Synthesis  of  Small  Molecule  Inhibitors  of  Protein  Kinase  D  and  Botulinum  Neurotoxin  A1  James  Johnson  

Wipf  Group  Topic  seminar  9/7/13  

1  

Synthesis  of  Small  Molecule  Inhibitors  of  Protein  Kinase  D  and  How  I  learned  to  Love  PMB  Protecting  Groups.  James  Johnson  

Wipf  Group  Topic  seminar  9/7/13  

2  

Presentation  format  •  Protein  Kinase  D    •  Background    •  The  Protein  •  Synthesis  of  Targets  and  Analogs.  •  Summary  

•  Botulinum  Neurotoxin    •  Background  •  The  Protein  •  Synthesis  of  Targets  •  Summary  

3  

Protein  Kinases  

4  

•  Protein  Kinases  (PK)  are  used  in  cell  signal  transducKon  by  the  phosphorylaKon  of  proteins.  

•  Popular  pharmaceuKcal  targets  •  PK’s  involved  in  cell  growth,  

proliferaKon,  or  apoptosis  •  Gleevec,  Sutent,  Tykerb,  Sprycel,  

Tasinga  are  approved  Protein  Kinase  inhibitors  

   

Protein  Kinase  D  •  Protein  Kinase  D  (PKD)  is  a  family  of  diacylglycerol  (DAG)-‐sKmulated  serine/threonine  kinases.  

•  Part  of  superfamily  of  Ca+2  /  calmodulin  Kinases  (CAMK)  and  are  highly  homologous  to  the  myosin  light  chain  kinase.  

•  Originally  classified  as  a  subfamily  of  the  Protein  Kinase  C  family.  

•  PKD  isoforms:  PKD1  (PKCµ),  PKD2,  PKD3  (PKCν)  

5  

Biochem.  J.  2010,  429,  565-‐572  Science  2002,  298,  1912-‐1934  

O R2

O

O

OH

R1

OR1 OleoylR2 Palmitoyl

DAG

OH

OH H

OO

HH

HOO

O

O

R

Phorbol ester

PKD  

Mol  Cancer  Res  2011  9:985-‐996  

6  

7  

Trends  Pharmacol.  Sci.  2006,  27  (6),  317-‐323.  EMBO  reports  2011,  12,  785-‐796  

PKD  and  signaling  

HTS  IMAP-‐FP  Assay  

8  

•  IMAP  (Immobilized  Metal  Affinity  PhosphorylaKon)-‐FP  or  TR-‐FRET.  

•  Small  trivalent  NanoparKcles  bound  to  ATP    •  Non  radioacKve.  Can  be  analyzed  by  FP  or  TR-‐

FRET  •  Higher  FP  with  bound  pepKde  due  to                    

lower  anisotropy.  

2013  Molecular  Devices  

Biological  Assay  •  In  Vitro  Radiometric  Kinase  Assay  •  Measures  Kinase  binding  using  P32  labeled  ATP.  

•  Cell  based:  •  MTT  ((3-‐(4,5-‐dimethylthiazol-‐2-‐yl)-‐2,5-‐diphenyltetrazolium  bromide)  •  Measures  cell  viability  

•  WST-‐1  (water  soluble  tetrazolium)    •  Measures  cell  viability  

9  

Clontech:  WST-‐assay,    

NN+N NS

N

Br-MTT

NN+N N

I

O2N

NaO3S

SO3Na

WST-1

NN

N

I

O2N

NaO3S

SO3NaHN

WST-1 formazan

Known  PKD  Inhibitors  

 (BBA)  Reviews  on  Cancer  2010,  1806  (2),  183-‐192.  J.  Biol.  Chem.  2008,  283:33516-‐33526,  ACS  Med.  Chem.  LeH.  2011,  2,  154-‐159.      

