Kinase 2014: Past, Present and Beyond

New Kinase Inhibitors Targeting theNew Kinase Inhibitors Targeting theNew Kinase Inhibitors Targeting the New Kinase Inhibitors Targeting the Clinical Acquired Resistance Related Clinical Acquired Resistance Related

MutantsMutantsK DiKe Ding

Guangzhou Institutes of Biomedicine and Health, CASg ,

May.19, 2014

(Cambridge UK)

中国科学院广州生物医药与健康研究院 GIBH.CAS

(Cambridge, UK)1

Protein Protein KinasesKinases Are Important Targets for Drug DiscoveryAre Important Targets for Drug Discovery

PProtein kinase

• Cell growth/ proliferation• Differentiation

Dephospho Ph h

P

ATP

kinase• Viability/survival• Homeostasis• Effector function (e gDephospho

proteinsPhosphoproteins

(Modified functions)

Effector function (e.g.cytotoxicity, cytokineproduction)• Cell death

◆About 518 protein kinases were found; an estimated 30,000 genes in humans; about 1.7% of thehuman genome encodes protein kinases

(Modified functions)

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%

中国科学院广州生物医药与健康研究院 GIBH.CAS2

to 30% of all targets screened in the pharmaceutical industres today.

Kinase Inhibitors Have Achieved Significant Benefits Kinase Inhibitors Have Achieved Significant Benefits for Treatment of Multiple Cancers, However ……for Treatment of Multiple Cancers, However ……p ,p ,

Launched time Generic name Company Targets Indication

2001 Imatinib Novartis Bcr-Abl CMLB Abl

2007 Nilotinib Novartis Bcr-Abl CML

2006 Dasatinib BMS Bcr-Abl CML

Bcr-AblT315I

2002 Gefitinib Astrazenec EGFR NSCLC

2005 Erlotinib OSI Pharm. EGFR NSCLC

2007 L i ib GSK EGFR b i

EGFRT790M

2007 Lapatinib GSK EGFR breast carcinoma

2005 Sorafenib Bayor VEGFR renal carcinoma

2005 Sunitinib Pfizer VEGFR GIST ALK2005 Sunitinib Pfizer VEGFR GIST

2011 Crozotinib Pfizer ALK NSCLC

2011 Icotinib BetaPharm EGFR NSCLC

ALKL1196M

2012 Ponatinib Ariad Pharm Bcr-Abl CML

2013 AfatinibBoehringer

EGFR NSCLC

中国科学院广州生物医药与健康研究院 GIBH.CAS

2013 AfatinibIngelheim

EGFR NSCLC

2013 Ibrutinib Pharmacyclics BtK CLL 3

One of Our Major Research Interests Is: One of Our Major Research Interests Is:

Design and synthesize new kinase inhibitors targeting the clinically acquired

resistance related mutants which may serve as novel therapeutic agentsresistance related mutants, which may serve as novel therapeutic agents.

O O

OHNH2

OHNH2S

Ile 315 Met 318GZD824

Structure information Critical amino acids

Riociguat, Bayer ,

registered,

NNNH2

CO2MeNH2N

NHN

Nregistered,hypertension

N NN

Fand other 60s

N

P i il i t t D lik

中国科学院广州生物医药与健康研究院 GIBH.CAS4

Privileging structures Drug-like core

Recent Research SummaryRecent Research Summary

Di Molecular T f T t D l t St

Recent Research Summary Recent Research Summary

Diseases Targets Types of Target Development Stage

CML Bcr-AblT315I Kinase IND enabling (li d )CML Bcr Abl Kinase (licensed out)

NSCLCs EGFRT790M Kinase Candidate selection

Cancer/ or fibrosis DDR1 Kinase Candidate selection

Melanoma/colon B-RafV600E/EGFR i d i i iMelanoma/colon cancer

B Raf EGFR dual inhibition Kinase Lead optimization

TNBC/metabolic ERR Orphan NHR IND enablingdiseases ERR Orphan NHR (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.CAS5

interaction

Design and synthesis of new Design and synthesis of new BcrBcr--Abl Abl

inhibitors targeting the T315I mutantinhibitors targeting the T315I mutant

中国科学院广州生物医药与健康研究院 GIBH.CAS6

BackgroundBackground：：the Unmet Clinical Needsthe Unmet Clinical Needs

• Imatinib achieves significant clinical benefit for CML management . However,

clinically acquired resistance becomes a major challenge (accelerated phase

andblast phase, 50-80%）p

• Bcr-Abl mutation is the primary mechanism for the resistance. More than

100 resistance related Bcr Abl mutants have been identified100 resistance-related Bcr-Abl mutants have been 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.CAS7

Background: Background: the structure base for molecule designthe structure base for molecule design

• The mechanism for Bcr-Abl T315I mutation induced resistance :h drogen bond loss steric hindrancehydrogen bond loss, steric hindrance.

