Fragment-­‐based  Drug  Design  and  Iden2fica2on  of  HJC0123,  

A  Novel  Orally  Bioavailable  STAT3  Inhibitor  for  Cancer  Therapy    

Chen,  H.;  Yang,  Z.;  Ding,  C.;  Chu,  L.;    Zhang,  Y.;  Terry,  K.;  Liu,  H.;  Shen,  Q.;  Zhou,  J.  TX,  US  

EJMC.  62  (2013)  498-­‐507  

Fangfei  Qin  Wipf  Group  Current  Literature  

May  11,  2013  

N

OHN

N N

O O

SO OO2N

STX-­‐0119  inhibit  STAT3  dimeriza2on  

N

OHN S

O O

Sta`c  non-­‐pep2dic  STAT3  inhibitors  

inhibit  phosphoryla2on  

MCF-­‐7:  0.1  µm  MDA-­‐MN-­‐231:  0.29  µm  Orally  bioavailable    

N

OH

Cl N N

O O

SO O NBr

CN

Fragment-­‐Based  Drug  Design  

1  

Research  Scheme  

Known  cps  

Design  

Synthesis  

Primary  Biological  Evalua2on  

Molecular  Docking  

Further  Op2miiza2on  

Further  Biological  Evalua2on  

SAR  study  

2  

Let’s  Start  with  A  Story…  

•  In  2004,  when  I  was  14  years  old…  

HN

OHO

Paracetamol:analgesic and antipyretic

250 mg

N

O

N

Cl

NH

O

O N

N

HO

My imagination

NH2

Amantadine:antiviral100 mg

N

NO

O

N

N

Caffeine:CNS stimulant

15 mg

Cl

N

N

Chlorphenamine:first-generation alkylamine antihistamine

2 mg

Ingredients  

Compound  Paracetamol    and  Amantadine    Hydrochloride  Capsules    

3  

Fragment-­‐Based  Drug  Design  (FBDD)    

J.  Med.  Chem.,  2004,  47  (14),  3463–3482  

Nat.  Rev.  Drug  Discov.2004,  3(8),  660-­‐672  

Fortunately…  

June,  2004   August,  2004  

[1]  D.A.  Erlanson,  R.S.  McDowell,  T.  O’Brien,  Fragment-­‐based  drug  discovery,  J.  Med.  Chem.  47  (2004)  3463-­‐3482.  [2]  D.C.  Rees,  M.  Congreve,  C.W.  Murray,  R.  Carr,  Fragment-­‐based  lead  discovery,  Nat.  Rev.  Drug  Discov.  3  (2004)  660e672.  [3]  P.J.  Hajduk,  Fragment-­‐based  drug  design:  how  big  is  too  big?  J.  Med.  Chem.  49  (2006)  6972e6976.  [4]  K.  Babaoglu,  B.K.  Shoichet,  Deconstruc2ng  fragment-­‐based  inhibitor  discov-­‐  ery,  Nat.  Chem.  Biol.  2  (2006)  720e723.  

Fragment-­‐based  drug  design  (FBDD)  is  a  promising  approach  for  the  genera2on  of  lead  molecules  with  enhanced  ac2vity  and  especially  drug-­‐like  proper2es  against  therapeu2c  targets.  

July,  2004  

My  birthday!  

1  month  before   1  month  Later  

4  

STAT3  Signaling  Pathway  VS  Cancer  Therapy  

Inhibit  STAT3  dimeriza2on:  PY*LKTK,  STX-­‐0119,  S31-­‐201  

Inhibit  phosphoryla2on:  Sta`c  

Inhibit  DNA  binding:  CAP-­‐1,  IS3  295  Inhibit  transcrip2on:  

niclosamide  

Inhibit  JAK:  WP1066,  AG490  

5  

STAT3  Inhibitors  

H-­‐Pro-­‐Tyr-­‐(PO₃H₂)-­‐Leu-­‐Lys-­‐Thr-­‐Lys-­‐Ala-­‐Ala-­‐Val-­‐Leu-­‐Leu-­‐Pro-­‐Val-­‐Leu-­‐Leu-­‐Ala-­‐Ala-­‐Pro-­‐OH.  CF3CO2H  

