U.S. patent application number 14/744042 was filed with the patent office on 2016-12-22 for oncogenic ros1 kinase inhibitor.
The applicant listed for this patent is Macau University of Science and Technology. Invention is credited to Lai Han LEUNG, Liang LIU, Lian Xiang LUO, Xiao Jun YAO, Yan Ling ZHOU.
Application Number | 20160367546 14/744042 |
Document ID | / |
Family ID | 53716627 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160367546 |
Kind Code |
A1 |
YAO; Xiao Jun ; et
al. |
December 22, 2016 |
ONCOGENIC ROS1 KINASE INHIBITOR
Abstract
The present invention provides a compound that inhibits activity
of oncogenic ROS1, a composition comprising said compound. The
present invention also provides the use of said composition for
treating cancer.
Inventors: |
YAO; Xiao Jun; (MO, MO)
; LEUNG; Lai Han; (MO, MO) ; LUO; Lian Xiang;
(MO, MO) ; ZHOU; Yan Ling; (MO, MO) ; LIU;
Liang; (MO, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Macau University of Science and Technology |
Macau |
|
MO |
|
|
Family ID: |
53716627 |
Appl. No.: |
14/744042 |
Filed: |
June 19, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4745 20130101;
A61P 35/00 20180101 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. A method of treating cancer comprising administering a
composition comprising a compound of Formula I: ##STR00003## to a
ROS1 positive non-small cell lung cancer patient.
5. (canceled)
6. (canceled)
7. A method of inducing apoptosis in non-small cell lung cancer
cells comprising contacting a composition comprising a compound of
Formula I: ##STR00004## to said non-small cell lung cancer
cells.
8. The method of claim 7, wherein said non-small cell lung cancer
cells have an oncogenic ROS1 gene resulted from chromosome
rearrangement and/or gene fusion.
Description
FIELD OF INVENTION
[0001] The present invention relates to compound that inhibits ROS1
kinase activity, composition comprising said compound and the use
of said compound for treating cancer.
BACKGROUND OF INVENTION
[0002] Receptor tyrosine kinases (RTKs) are vital mediators of
extracellular signals, which direct cell growth, survival and
motility pathways. Several pathogenesis including chromosomal
rearrangements, gene amplification, and point mutations result in
abnormal and constitutive RTK activation which is in turn
responsible for initiation and progression of many cancers,
including non-small cell lung cancer (NSCLC). The first targetable
fusion gene of RTK identified in NSCLC is Echinoderm microtubule
associated protein like 4 ((EML4)-ALK). Oncogenic c-ros oncogenel
(ROS1) RTK is later reported to be fused with other forms of lung
constitutive expressed 5' or 3'-fusion gene partners in NSCLC.
Approximately 1.about.2% of NSCLC patients harbor multiple kinds of
ROS1 chromosome rearrangement. Recent developments in
targeted-based therapies have led to a major paradigm shift in
oncology. Small-molecule tyrosine kinase inhibitors are applied to
treat cancer patients who have tyrosine kinase gene fusions.
Several tyrosine kinase inhibitors have been shown to have
promising effect in the clinical practice. For example, Crizotinib,
a potent ATP-competitive small molecule inhibitor of ALK, have now
been approved by the FDA for treating NSCLC patients that are ALK
rearrangement-positive. Crizotinib displays marked anti-tumor
activity both in vitro and in vivo as well as in clinical
practices. Since the tyrosine kinase domains of ALK and ROS1 are
very similar, with 77% identity within the ATP-binding sites, most
ALK inhibitors have cross activity against ROS1. In one early
clinical trial of crizotinib to treat NSCLC patients harboring ROS1
rearrangements, the objective response rate was 72%, the median
duration of response was 17.6 months and median progression-free
survival was 19.2 months. Although most patients with ROS1-positive
NSCLC exhibit substantial clinical benefit from Crizotinib, the
efficacy of Crizotinib is limited by the development of acquired
drug resistance. Accordingly, there is a need for new compound
targeting ROS1 for cancer treatment.
SUMMARY OF INVENTION
[0003] The first aspect of the present invention relates to a
compound having structure of Formula I,
##STR00001##
[0004] The second aspect of the present invention relates to a
composition comprising the compound of Formula I. One embodiment of
the second aspect of the present invention relates to use of the
composition comprises the compound of Formula I to inhibit activity
of oncogene receptor tyrosine kinase (ROS1). The composition
inhibits ROS1 kinase activity and suppresses phosphorylation of
ROS1, as well as anti-apoptotic and growth signaling downstream to
ROS1.
