Kinase 2014: Past, Present and Beyond
New Kinase Inhibitors Targeting the
Clinical Acquired Resistance Related
Mutants
K Ding
Ke
Di
Guangzhou
g
Institutes of Biomedicine and Health,, CAS
May.19, 2014
(Cambridge UK)
(Cambridge,
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Protein Kinases Are Important Targets for Drug Discovery
• Cell growth/ proliferation
Protein
kinase
Dephospho
proteins
P
• Differentiation
• Viability/survival
• Homeostasis
ATP
Phospho
Ph
h
proteins
(Modified functions)
(e g
• Effector function (e.g.
cytotoxicity,
cytokine
production)
• Cell death
◆About 518 protein kinases were found; an estimated 30,000 genes in humans; about 1.7% of the
human genome encodes protein kinases
◆ Protein kinases are the fourth largest gene family in humans:
C2H2 zinc finger proteins (3%)
G-protein coupled receptors (2.8%)
Major histocompatibility (MHC) complex protein family (2.8%)
◆ Protein kinases are one class of the most popular targets for drug discovery, accounting for 25%
to 30% of all targets screened in the pharmaceutical industres today.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Kinase Inhibitors Have Achieved Significant Benefits
for Treatment of Multiple
p Cancers,, However ……
Launched time
Generic name
Company
Targets
Indication
2001
Imatinib
Novartis
Bcr-Abl
CML
2007
Nilotinib
Novartis
Bcr-Abl
CML
2006
Dasatinib
BMS
Bcr-Abl
CML
2002
Gefitinib
Astrazenec
EGFR
NSCLC
2005
Erlotinib
OSI Pharm.
EGFR
NSCLC
2007
L
Lapatinib
i ib
GSK
EGFR
b
breast
carcinoma
i
2005
Sorafenib
Bayor
VEGFR
renal carcinoma
2005
Sunitinib
Pfizer
VEGFR
GIST
2011
Crozotinib
Pfizer
ALK
NSCLC
2011
Icotinib
BetaPharm
EGFR
NSCLC
2012
Ponatinib
Ariad Pharm
Bcr-Abl
CML
2013
Afatinib
Boehringer
Ingelheim
EGFR
NSCLC
2013
Ibrutinib
Pharmacyclics
BtK
CLL
Bcr-Abl
B
Abl
T315I
EGFR
T790M
ALK
L1196M
中国科学院广州生物医药与健康研究院 GIBH.CAS
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One of Our Major Research Interests Is:
Design and synthesize new kinase inhibitors targeting the clinically acquired
resistance related mutants,
mutants which may serve as novel therapeutic agents.
agents
O
O
OH
OH
NH2
Ile 315
Structure information
NH2
Met 318
GZD824
Critical amino acids
N CO2Me
H2N
H
N
NH2
N
N
N
N
S
N
Riociguat,
Bayer ,
registered,
hypertension
N
N
and other 60s
F
P i il i structures
Privileging
t
t
D
Drug-like
lik core
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Recent Research Summary
Di
Diseases
Molecular
Targets
T
Types
off T
Targett
D l
Development
t St
Stage
CML
Bcr-Abl
Bcr
AblT315I
Kinase
IND enabling
(li
(licensed
d out))
NSCLCs
EGFRT790M
Kinase
Candidate selection
Cancer/ or fibrosis
DDR1
Kinase
Candidate selection
Melanoma/colon
cancer
B RafV600E/EGFR
B-Raf
dual inhibition
Kinase
i
Lead
d optimization
i i i
TNBC/metabolic
diseases
ERR
Orphan NHR
IND enabling
(licensed out)
Cancer
P53-MDM2
Protein-protein
interaction
Phase I (Sanofi)
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Design and synthesis of new Bcr
Bcr--Abl
inhibitors targeting the T315I mutant
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Background：
Background
：the Unmet Clinical Needs
•
Imatinib achieves significant clinical benefit for CML management . However,
clinically acquired resistance becomes a major challenge (accelerated phase
phase, 50-80%）
andblast p
•
Bcr-Abl mutation is the primary mechanism for the resistance. More than
100 resistance
resistance-related
related Bcr-Abl
Bcr Abl mutants have been identified
identified.
•
The “gatekeeper” T315I is most common mutation. The second-generation
inhibitors (i.e. nilotinib and dasatinib) are not capable of inhibiting Bcr-Abl
T315I mutant.