PY*LKTK  

Inhibit  STAT3  dimeriza2on  HNO

N

N N

O O

STX-0119

O2N SO O

Stattic

 Inhibit  phosphoryla2on   Inhibit  DNA-­‐  binding  

H2C NH2

H2C NH2

Pt

Cl

NO2

ClCl

CPA-1

Pt-Cl Cl--Cl Cl-

-Cl Cl-2H+ + + CH

OH

CH2CH3

N N CH

CH2

OHCH3

IS3 295

HO

HO

O

HN

OO SO O

S31-201

Inhibit  transcrip2onal  func2on  of  STAT  3  HNO

N

Br

CN

WP1066

HO

HOCN

CONHCH2

AG4906  

Me  too,  Me  berer!  

JAK  HNO

N

Br

CN

WP1066

phosphoryla2on   O2N SO O

Stattic

dimeriza2on  

HNO

N

N N

O O

STX-0119

transcrip2on  

HNO

N

Br

CN

WP1066

Heterocyclic  ring  

Amide  linker  

Heterocyclic  ring  

OHN S

O O

4

CNN

OHN S

O O

5

N

OHN

8

OHN

HO

Cl

9

SO OH

NO

CN

N

Br10

SO OH

NO

HO

Cl

12

MW=120~251  7  

Synthe2c  Route  

R1

O

OHS

O OH2N+ SO OH

N

R1

Oa

R1

O

OH+ H2N

a

HNR1

O

1, 4:

2, 5:

R1=

R1=

NC

N

2, 5: R1=N

OH

Cl

6, 9: R1=a) HBTU, DIPEA, CH2Cl2, rt, 39-94%

SO OH

NO

CN

N

Br

4

N

Br

O

piperidine,EtOH, 90 °C72%4, 5

8, 9

1, 2

2, 6 7

3 10

OH

Cl

O

OH

OH

Cl

O

Cl

OH

Cl

O

NHR2c, db

6 11 12, 13

SO O

N N

O O

12. R2=

13. R2=

(b) SOCl2, toluene, reflux; (c) R2NH2, pyridine, DMF, 0 °C to rt; (d) 1 N LiOH (aq.), THF, H2O, 0 °C to rt, 39-50% (three steps). 8  

Biological  Evalua2on—SAR  Study  

NNC

NBr

CN

OH

Cl

A =

A1 A2 A3 A4

SO O

N N

O O

B1 B2 B3

B =

A NH

OB

B1>B3  

A1>A2  

A1>A4  

9  

Molecular  Docking  Studies    

A1,  B1:  STAT3-­‐SH2  domain    

Surface  of  the  electrosta2c  map.  

Residues  of  STAT3.  

Generated  using  Pymol.   Predicted  binding  mode  for  compound  5  1.  S.  Becker,  B.  et.  al,  Nature  394  (1998)  145~151.  2.  O.  Tror.  et.  al,  J.  Comput.  Chem.  31  (2010)  455~461.  3.  K.  Matsuno,  et.  al.  ACS  Med.  Chem.  Ler.  1  (2010)  371~375.    4.  J.  Turkson,  et.  al.  J.  Biol.  Chem.  280  (2005)  32979~32988.    5.  H.  Song,  et.  al,  Proc.  Natl.  Acad.  Sci.  U.  S.  A.  102  (2005)  4700~4705.    