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HN

NN

O

Gö 6983PKD1 IC50: 20 uM

N

Me2N

HN

NH

OO

HO

O

NH

O

R2

R4

NZ

O

R1

R3

R5HO

S

NHS

O N

NMeO

S

NHS

O

CID755673IC50:

Rad .18 uMCell 11.8 uM

kb-NB142-70IC50:

Rad .028 uMCell 2.2uM

kmg-NB4-23IC50Rad .12 uMCell 6.8 uM

CNGö 6976PKD1 IC50: 20 nM

N

N

NH

N

H2N

O

NH

BPKDiPKD1 IC50: 1 nM

NHN

NHNHN

O

3,5-diarylpyrazolePKD1 IC50: 2.3 uM

N

N

HN

NH2

HO

NN

CRT0066101PKD1 IC50: 1 nM

MeO

OH

HO

OHResveratrolPKD1 IC50: 200 uM

Synthesis  of  1-‐NA-‐PP1  

11  

N

N

Cl

Cl

O

Cl

N

N

R1

HN NH2

Cl

Cl

NN

R1

2h, -78 °C

Br2

NH

HN

O O

OPOCl3, DMF

100 °C, 2 d57%

NEt3, THF

N

NCl

NHR2

NN

R1

BrR3B(OH)2

Pd2(dba)3Phosphine, Dioxane, 80 °C

N

NCl

NHR2

NN

R1

R310% Pd/C

NH4HCO2, MeOH, rt

N

N

NHR2

NN

R1

R3

H

19 examples23-85%

H2O, reflux

R1 = Me 59%R1 = t-Bu 81%

R2NH2NEt3, THF, rt

R1 = Me, R2= H (81%), Me (91%) R1 = t-Bu, R2= H (98%), Me (90%)

N

NCl

NHR2

NN

R1

R1 = Me, R2= H (40%), Me (88%) R1 = t-Bu, R2= H (84%), Me (71%)

25-35%

N

N

NH2

NN

PLoS  ONE  2013,  In  Press  

Analog  diversity  

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N N

Cl

Cl Cl

O

N

N

NH

Cl NN

N

N

NH2

Cl NN

N

N

NH2

NN

N

N

NH2

NN

F

N

N

NH2

NN

Cl

N

N

NH2

NN

O

N

N

Cl

NN

Cl

N

N

Cl

Cl NN

N

N

NH

NN

Cl

N

N

NH2

NN

N

N

NH

NN

N

N

NH

NN

O

N

N

NH2

NN

N

N

NH

NN

O

N

N

NH2

NN

O

N

N

NH

NN

N

N

NH

NNN

N

NH

NN

OCF3

N

N

NH

NN

F

N

N

NH2

NN

O

Cl

N

N

NH

Cl NN

O

N

N

NH

Cl NN

N

N

NH

Cl NN

OCF3

N

N

NH

Cl NN

F

1-‐NA-‐PP1  Activity  

13  

10 100 1000 100000

20

40

60

80

100

PKD2PKD3

PKD1

[1-NA-PP1] nM

% a

ctiv

ity

IC50 = 0.155 µM

IC50 = 0.133 µM IC50 = 0.104 µM

1-‐NA-‐PP1  

Cells  (PKD2)   %Kinase  Inhibi8on:  IC50  uM  

HCT116   0%  :  11.5  ±  1.6  

RKO   0%  :  6.3  ±  0.6  

Manuj  Tandon  PKD  acKvity  in  1-‐NA-‐PP1  using  a  radiometric  in  vitro  kinase  assay    John  Schmitz  cellular  data  and  acKvity  for  PKD2  WST-‐1  viability  assay    

Future  goals  •  New  Scaffold  Development  through  homology  modeling  and  in-‐silico  ligand  screening.  

•  Produce  a  potent,  isoform  specific  PKD  inhibitor.  

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Presentation  format  •  Protein  Kinase  D    •  Background    •  The  Protein  •  Synthesis  of  Targets  and  Analogs.  •  Summary  

•  Botulinum  Neurotoxin    •  Background  •  The  Protein  •  Synthesis  of  Targets  •  Summary  

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Botulinium  Neurotoxin  A1  

DOI:10.2210/pdb3bta/pdb  

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Overview  •  Background  •  History  •  IntoxicaKon  •  Uses  

•  Protein  •  MOA  

•  Previous  Inhibitors    •  Development  of  inhibitors  around  pharmacophore  •  New  inhibitors  •  Synthesis  of  New  Inhibitors  

17  

Botulinum  Neurotoxin  •  Produced  by  the  spore  forming  gram-‐posiKve  bacteria  Clostridium  Botulinum.  