•
Bcr-Abl T315I-induced drug resistance remains an unmet clinical challenge
for CML treatment.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Background: the structure base for molecule design
• The mechanism for Bcr-Abl T315I mutation induced resistance :
h drogen bond loss,
hydrogen
loss steric hindrance.
hindrance
• Ile315 becomes the most critical residue for new Bcr-AblT315I inhibitor
d i
design.
Imatinib
Thr315
Ch
Chemcial
i l Formula:
F
l C29H31N7O
Ile(315)
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Background: the structure base for molecule design
• Met318 is the 2nd key residue for inhibitor design.
imatinib
bosutinib
nilotinib
ponatinib
中国科学院广州生物医药与健康研究院 GIBH.CAS
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“Critical Amino Acid” based design of GZD824
GZD824
O
HN
O
N
N
O
N
HN
S
N
N
N
N
G S K -3 5 6 2 7 8 , G S K
P h a se I,
H u n g tin g to n 's d is e a s e a
E ta zo la te , B M S
P h a se I I
PD & AD
N
H 2N
N
C O 2M e
NH2
N
N
R io c ig u a t B a y e r
ÁÙ´² ½áÊø ´ý Åú
h y p e r te n s io n
N
N
N
F
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Chemical synthesis of GZD824
Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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GZD824 potently inhibits the T315I mutant
Biological activities（nM）
Clinical drugs
GZD824
imatinib
nilotinib
dasatinib
>1,000
1 000
>1,000
1 000
>1,000
1 000
0 71
0.71
Abl T315I
>5,000
>700
>1,400
0.68
Ba/F3 cell s（IC50） Abl T315I
>10,000
>15,000
>3,000
7.1
Normal cells（IC50） HL-7702
>1000
>1000
>1000
>1000
Bi di affinity（K
Binding
ffi it （Kd） Abl T315I
Kinase inhibition
（IC50）
Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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GZD824 also inhibits other resistant mutants
The binding affinities of GZD824 with different Bcr-Abl proteins
c-Abl
Binding affinity (Kd nM)
Non-phosphorylated form
Phosphorylated form
Wild type
0.32
0.34
T315I
0.71
3.20
Q252H
0.46
NA
E255K
NA
0 28
0.28
F317I
1.8
NA
H396P
0 18
0.18
NA
M351T
NA
0.23
Ren X.;
Ren,
X ; Ding,
Ding K.*
K * J.
J Med.
Med Chem.
Chem 2013,
2013 56 (3),
(3) pp 879–894.
879 894
中国科学院广州生物医药与健康研究院 GIBH.CAS
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D824 inhibits growth of cells with resistant mutants
Kinase inhibition (IC50,nM)
Bcr-Abl
Imatinib
Dasatinib
GZD824
Wild type
98 2
98.2
0 26
0.26
0.34
T315I
5155
1450
0.68
E255K
485.8
0.21
0.27
G250E
359.9
0.25
0.71
Q252H
115.0
0.24
0.15
H396P
173.9
0.30
0.35
M351T
114.3
0.22
0.29
Y253F
749 6
749.6
0 17
0.17
0 35
0.35
Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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D824 inhibits growth of BcrBcr-Abl positive cells
.
The cell growth inhibitory activities of GZD824 against different cells
organ
cell lines
GZD824 (X±SD, nM)
TAXOL (X±SD, nM)
K-562
K-562R
0.2±0.1
5.1±0.5
4.5±0.7
7.0±0.5
KU812
0.1±0.0
5.3±3.2
SUP-B15
2.5±1.0
8.0±3.4
U-937
390.2±153.4
2.3±0.2
MOLT4
26.3±10.2
2.7±0.9
HL-60
348.9±158.2
5.8±1.5
HL-7702
1871± 901
4.0±0.6
(Q252H)
Blood
Normal cells
Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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D824 demonstrates promising in vivo efficacy
Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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D824 demonstrates promising in vivo efficacy
GZD824 efficiently prolongs animal survival in an allograft tumor model using
Ba/F3 cells co-expressing
co expressing Bcr-Abl
Bcr Abl T315I and luciferase
luciferase.