Quinoline  ring  could  fit   effec2vely   into  the   hydrophobic  clex  around  Ile634    

Hydrogen  bond  

10  

SAR  Con2nued—Hydrophobic  Groups  

N

OHN S

O O

5

N

19

OHN S

OO

N

OHN S

OO

20

N

OHN S

OO

21

N

OHN S

OO

22

N

OHN S

OO

23

Hydrophobic  groups  

Amide  linker  

Heterocyclic  ring  

Keep  for  hydrogen  bond  

Change  for  further  study  

5   19   20   21   22   23  

IC50  (μM)  

Breast  cancer  ER-­‐Posi2ve  

MCF-­‐7     0.1   0.65   >10   >10   3.78   2.97  

Breast  cancer  ER-­‐Nega2ve    

MDA-­‐MB-­‐231    

0.29   0.45   >10   >10   1.85   6.21  

Pancrea2c  cancer   AsPC1     1.25   0.12   >10   ND   1.3   6.97  

Panc-­‐1     0.26   0.31   >10   ND   3.35   7.92   11  

Physicochemical  Analysis:  cpd  5—Most  Desirable    

12  

Cellular  Biological  Characteriza2on—cpd  5    

Fig.  S1:  Effect  of  5  (HJC0123)  on  cell  growth  and  cellular  morphological  change.  Significantly  inhibited  cell  prolifera2on  and  induced  apoptosis.     13  

Effect  on  Promoter  Ac2vity    

Fig  4:  Compound  5  (HJC0123)  inhibited  the  STAT3  mediated  luciferase  reporter  ac2vity  in  MDA-­‐MB-­‐231  cells.  

Inhibit:  65%  at  5  μM  

cpd  5  acts  as  a  potent  small-­‐  molecule  inhibitor  of  STAT3  ac2va2on    

14  

Inhibitory  Ac2vity  against  STAT3  Pathway    

Fig.  5.  Western  blot  analysis  of  biochemical  markers  for  apoptosis  induc2on  and  inhibi2on  of  STAT3  ac2vity  by  compound  5  (HJC0123)  in  the  MDA-­‐MB-­‐231  cell  line.  

Promote  apoptosis  

Inhibit  STAT  3  signaling  

Loading  control  

Expression  reduced  

15  

Flow  Cytometry—cpd  5    

Figure  S2.    Induc2on  of  apoptosis  on  MDA-­‐MB-­‐231  cells  by  HJC0123.  

Ac2vated  apoptosis      dose-­‐dependent  manner      

16  

Cell  Cycle  Distribu2on  Analysis  —  cpd  5    

Figure  S3.    Changes  of  cell  cycle  distribu2on  in  MDA-­‐MB-­‐231  cells  axer  treatment  with  HJC0123.    

Arrested    S  phase        dose-­‐dependent  manner      

17  

In  Vivo  Biological  Characteriza2on—cpd  5    

Fig.  6.  In  vivo  efficacy  of  compound  5  (HJC0123)  in  inhibi2ng  growth  of  xenograx  tumors  (Breast  cancer  MDA-­‐MB-­‐231)  in  mice  (p.o.).   18  

Assay  Summary  

Assay  

Cell  level (ac2vity  test)  

ability  to  arrest  cells   promoted  the  apoptosis  of  cancer  cells  

cancer  cell  prolifera2on  inhibited  cell  

prolifera2on  and  induced  apoptosis    

Molecular  level (pharmacological  

elucida2on)  

Transient  transfec2on  and  dual  luciferase  reporter  assays  

inhibited  the  STAT3  promoter  ac2vity    

Western  blot    

STAT3  expression  reduced    

pSTAT3  at  Tyr-­‐705  suppressed    

In  vivo   an2tumor  

xenograx  tumors  suppressed      

No  significant  toxicity    

compound  5  is  a  potent,  

efficacious  and  orally  

bioavailable  an2-­‐cancer  

drug      

19  

Conclusion  FBDD    

six  privileged  fragments    

compounds  5,  12,  and  19    

advanced  chemical  leads    

 Inhibit  STAT3  promoter  ac2vity  

In  vitro   In  vivo  

down-­‐regulate  pSTAT3  

compound  5    most  potent    

increase  cleaved  caspase-­‐3  

inhibit  cell  cycle  progression  and  promote  apoptosis  

suppress  ER-­‐  nega2ve  breast  cancer    MDA-­‐MB-­‐231  xenograx  tumor  growth    

an  efficacious  and  orally  bioavailable  drug  candidate  for  human  cancer  therapy.    

Future  work:  Further  modifica2on  &  more  extensive  mechanis2c  study  con2nuing   20