoline scaffold of 25a forms a
369.2 nM) against the EGFRL858R/T790M/C797S mutant. Both the
reference21 and our molecular docking simulation (gscore =
−8.2 kcal/mol) indicated that compound 2 could extend into
the ATP binding pocket of EGFR (Figure 2A). Inspired by the
binding model of EAI045, we attempted to modify 2 to
occupy both the ATP binding site and the allosteric site of the
EGFR kinase, with the aim of enhancing the binding affinity of
the inhibitor to EGFRL858R/T790M/C797S, to effectively compete
with ATP and thus overcome resistance. Then, 25a was
developed (Figure 2B). Based on 25a, we designed and
synthesized a series of novel, highly potent, and noncovalent
reversible inhibitors of EGFRL858R/T790M/C797S and explored
their structure−activity relationships. In this study, one of the
most promising compounds is 25g (Figure 2B), which
inhibited the enzymatic activity of EGFRL858R/T790M/C797S
with an IC50 value of 2.2 nM. Cell proliferation assays showed
that 25g effectively and selectively inhibited the growth of
EGFRL858R/T790M/C797S-dependent cells. These findings may
help overcome acquired resistance to third-generation EGFR
inhibitors.
Figure 3. Docked pose of compound 25a. The EGFR protein (PDB:
5d41) is shown as a gray cartoon, and the key residues are shown as
blue sticks. Key H-bonds are displayed as black dashes and measured
by distances. The figure was generated using Pymol 1.3.
RESULTS AND DISCUSSION
■
The docking model of 2 with EGFRT790M/V948R shows that 2
binds at the ATP binding site of EGFR with its phenyl group
occupying a position similar to that of the thiazole moiety of
EAI001 (Figure 2A). EAI001 binds as a “Y shaped”
configuration in the allosteric site.22 Modifying 2 to occupy
both the ATP binding site and the allosteric site may be a
promising way to increase the bioactivity against the
EGFRL858R/T790M/C797S triple mutant.
hydrogen bond with residue Met793 in the hinge region; (2)
the “Y-shaped” R1 group oxoisoindolin-2-phenylacetamide
extends into the EGFR kinase allosteric site with hydrophobic
interaction; and (3) the alkoxy side chain R2, R3 of the
quinoline scaffold faces toward the solvent-exposed region.
We then optimized 25a mainly from three aspects: (1) the
allosteric region; (2) the hinge region; and (3) the solvent-
exposed region.
To facilitate the occupation of the allosteric site of EGFR,
different hydrophobic groups were introduced to the R1
Figure 2. (A) Overlaid model of the docked pose of compound 2 (yellow) and the bound conformation of the allosteric inhibitor EAI001 (cyan)
948R
in EGFRT790M/V
(PDB: 5d41), key residues are shown as gray sticks; (B) Chemical structures of compounds 25a and 25g; (C) The 2D
interactions diagram for the quinazoline scaffold to show strategies of structural modifications, with chemical structures of the representative
compounds. n = 0, 1; Z = −CH3, −CH(CH3)2, −Ph.
B
DOI: 10.1021/acsmedchemlett.8b00564
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In the allosteric region, “the Y-shaped” group oxoisoindolin-
2-phenylacetamide was introduced at the R1 position (Figure
2C). Compound 25b was first synthesized, and the “Y-shaped”
group was attached to the ortho position (5′-position) of
anilino-quinazoline. Compound 25b displayed an IC50 value of
37.1 nM against EGFRL858R/T790M/C797S, a 4-fold decrease
compared to that of 25a. Compounds 25c and its isomer 25d
exhibited IC50 values of 7.9 nM and 19.2 nM, respectively,
against EGFRL858R/T790M/C797S. This result indicates that the S-
enantiomer is preferred over R, but both are acceptable. Then,
a fluorine atom was introduced to different positions of the
phenyl to acquire 25e−25g. Kinase assay results showed that
25g was the most potent, increasing the inhibitory activity by
over 4-fold compared with 25a (IC50 = 2.2 nM). The
introduction of the two fluorine atoms plays a crucial role in
strengthening the binding affinity. Replacing the phenyl group
of 25a within a cyclohexane group led to compound 25h.
Compound 25h displayed less potent inhibitory activity, with
an IC50 value of 179.6 nM against EGFRL858R/T790M/C797S, a
significant decrease in activity compared with 25a, suggesting
that the π−π stacking interaction between the phenyl of the
“Y-shaped” group and residue Phe856 of the hydrophobic
allosteric cavity plays an important role in maintaining the
bioactivities of this series against EGFRL858R/T790M/C797S
(Figure 3).