[0005] The third aspect of the present invention relates to a
method of treating cancer comprising administering a composition
comprises a compound of Formula I to a subject in need thereof. The
subject in need thereof is a subject having an abnormality in ROS1
gene. The abnormality of ROS1 gene may be ROS1 chromosome
rearrangement and/or ROS1 gene fusion. The subject in need thereof
is ROS1-positive NSCLC individual. In one embodiment of the third
aspect of the present invention, the cancer treated is non-small
cell lung cancer (NSCLC).
[0006] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described.
[0007] The invention includes all such variation and modifications.
The invention also includes all of the steps and features referred
to or indicated in the specification, individually or collectively,
and any and all combinations or any two or more of the steps or
features.
[0008] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood to one of ordinary
skill in the art to which the invention belongs.
[0009] Other and advantages of the invention will be apparent to
those skilled in the art from a review of the ensuing
description.
BRIEF DESCRIPTION OF DRAWINGS
[0010] FIG. 1 shows the cell viability of HCC78 cells (FIG. 1A) and
BEAS-2B cells (FIG. 1B) after 72 hours treatment with compound of
Formula I of the present invention.
[0011] FIG. 2 shows florescence images (FIG. 2A) and cytogram
pattern (FIG. 2B) of HCC78 cells having treated with compound of
Formula I of the present invention.
[0012] FIG. 3 shows rate of apoptosis of HCC78 cells having treated
with compound of Formula I of the present invention.
[0013] FIG. 4 is a western blot showing the binding affinity of the
compound of Formula I of the present invention to ROS1 and other
signaling molecules downstream to ROS1.
[0014] FIG. 5 shows 3D schematic representation of the compound of
Formula I binds to ROS1 (FIG. 5A) and (FIG. 5B).
DETAILED DESCRIPTION OF INVENTION
[0015] The present invention is not to be limited in scope by any
of the specific embodiments described herein. The following
embodiments are presented for exemplification only.
[0016] The present invention provides a compound of Formula I,
##STR00002##
[0017] The present compound of Formula I is a potent inhibitor of
ROS1 oncogene receptor tyrosine kinase (ROS1). The present compound
binds to ROS1 and inhibits kinase activity of ROS1 and suppresses
ROS1 phosphorylation. Anti-apoptotic and growth signallings
downstream of ROS1 are also inhibited by the compound of Formula I
of the present invention.
[0018] The second aspect of the present invention relates to a
composition comprising the compound of Formula I. One embodiment of
the second aspect of the present invention relates to use of the
composition comprises the compound of Formula I to inhibit activity
of ROS1 oncogene receptor tyrosine kinase (ROS1). The composition
inhibits ROS1 kinase activity and suppresses phosphorylation of
ROS1, as well as anti-apoptotic and growth signaling downstream to
ROS1.
[0019] The third aspect of the present invention relates to a
method of treating cancer comprising administering a composition
comprises a compound of Formula I to a subject in need thereof. The
subject in need thereof is a subject having an abnormality in ROS1
gene. The abnormality of ROS1 gene is oncogenic result by ROS1
chromosome rearrangement and/or ROS1 gene fusion. The subject in
need thereof is a ROS1-positive NSCLC individual. In one embodiment
of the third aspect of the present invention, the cancer treated is
non-small cell lung cancer (NSCLC). The ROS1 gene rearrangements in
NSCLC have been widely studied in large scale screening study
through RT-PCR and DNA sequencing in NSCLC cell lines and NSCLC
patients. The ROS1 gene rearrangements are readily known in the
art. One common example of ROS1 gene rearrangements involve fusion
of SLC34A2-ROS1 and CD74-ROS1. The CD74 gene encodes a type 2
transmembrane protein that fuses with ROS1 to generate a CD74-ROS1
transcript found to be the most common form of all ROS1 fusion
genes in NSCLC, accounting for about 40% of all ROS1 fusions genes
in NSCLC.