• Ile315 becomes the most critical residue for new Bcr-AblT315I inhibitord idesign.

ImatinibCh i l F l C H N O

中国科学院广州生物医药与健康研究院 GIBH.CAS8

Thr315 Chemcial Formula: C29H31N7O Ile(315)

Background: Background: the structure base for molecule designthe 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.CAS9

“Critical Amino Acid” based design of GZD824“Critical Amino Acid” based design of GZD824

GZD824

N NN

O

O H NN

N NN

S

H N

O

N

E tazo la te , B M SP h ase II

P D & A D

N H

C O 2M eNH 2N

G S K -3 56278 , G SKP hase I,

H u ngtington 's d iseasea

R io cig uat B ayer ÁÙ́ ² ½áÊø ´ý Åúh yp ertension

N NN

NNN H 2

中国科学院广州生物医药与健康研究院 GIBH.CAS10

F

Chemical synthesis of GZD824Chemical synthesis of GZD824

中国科学院广州生物医药与健康研究院 GIBH.CAS11

Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.

GZD824 potently inhibits the T315I mutantGZD824 potently inhibits the T315I mutant

Clinical drugsBiological activities（nM）

Clinical drugsGZD824

imatinib nilotinib dasatinib

Bi di ffi it （K ）Abl T315I 1 000 1 000 1 000 0 71Binding affinity（Kd）Abl T315I >1,000 >1,000 >1,000 0.71

Kinase inhibition（IC ）

Abl T315I >5,000 >700 >1,400 0.68（IC50）

Abl >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

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.CAS12

GZD824 also inhibits other resistant mutantsGZD824 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 28E255K NA 0.28

F317I 1.8 NA

H396P 0 18 NA

Ren X ; Ding K * J Med Chem 2013 56 (3) pp 879 894

H396P 0.18 NA

M351T NA 0.23

中国科学院广州生物医药与健康研究院 GIBH.CAS13

Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.

D824 inhibits growth of cells with resistant mutantsD824 inhibits growth of cells with resistant mutants

Kinase inhibition (IC50,nM)

Bcr-Abl Imatinib Dasatinib GZD824

Wild type 98 2 0 26 0.34Wild type 98.2 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 0 17 0 35

中国科学院广州生物医药与健康研究院 GIBH.CAS14Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.

Y253F 749.6 0.17 0.35

D824 inhibits growth of D824 inhibits growth of BcrBcr--AblAbl positive cellspositive cells

The cell growth inhibitory activities of GZD824 against different cells.organ cell lines GZD824 (X±SD, nM) TAXOL (X±SD, nM)

K-562 0.2±0.1 5.1±0.5K-562R(Q252H)

4.5±0.7 7.0±0.5

BloodKU812 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

Normal cells HL-7702 1871± 901 4.0±0.6

中国科学院广州生物医药与健康研究院 GIBH.CAS15

Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.

D824 demonstrates promising D824 demonstrates promising in vivo in vivo efficacyefficacy

中国科学院广州生物医药与健康研究院 GIBH.CAS16

Ren, X.; Ding, K.* J. Med. Chem. 2013, 56 (3), pp 879–894.

D824 demonstrates promising D824 demonstrates promising in vivo in vivo efficacyefficacy

GZD824 efficiently prolongs animal survival in an allograft tumor model using Ba/F3 cells co expressing Bcr Abl T315I and luciferase

Day 0Ba/F3 cells co-expressing Bcr-Abl T315I and luciferase.