•  Toxicity  less  than  1  ng/kg  in  humans  is  the  lethal  dose  •  From  a  family  that  contains  7  serotypes  A-‐G.  •  The  genus  of  Clostridium  contains  several  toxic  members  i.e.  C.  tetani  and  C.  perfringens.  

•  Typical  symptoms  include:  •  Muscle  Weakness  •  Flaccid  Paralysis  •  Respiratory  Failure  

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Mov.  Disord.  2004,  19:  S2–S6..    

History  •  First  discovered  by  J.  Kerner  in  1822  as  “Sausage  Poisoning”  or  “Faly”  Poisoning.  Became  known  as  “Kerner’s  disease”  

•  1895  E.  Ermengem  isolated  and  anaerobic  rod-‐shaped  bacteria  gave  the  name  Clostridium  Botulinum  

•  1944  E.  Schantz  discovers  methods  for  large  scale  purificaKon  of  BoNT/A1  

•  1989  BoNT/A  first  biological  toxin  to  be  approved  by  the  FDA  as  BOTOX  

Perspect  Biol  Med,  1997,  40,  317-‐327  Mov.  Disord.  2004,  19:  S2–S6..  

19  

BoNT’s  •  TherapeuKc:  •  Botox,  Dysport,  Myobloc  are  forms  of  Botulinum  A  and  B  approved  by  the  FDA  in  1991  

•  Must  be  applied  intramuscular  •  Can  be  used  to  treat  MS,  urinary  inconKnence,  Chronic  Migraine,  Upper  limb  spasKcity,  Axillary  hyperhidrosis,  and  Blepharospasm.  

•  Biological  Warfare:  •  Saddam  Hussein  produced  large  quanKKes  of  the  C.  Botulinum  during  Gulf  War  

•  ~300g  could  kill  the  enKre  populaKon  in  the  United  States  

hlp://www.fda.gov/Drugs/DrugSafety/  Movement  Disorders  1997,  12,  1013-‐1018  

20  

Methods  of  Intoxication  •  Food-‐borne  •  Infant  botulism/hidden  botulism  •  Wound  botulism  •  Inadvertent  botulism  •  IntenKonal  botulism  

21  

BoNT/A    the  Protein  •  1296  amino  acids  long  –  divided  into  3  domains  each  50kDa  •  CatalyKc  domain  50kDa  Light  chain  •  TranslocaKon  domain  50  kDa  Heavy  chain  N-‐terminal  domain  •  Binding  domain  50kDa  Heavy  chain  C-‐terminal  domain  •  Stable  to  pH  3-‐5  •  LC  and  HC  linked          disulfide  bridge  

Nat.  Struct.  Biol.  1998,  5,  898  –902,  Trends  Biochem.  Sci.  2002,  57,  552  

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Protein  Binding  site  •  Binding  site  on  LC  •  Each  serotype  of  BoNT  cleaves  a  SNARE  protein    •  VAMP(vesicle  associated  membrane  protein)  1+2  (synaptobrevins  

•  BoNT  B,  D,  F,  G  •  SNAP-‐25  (Synaptosomal  associated  protein-‐25)  

•  BoNT  A,  C,  E  •  Syntaxin    

•  BoNT  C    

23  

Science  2012,  335,  928-‐929    

Mechanism  of  Action  

ACS  Chem.  Biol.  2006,  1,  359-‐369.  Annu.  Rev.  Cell  Dev.  Biol.  2003.  19:493–517    

24  

Normal  Neurotransmiler  release.    •  Acetyl  Choline  (ACh)  is  a  neurotransmiler  

involved  in  muscle  contracKon    •  Stored  in  vesicles  that  upon  polarizaKon  

move  to  the  membrane  SNARE  proteins  that  facilitate  exocytosis  to  the  neruomuscular  juncKon  in  the  peripheral  nervous  system.  

•  SNARE  proteins  are  Soluble  N-‐ethylmaleimide-‐sensiKve  factor  Alachment  protein  Receptor.  

•  SNAREs  consist  of:  •  SNAPs,  Syntaxin,  and  Vamps  

 

N O

OAcetylcholine

Mechanism  of  Action  

25  

ACS  Chem.  Biol.  2006,  1,  359-‐369.    