Day 0
Day 7
Vehicle 100 mg/kg 1 mg/kg
Imatinib
2 mg/kg
5 mg/kg
10 mg/kg 20mg/kg
GZD824
中国科学院广州生物医药与健康研究院 GIBH.CAS
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D824 displays good drug
drug--like properties
The drug-like
g
properties
p
p
of GZD824：
1）PK profile: F = 48.7%；T1/2 = 8-10 hrs；no inhibition against
CYPs at high concentrations (over 1000 folds)；
2）S f
2）Safety:
H G IC50 > 50 M；safety
HerG，IC
M
f
window
i d > 10 ffolds；
ld
3）Currently is under IND enabling investigation (Guangzhou
Dunjian Pharm.)
1.
Ren, X.; Ding, K.* etc. J. Med. Chem. 2013, 56 (3), pp 879–894
2.
Li, Y.; Ding, K.* etc. J. Med. Chem. 2012, 55 (22), 10033-10046.
3.
Ke Ding, Deping Wang, Duanqing Pei, Zhang Zhang, etc. Chinese patent granted, # ZL 201010216603.7 ;
PCT application：WO 2012/000304
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Discovery of Novel Selective EGFRT790M
Mutant Inhibitors Overcoming the
Clinically Acquired Resistance against
Gefinitib
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Background：
Background
：EGFR Inhibitor & NSCLC
•
EGFR is a well validated target for anticancer drug discovery。
•
2003，FDA approved Gefitinib (Iressa) for the treatment of NSCLC patients with
EGFR activating mutation (L858R, del E746_A750). The first exapmle for
ppersonalized medicine.
•
2004，Erlotinib was approved by FDA .
•
2011 Icotinib was approved by SFDA.
2011，Icotinib
SFDA
•
2013，the irresible 2nd inhibitor Gilotrif (BIBW-2992) was approved
EGFR mutation
NSCLC
patients
(L858R,del E746_A750)
EGFR wild type
(or other mutations)
Other
therapies
中国科学院广州生物医药与健康研究院 GIBH.CAS
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EGFRT790M Mutation & Acquired Resistance
•
The first generation inhibitors, gefinitib and erlotinib have achieved
significant clinical benefits.
benefits However,
However emerging acquired resistance to them
has become a major clinical challenge.
•
EGFRT790M mutation is the primary mechanism for the resistance against
Iressa in NSCLC patients（50% of resistant NSCLC patients）
•
EGFRT790M mutation only moderately affects the binding of gefinitib and
erotinib, but significantly enhances the binding affinity for ATP with EGFR.
EGFRWT
Km = 5.2 M
Gefitinib Insensitive
EGFRL858R
Km = 148 M
EGFRL858R/T790M
Km = 8.4 M
Gefitinib Sensitive
Gefitinib Resistant
中国科学院广州生物医药与健康研究院 GIBH.CAS
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EGFRT790M Mutation & Acquired Resistance
•
The 2nd generation irreversible inhibitors: effective in animal models, but
low MTD in clinical investigation; dose-limit toxicity. The drug
concentrations are not high enough to inhibit EGFRT790M at MTD,
MTD poor
efficacy. Most of the clinical trials are halted.
•
ey p
problem:
ob e : noo se
selectivity
ect v ty ove
over EGFR
G WT.
Key
F
O
O
HN
HN
SH
--SH
O
N
N
Cl
N
N
N
HN
H
N
O
O
Cl
CN
O
N
Neratinib
Canertinib
F
F
H
N
N
O
O
HN
Cl
N
H
N
N
O
HN
Cl
N
O
N
N
O
Dacomitinib
Afatinib
• EGFRT790M induced drug resistance remains an unmet
clinical challenge for NSCLCs treatment!!
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Selective EGFRT790M Inhibitors Overcoming Resistance
•
Inhibitors selectively targeting EGFRT790M mutants become a novel attractive strategy
for clinical management
g
of NSCLC ppatients with acquired
q
resistance. However:
•
EGFRWT and the EGFRT790M mutants share highly similar 3-dimensional structures and
have almost identical binding affinities with ATP.