Day 7

100 mg/kgImatinib

Vehicle 1 mg/kg 10 mg/kg 20mg/kg5 mg/kg2 mg/kg

中国科学院广州生物医药与健康研究院 GIBH.CAS

Imatinib GZD82417

D824 displays good drugD824 displays good drug--like propertieslike properties

The drug-like properties of GZD824：g p p

1）PK profile: F = 48.7%；T1/2 = 8-10 hrs；no inhibition against

CYPs at high concentrations (over 1000 folds)；

2）S f H G IC 50 M f i d 10 f ld2）Safety: HerG，IC50 > 50 M；safety window > 10 folds；

3）Currently is under IND enabling investigation (Guangzhou3）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 ;

中国科学院广州生物医药与健康研究院 GIBH.CAS18

PCT application：WO 2012/000304

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.CAS19

BackgroundBackground：：EGFR Inhibitor & NSCLCEGFR Inhibitor & NSCLC• EGFR is a well validated target for anticancer drug discovery。

• 2003，FDA approved Gefitinib (Iressa) for the treatment of NSCLC patients with2003，FDA approved Gefitinib (Iressa) for the treatment of NSCLC patients with

EGFR activating mutation (L858R, del E746_A750). The first exapmle for

personalized medicine.p

• 2004，Erlotinib was approved by FDA .

• 2011 Icotinib was approved by SFDA• 2011，Icotinib was approved by SFDA.

• 2013，the irresible 2nd inhibitor Gilotrif (BIBW-2992) was approved

EGFR mutation)NSCLC

patients

(L858R,del E746_A750)

EGFR wild type Other

中国科学院广州生物医药与健康研究院 GIBH.CAS20

(or other mutations) therapies

EGFREGFRT790M T790M Mutation & Acquired ResistanceMutation & Acquired Resistance

• The first generation inhibitors, gefinitib and erlotinib have achievedsignificant clinical benefits However emerging acquired resistance to themsignificant clinical benefits. However, emerging acquired resistance to themhas become a major clinical challenge.

• EGFRT790M mutation is the primary mechanism for the resistance against• EGFR mutation is the primary mechanism for the resistance againstIressa in NSCLC patients（50% of resistant NSCLC patients）

• EGFRT790M mutation only moderately affects the binding of gefinitib andEGFR mutation only moderately affects the binding of gefinitib anderotinib, but significantly enhances the binding affinity for ATP with EGFR.

EGFRWT

Km = 5.2 MEGFRL858R

Km = 148 MEGFRL858R/T790M

Km = 8.4 M

Gefitinib Insensitive Gefitinib Sensitive Gefitinib Resistant

中国科学院广州生物医药与健康研究院 GIBH.CAS21

EGFREGFRT790M T790M Mutation & Acquired ResistanceMutation & Acquired Resistance

• The 2nd generation irreversible inhibitors: effective in animal models, butlow MTD in clinical investigation; dose-limit toxicity. The drugconcentrations are not high enough to inhibit EGFRT790M at MTD poorconcentrations are not high enough to inhibit EGFRT790M at MTD, poorefficacy. Most of the clinical trials are halted.

• Key problem: no selectivity over EGFR WT.ey p ob e : o se ect v ty ove G .

SHN

HNHN Cl

F

O

CNHN

HN Cl

N

ON

--SH NONO

Canertinib

NOO

N

Neratinib

FHN Cl

F

N

N

O

HN

N

HN Cl

ON

NHN

O

HN Cl

ON

O

• EGFRT790M induced drug resistance remains an unmet!!

DacomitinibO

Afatinib

中国科学院广州生物医药与健康研究院 GIBH.CAS

clinical challenge for NSCLCs treatment!!22

Selective EGFRSelective EGFRT790MT790M Inhibitors Overcoming ResistanceInhibitors Overcoming Resistance

• Inhibitors selectively targeting EGFRT790M mutants become a novel attractive strategy

for clinical management of NSCLC patients with acquired resistance. However:g p q

• EGFRWT and the EGFRT790M mutants share highly similar 3-dimensional structures and

have almost identical binding affinities with ATP.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 But the selectivity is relatively low (25-40 folds)currently in phase I/II trial. But the selectivity is relatively low (25-40 folds).

中国科学院广州生物医药与健康研究院 GIBH.CAS

W. Zhou et al. Nature 462(2009) 1-70-107423

“Critical Amino Acid” based design of EGFR “Critical Amino Acid” based design of EGFR T790MT790M

inhibitorsinhibitorsinhibitorsinhibitors

Met793Met790Met790

D101

Xu T ; Ding, K.* Angew. Chem. Int. Edt. 2013 DOI: 10 1002/anie 201302313.