[0020] The compound of formula I of the present invention is shown
to be highly cytotoxic and selective to cancer cells. The present
compound of Formula I has little effect on healthy normal
non-cancerous cells. To show the highly cytotoxic and selective
properties of the present compound of Formula I, HCC78 NSCLC cells
and normal lung epithelial cells (BEAS-2B) are treated with the
compound of the present invention and effects are observed. HCC78
NSCLC cells are non-small cell lung cancer cells with a ROS1 gene
fusion. HCC78 cells are obtained from the ATCC and cultured in
environment of 5% CO2 at 37.degree. C. in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 units/mL
penicillin, and 100 .mu.g/mL streptomycin, The compound of Formula
I is dissolved in DMSO. Using a MTT assay, 3000 HCC78 or BEAS-2B
cells are seeded on 96-well plates, cultured overnight for cell
adhesion, and treated with DMSO or various concentrations of the
present compound of Formula I for 72 h. Triplicate are performed.
10 .mu.L of MTT (5 mg/mL; Sigma) are added to each well, and
incubation continues for another 4 hours. Then the dark blue
crystals are dissolved in 100 .mu.L of the resolved solution (10%
SDS and 0.1 mM HCL). The absorbance is measured at 570 nm by a
microplate reader (Tecan, Morrisville, N.C., USA). The cell
viability is calculated relative to untreated controls, with
results based on at least 3 independent experiments. The MTT assay
shows that compound of Formula I is a potent inhibitor of HCC78
cells with a IC.sub.50 of G341-0312 is 1.62 .mu.M.+-.0.39 .mu.M,
while it shows much lower cytotoxicity on normal lung epithelial
cells (BEAS-2B) after 72 hr treatment, with a IC.sub.50 in BEAS-2B
is 6.08 .mu.M.+-.1.16 .mu.M (FIG. 1A and FIG. 1B). The present
compound of Formula I exerts a 3.75 fold cytotoxicity towards
cancer cells than normal healthy cells. The present compound of
Formula I is highly selectively towards cancer cells.
[0021] The compound of Formula I of the present invention, as a
potent ROS1 inhibitor, induces apoptosis in cancerous cells.
Apoptosis assay is performed on HCC78 cells to demonstrate the
potent ROS1 inhibitory effect of the present invention. HCC78 cells
(1.0.times.10.sup.5 cells/well) are allowed to attach to a 6-well
plate for 24 h, and the cells are treated with the various
concentrations of the present compound for an additional 72 h. At
the end of incubation, the cells are harvested by trypsinization
and washed twice with ice-cold PBS. After centrifugation and
removal of the supernatants, cell pellets are resuspended in 100
.mu.L 1.times. Annexin-binding buffer. 2 .mu.l Annexin-V FITC and 2
.mu.l PI (100 .mu.g/ml) are added and incubated in the dark at room
temperature for 15 min, before further addition of 400 .mu.l of
1.times. Annexin-binding buffer. The stained cells are analyzed
quantitatively using a flow cytometer (BD Biosciences, San Jose,
Calif., USA). FIG. 2A shows fluorescence images of HCC78 cells
having treated with the present compound of Formula I (G341-0312)
at 1.25 .mu.M, 2.5 .mu.M and 5 .mu.M; 2.5 .mu.M Crizotinib (a known
ROS1 inhibitor as positive control) and DMSO (control, negative
control). The results show that HCC78 cells having treated with the
present compounds detach from the surface and are small at 2.5
.mu.M of G341-0312. Such cell morphology indicates apoptosis. For a
more quantitative view, flow cytometry analysis is performed. As
seen in FIG. 2B and FIG. 3, the present compound of Formula I
exhibits anti-cancer ability through induction of apoptosis on
HCC78 cells in a concentration dependent manner. A significant
apoptosis level is observed in HCC78 cells having treated with the
compound of the present invention.
[0022] The compound of Formula I of the present invention also
suppresses ROS1 phosphorylation and anti-apoptotic and growth
signaling pathways that are downstream to ROS1. Previous studies
demonstrate that ROS1 fusion kinases signal is activated through
the tyrosine phosphatase Src homology-2 domain containing protein
tyrosine phosphatase-2 (SHP2) and causes activation of the
downstream MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT3 signaling axes.
Together, these downstream signaling pathways promote tumor cell
survival and proliferation. Therefore, inhibition of these
downstream signaling pathways suppresses growth and proliferation
of cancer cells and results in anti-cancer effect. The effect of
the compound of Formula I on ROS1 phosphorylation as well as
signaling biomolecules downstream of ROS1 are studied.