Figure 4. Docked pose of compound 25g. The EGFR protein (PDB:
5d41) is shown as a gray cartoon, and the key residues are shown as
blue sticks. Key H-bonds are displayed as black dashes and measured
by distance. The figure was generated using Pymol 1.3.
In the hinge region, only one hydrogen-bond interaction can
form between the quinazoline scaffold of 25a and Met793
(Figure 2C). To enhance the binding strength, we proposed
that another hydrogen bond might be formed between the
compound and Met793 by introducing a substituent at the R
position containing a hydrogen bond donor such as −NH2;
thus, 25j was synthesized. Kinase assay results showed that 25j
displayed no inhibitory activity against either T790M/L858R
or L858R/T790M/C797S mutant, indicating that “-NH2” was
not tolerated at the R position.
Figure 5. Kinase profile of 25g cross 37 kinases. *EGFRLTC
abbreviation for EGFRL858R/T790M/C797S
:
.
Table 1, 25g exhibited better antiproliferative effects against
BaF3-EGFRL858R/T790M/C797S cell lines than AZD9291 (IC50:
In the solvent-exposed region, to investigate the effect of the
hydrophilic tail on bioactivity against EGFRL858R/T790M/C797S
,
we performed structural derivatization using piperidine,
morpholine, and alkyl chains at R2 or R3 to synthesize
compounds 25i and 25k−o. The kinase inhibitory activities of
these compounds were lower than that of 25a, which has an
IC50 value of 9.3 nM against EGFRL858R/T790M/C797S (Table S2,
Supporting Information).
After a series of optimizations, compound 25g, with the best
kinase inhibitory activity against EGFRL858R/T790M/C797S, was
obtained (IC50 = 2.2 nM). Like 25a, docking simulations
suggested that 25g also occupies both the ATP binding site
and the allosteric site (Figure 4), exhibiting similar
intermolecular interactions.
a
Table 1. In Vitro EGFR Antiproliferative Activity
Antiproliferative IC50 (μM)
Cells
25g
AZD9291
Brigatinib
BaF3
7.68 0.73
5.11 0.49
3.93 0.38
0.03 0.01
1.44 0.03
7.31 0.98
0.42 0.09
ND
BaF3-EGFRL858R/T790M/C797S 0.64 0.14
b
H1975
A431
3.03 0.49
1.24 0.16
b
ND
a
Antiproliferative activity was examined by the Resazurin assay or the
SRB assay. Date are averages of at least three independent
determinations and reported as the means
SD (standard
b
deviations). Not determined.
The synthetic procedures of all compounds are shown in the
Supporting Information (Schemes S1−S3).
The kinase profile of 25g across 37 kinases is shown in
Figure 5. Treatment with 25g at 100 nM demonstrated more
than 90% inhibition of EGFRL858R/T790M/C797S as well as the
EGFR family of kinases (ErbB2 and ErbB4) and BLK, and it
exhibited moderate potency against Src, Abl, PDGFs, Txk,
AXL, BTK, and VEGFR2 with inhibition rates from 50% to
75%, However, 25g showed less than 50% inhibition of 25
other kinases (VEGFR1, 3, EPH-A2, -B2, IGF1R, FGFR1-4,
JAK1, 3, Trk-A, -B, -C, ALK, IKK, RET, C-kit, and so on),
indicating that 25g showed a favorable selectivity.
0.64 μM vs 3.93 μM) and EAI045 (IC50 > 10 μM, Table S5,
Supporting Information). Meanwhile, 25g showed moderate
antiproliferative effects against the parental BaF3 cells (IC50
=
7.68 μM), H1975 (IC50 = 3.03 μM) cells, and A431 cells (IC50
= 1.24 μM). Therefore, 25g was selected as the
EGFRL858R/T790M/C797S inhibitor for further study of cell
signaling pathways.