中国科学院广州生物医药与健康研究院 GIBH.CAS

Xu, T.; Ding, K. Angew. Chem. Int. Edt. 2013, DOI: 10.1002/anie.201302313. 24

The compound displays great selectivity on EGFR The compound displays great selectivity on EGFR T790MT790M

mutant over the wildmutant over the wild--typetype kinasekinaseBinding affinities（Kd）：

EGFR WT 310 M

mutant over the wildmutant over the wild type type kinasekinase

EGFR WT = 310 nM

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 ) = 119.2d ( ) d ( )

Kd (EGFRWT)/ Kd (EGFRT790M ) = 238.5

No obvious inhibition against 455 different kinases

中国科学院广州生物医药与健康研究院 GIBH.CAS

No obvious inhibition against 455 different kinasesevaluated as 100nM.

25

The compound displays great selectivity on EGFR The compound displays great selectivity on EGFR T790MT790M

mutant over the wildmutant over the wild typetype kinasekinase

Cells lines EGFR status Anti-proliferation (IC50, M)

mutant over the wildmutant over the wild--type type kinasekinase

p 50

H1975 L858R/T790M 0.086±0.018H332 WT >30A549 WT >30

H1299 WT >30H1703 WT >30H661 WT >3095D WT >3095D WT >30

H358 WT >30

HCC827 del E746 A750 0.049±0.027_

HL-7702 WT >30HLF-1 WT 10.50±1.46

中国科学院广州生物医药与健康研究院 GIBH.CAS

The most selective EGFRT790M inhibitor reported to date. 26

The compound potently inhibit the activation of The compound potently inhibit the activation of EGFR signal pathwayEGFR signal pathway

0 0 016 0 08 0 4 2 0

3d

0 0 01 0 1

Gfb

0 0 016 0 08 0 4 2 0

3g

0 0.016 0.08 0.4 2.0 0 0.01 0.1

EGFR

0 0.016 0.08 0.4 2.0

pEGFR

Akt

pAkt

Erk

pErkpErk

GAPDH

中国科学院广州生物医药与健康研究院 GIBH.CAS27

The compound doseThe compound dose--dependently dependently inhibit the inhibit the migration and invasion of NCImigration and invasion of NCI--1975 NSCLC cells1975 NSCLC cellsgg

中国科学院广州生物医药与健康研究院 GIBH.CAS28

The compound doseThe compound dose--dependently dependently inhibit the colony inhibit the colony formation of NCIformation of NCI--1975 cells1975 cells

中国科学院广州生物医药与健康研究院 GIBH.CAS29

New compounds are obtained with improved PK New compounds are obtained with improved PK profilesprofiles

A D Animal Dose AUC (0-∞) C

profilesprofiles

Cpd. A.D.route

Animal No. level

mg/kg

AUC (0-∞) mg/L*h T1/2 (h) Tmax (h) Cmax

g/L F(%)

PO ♂4 25 1640 1.695 0.88 10405-1 30.0

IV ♂4 5 2491 0.617 0.03 3975

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.

中国科学院广州生物医药与健康研究院 GIBH.CAS30

The new compounds shows good The new compounds shows good in vivo in vivo efficacyefficacy

中国科学院广州生物医药与健康研究院 GIBH.CAS31

SummarySummary

Starting From the Unmet Clinical Needs：

Obtained the Bcr-Abl T315I inhibitor GZD824 as new

candidate to manage the clinically acquired resistance

against Imatinib;g ;

Designed and synthesized highly selective EGFRT790M

inhibitors which may potentially used to manage the

clinically acquired resistance against Gefitinibclinically acquired resistance against Gefitinib .

中国科学院广州生物医药与健康研究院 GIBH.CAS32

Acknowledgements Acknowledgements • Collaborators

1）Biologists/Pharmacologists：SIMM、SYSU、GIBH etcSIMM、SYSU、GIBH etc.

2) Medicinal Chemistry：Graduate

students and Ras.

• Financial SupportFinancial SupportNSFC, 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.CAS33

Thank You！

中国科学院广州生物医药与健康研究院 GIBH.CAS34