[0023] The cells are planted on 6-well plate, allowed to attach for
24 hours, and treated with the various concentrations of compound
of Formula I for 72 hrs. Cells are washed twice with cold PBS then
lysed in RIPA lysis buffer containing protease and phosphatase
inhibitors. Protein concentration of the cell lysates are measured
using the Bio-Rad protein Assay kit (Bio-Rad, Philadelphia, Pa.,
USA). After equalizing the protein concentrations of the samples,
5.times. laemmli buffer is added and boiled at 100.degree. C. for 5
min. Equal amounts of protein samples (30 .mu.g) are subjected to
SDS-PAGE of a 10% gel. The separated proteins are transferred to a
nitrocellulose (NC) membrane, which is then exposed to 5% non-fat
dried milk in TBS containing 0.1% Tween 20 (0.1% TBST) for 1 h at
room temperature, followed by overnight incubation at 4.degree. C.
with primary anti-bodies to GAPDH, phospho-AKT, AKT, photspho-ROS1,
ROS1, phospho-ERK, ERK, photspho-STAT3, STAT3. After washing 3
times by TBST (5 mins/time), the membranes are incubated for 1 hour
at room temperature with the secondary fluorescent antibodies
(1:10000 dilutions) to rabbit or mouse. The signal intensity of the
membranes is detected by an LI-COR Odessy scanner (Belfast, Me.,
USA).
[0024] Treatment of HCC78 cells with compound of Formula I
(G341-0312) leads to a dose-dependent decrease of ROS1
phosphorylation as well as its downstream signaling molecules
Erk1/2, STAT3 and AKT signaling, further supporting the anti-cancer
effect of G341-0312 (FIG. 4). Crizotinib is used as positive
control.
[0025] The binding mechanism of the present compound of Formula I
to ROS1 is studied. Molecular docking calculation is performed to
study the interaction between compound of Formula I and ROS1 by
Induced Fit Docking module in Schrodinger software (Schrodinger,
Inc., New York, N.Y., 2009). Compound of Formula I is prepared and
optimized in the LigPrep module. During the induced fit docking,
centroid of the crizotinib is defined as the active site and the
pose of ligand is valued with XP docking score. The pose with the
highest score is selected for further analysis. The 3D structure of
ROS1 is derived from the PDB database (PDB ID: 3ZBF) and prepared
using the Protein Preparation Wizard.
[0026] The present compound of Formula I has a similar binding
mechanism to ROS1 as Crizotinib. The docking scores of the present
compound and Crizotinib to ROS1 are -11.157 and -9.674 Kcal/mol,
respectively. The present compound is shown to have a better
binding affinity to ROS1 than Crizotinib. As seen in FIG. 5A, the
pyridine groups of the both compounds have hydrogen bond to Met2029
in the hinge region. The phenyl group and the anisole group of
compound of Formula I forms extra hydrophobic interaction with the
hinge and G-loop, respectively. As shown in FIG. 5B, residues Leu
2028, Met2029, Glu2030, Gly2032, Asp2033 in the hinge region have
contact with the compound of Formula I, while the residues Leu1951,
Leu1959 in the G-loop form hydrophobic interaction with the present
compound. Other residues, such as Lys1980, Asp2102, Leu2086, also
make a great contribution to the binding of the present
compound.
[0027] In the above examples, differences are analyzed by on-way
ANVOA. All statistical analyses are carried out using Graph
Prim5.0. P<0.05 was considered significant.
INDUSTRIAL APPLICABILITY
[0028] The present invention provides a new inhibitor, compound of
Formula I, that can specific target oncogenic ROS1 kinase, which
exhibits potent anti-cancer activity, and notably in NSCLC cell
with ROS1 fusion gene. The compound of Formula I of the present
invention significantly suppresses ROS1 phosphorylation as well as
its downstream anti-apoptotic and growth signaling biomolecules,
including PI3K/AKT, MEK/ERK and JAK/STAT3 signaling pathways. The
present compound shows relatively low toxicity to normal lung
epithelial cells, which can be used as anti-cancer drug for
targeting subgroup of cancer patients who harbor different forms of
ROS1 chromosomal rearrangement.
* * * * *