The inhibitory activity of the representative compound 25g
against the phosphorylation of EGFR was further confirmed by
Western blot analysis. As shown in Figure 6, compound 25g
dose-dependently inhibited the phosphorylation of EGFR in
BaF3-EGFRL858R/T790M/C797S cells, while AZD9291 was unable
Then, we tested the antiproliferative activities of 25g against
a panel of cell lines with different EGFR status. As shown in
C
DOI: 10.1021/acsmedchemlett.8b00564
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ACS Medicinal Chemistry Letters
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The results of kinase activity assays for all the
synthesized compounds and the methods used for
docking simulations and chemical and biological assays
(PDF)
The docked model of 25a with EGFR (PDB)
The docked model of 25g with EGFR (PDB)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: hlli@ecust.edu.cn (H.L.). Phone/Fax: +86-21-
64250213.
■
Figure 6. Bioactivity of 25g on the phosphorylation of EGFR in
BaF3-EGFRL858R/T790M/C797S cells.
*E-mail: hxie@jding.dhs.org (H.X.). Phone: +86-21-
50805897.
*E-mail: yfxu@ecust.edu.cn (Y.X.). Phone: +86-21-64251399.
ORCID
Shiliang Li: 0000-0003-4414-237X
Honglin Li: 0000-0003-2270-1900
to inhibit EGFR phosphorylation. Compound 25g showed
significant inhibitory effects against p-EGFR at a concentration
as low as 0.1 μM and almost completely inhibited the
phosphorylation of EGFR at a concentration of 1 μM, which
was comparable to the potency of the fourth-generation
allosteric inhibitor EAI045.
Author Contributions
§Q.L., T.Z., and S.L. contributed equally to this work. All
authors have given approval to the final version of the
manuscript.
In addition, we also evaluated the potency of 25g against
EGFR19Del/T790M/C797S, another triple mutant protein that
contains an exon 19 deletion activating mutation. The results
showed that 25g exhibited weak activity against the
EGFR19Del/T790M/C797S kinase, with an IC50 value of 331.3 nM
(Table S3) and weak antiproliferative activity against BaF3-
EGFR19D/T790M/C797S cells, with an IC50 value of 3.54 μM
(Table S4). Accordingly, 25g is more potent against
Funding
This work was supported by the National Key Research and
Development Program [2016YFA0502304 to H.L.]; the
National Natural Science Foundation of China (grant
81825020 to H.L., 81803437 to S.L.); the National Science
& Technology Major Project “Key New Drug Creation and
M a n u f a c t u r i n g P r o g r a m ” , C h i n a ( N u m b e r :
2018ZX09711002); and Fundamental Research Funds for
the Central Universities, Special Program for Applied Research
on Super Computation of the NSFC-Guangdong Joint Fund
(the second phase) under Grant No.U1501501. S.L. is
sponsored by the Shanghai Sailing Program (No.
18YF1405100). H.L. is sponsored by the National Program
for Special Support of Eminent Professionals and the National
Program for Support of Top-notch Young Professionals.
EGFRL858R/T790M/C797S than against EGFR19D/T790M/C797S
.
We calculated some drug-likeness properties and tested the
aqueous solubility of 25g (Table S7 and Table S8, Supporting
Information). The results showed that 25g has poor solubility.
Preliminary studies of in vivo pharmacokinetic (PK) properties
of compound 25g in rats following intravenous (IV) and oral
(PO) administration (Table S6, Supporting Information)
showed that 25g has a high clearance rate (CL = 960.8 mL/
h/kg) and poor oral bioavailability (F = 0.55%).
CONCLUSION
Notes
■
Based on a report of the EGFR allosteric site19 and an iterative
process of molecular docking, synthesis, and biological testing,
we developed a series of potent and noncovalent reversible
EGFR inhibitors that occupy both the ATP binding site and
the allosteric site. The structure−activity relationships of this
series of inhibitors were summarized against
EGFRL858R/T790M/C797S. One of the most promising com-
pounds, 25g inhibited the enzymatic activity of
EGFRL858R/T790M/C797S with an IC50 value of 2.2 nM. Western
blot results showed that 25g inhibits the phosphorylation of
EGFRL858R/T790M/C797S and downstream signal transduction at
the cellular level. Cell proliferation assays confirmed that 25g
effectively and selectively inhibited the growth of
EGFRL858R/T790M/C797S-dependent cells. With the aim of further
structural optimization to improve PK properties in the future,
this series of compounds might serve as a good basis for the
development of fourth-generation EGFR inhibitors of L858R/
T790M/C797S mutants.
The authors declare no competing financial interest.
ABBREVIATIONS
■
EGFR, epidermal growth factor receptor; NSCLC, nonsmall
cell lung cancer; PK, pharmacokinetic; IV, intravenous; PO,
oral.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00564.
■
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