•
The development of WZ4002 is halted due to IP issue; CO-1686 and AZD9291 are
currently in phase I/II trial
trial. But the selectivity is relatively low (25-40 folds).
folds)
W. Zhou et al. Nature 462(2009) 1-70-1074
中国科学院广州生物医药与健康研究院 GIBH.CAS
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“Critical Amino Acid” based design of EGFR T790M
inhibitors
Met793
Met790
D101
Xu T
Xu,
T.;; Ding, K.*
K. Angew. Chem. Int. Edt. 2013,
2013 DOI: 10.1002/anie.201302313
10 1002/anie 201302313
中国科学院广州生物医药与健康研究院 GIBH.CAS
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.
The compound displays great selectivity on EGFR T790M
mutant over the wildwild-type kinase
Binding affinities（Kd）：
EGFR WT = 310 nM
M
EGFR T790M = 1.3 nM
EGFR L858R/T790M = 2.3 nM
Kinase inhibition IC50 ）：
EGFR T790M = 4.55 nM
EGFR L858R/T790M = 2.18 nM
Selectivity（120-240 folds）：
Kd ((EGFRWT))/ Kd ((EGFR L858R/T790M ) = 119.2
Kd (EGFRWT)/ Kd (EGFRT790M ) = 238.5
No obvious inhibition against 455 different kinases
evaluated as 100nM.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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The compound displays great selectivity on EGFR T790M
mutant over the wild
wild--type kinase
Cells lines
EGFR status
Anti-proliferation
p
(IC50, 
M)
H1975
L858R/T790M
0.086±0.018
H332
WT
>30
A549
WT
>30
H1299
WT
>30
H1703
WT
>30
H661
WT
>30
95D
WT
>30
H358
WT
>30
HCC827
del E746_A750
0.049±0.027
HL-7702
WT
>30
HLF-1
WT
10.50±1.46
The most selective EGFRT790M inhibitor reported to date.
26
中国科学院广州生物医药与健康研究院 GIBH.CAS
The compound potently inhibit the activation of
EGFR signal pathway
3d
0 0
0.016
016 0
0.08
08 0
0.4
4 2
2.0
0
3g
0 0.016
0 016 0.08
0 08 0.4
0 4 2.0
20
Gfb
0
0 01
0.01
0
0.1
1
EGFR
pEGFR
Akt
pAkt
Erk
pErk
GAPDH
中国科学院广州生物医药与健康研究院 GIBH.CAS
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The compound dosedose-dependently inhibit the
migration
g
and invasion of NCINCI-1975 NSCLC cells
中国科学院广州生物医药与健康研究院 GIBH.CAS
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The compound dosedose-dependently inhibit the colony
formation of NCINCI-1975 cells
中国科学院广州生物医药与健康研究院 GIBH.CAS
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New compounds are obtained with improved PK
profiles
Cpd.
5-1
A.D.
A
D
route
Animal
No.
Dose
level
mg/kg
AUC (0-∞)
mg/L*h
T1/2 (h)
Tmax (h)
Cmax
g/L
PO
♂4
25
1640
1.695
0.88
1040
IV
♂4
5
2491
0.617
0.03
3975
F(%)
30.0
1. Xu, T.; and Ding, K.* Angew. Chem. Int. Edt. 2013, 52, 8387–8390.
2. Chang, S.; Ding, K. * J. Med. Chem. 2012, 55 (6), 2711-2723.
3. Han, C.; Ding, K.*; Ji, H.*; Tian, J. and Zhang, Y.* J. Med. Chem. 2013, 56, 4738–4748.
4. Xu, S.; Ding, K.* MedChemComm. 2012, 3, 1155-1159.
5. Xu. S. ; and Ding, K.* J. Med. Chem. 2013, revision.
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The new compounds shows good in vivo efficacy
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Summary
Starting From the Unmet Clinical Needs：
 Obtained the Bcr-Abl
T315I
inhibitor GZD824 as new
candidate to manage the clinically acquired resistance
against
g
Imatinib;;
 Designed and synthesized highly selective EGFRT790M
inhibitors which may potentially used to manage the
clinically acquired resistance against Gefitinib .
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Acknowledgements
• Collaborators
1）Biologists/Pharmacologists：
SIMM、SYSU、GIBH etc.
etc
2) Medicinal Chemistry：Graduate
students and Ras.
• Financial Support
NSFC, Ministry of S & T, CAS,
Guangdong province and Guangzhou
city.
中国科学院广州生物医药与健康研究院 GIBH.CAS
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Thank You！
中国科学院广州生物医药与健康研究院 GIBH.CAS
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