1) Binding  to  ganglioside  G1b  (GD1b,  GT1b,  and  GQ1b)  

2) InternalizaKon  of  toxin  receptor  complex  into  the  synapKc  vesicle  

3) AcidificaKon  of  the  synapKc  vesicle  causes  translocaKon  domain  HN  to  insert  into  Membrane  

4) TranslocaKon  of  BoNT  LC  across  membrane  

5) ReducKon  of  disulfide  in  cytosol  6) Hydrolysis  of  SNAP  25  –  inacKvaKon  of  snare  complex  leads  to  inability  for  Neuronal  exocytosis  of  acetylcholine    

 

26  

Mol.  Microbiol.1994,  13,  1  -‐  8    

Hydrolysis  of  SNAP-‐25  at  Gln197-‐Arg198  

Adapted  from  Structure  2008,  16,  1588–1597  JBC  2008,  283,  18883-‐18891      

27  

Gln197NH

O Arg198

Zn2+Glu 262

His 223His 227

O H

Tyr 366

HO H O

O Glu 224

SNAP 25

Gln197NH

O Arg198

Zn2+Glu 262

His 223His 227

O H

Tyr 366

HO H O

O Glu 224

SNAP 25

Gln197O

O

Zn2+Glu 262

His 223His 227

O H

Tyr 366

OO Glu 224

SNAP 25N

Arg198HHH

1st  Gen.  Inhibitors  of  BoNT’s  

•  InhibiKon  of  BoNT  monitored  by  HPLC  analysis  to  observe  hydrolysis  of  SNAPKde  at  residues  Gln197  and  Arg198.  Or  FRET  screening  of  large  library.    

•  HPLC  assay  using  SNAP-‐25  (141-‐206)  Heterocycles,  2009,  79,  487-‐520  Eur.  JOC  2012,  53,  374-‐379    

O

NH

OHCl

Cl2,4-Dichlorocinnamic

hydroxamic acid75% inhibition at 20 µM

NH

HN

NH HCl

O

HNNH HCl

NSC 24089875% inhibition at 20 µM

Ki = 10 µM

N

NH

HN

NH2

O

NNH

NSC 35775657% inhibition at 20 µM

NH

N

CN

I

OMe

Benzimidazoleacrylonitrile

IC50 = 7.2 µM

HN

OH

OH

OMe OH

NH

OH

HO

OMeOHMichellamine B62% inhibition at 20 µM

NCl

HN NH

HN

NClQ2-15

60% inhibition at 20 µM

28  

Development  around  Pharmacophore  

29  

•  Molecular  docking  of  pseudo-‐pepKde  like  mpp-‐RATKML  (Ki=331nM)  have  helped  idenKfy  key  interacKons  in  the  binding  site.  

•  Key  components:  •  Planar  linkers  •  Polar  chelaKng  funcKon  •  CaKonic  caps  

•  Recent  developments  in  non-‐metal-‐chelaKng  ligands  has  shown  a  new  hydrophobic  binding  pocket  that  can  be  accessed  by  different  aromaKc  linkers  

JBC  2007,  282,  5004-‐5014  Bioorg.  Med.  Chem.  Lel.  2009,  19,  5811-‐5813  

2nd  Gen.  Inhibitors  of  BoNT/A  

30  

NH

SN

NHNH

N

CWD-02180% inhibition at 20 µM

S

H2N

HN

NH2

NH

Ki = 10.8 µM

NH

N

CN

S S

Non competitive inhibitor of BoNT/AIC50 26 µM

NH

OHN

H2NNH2

N

HN N

Cl

x4 TFA

NH2

NNH

OHN

H2NHN

N

Cl

x4 TFA

Ki = .600 µM

Further  Development  3rd  Gen.  

31  

ACS  Med.  Chem.  Lel.  2010,  1,  301-‐305  Eur.  J.  Med.  Chem.  2012,  53,  374-‐379.  

HN

O

NH

O

O

HN N

NHN

HN

O

NH

NH

HN

N

NCl

Cl

Ki = .572 µM

HN

O

NH

O

O

HN N

NHN

HN

O

NH

Ki = 8.53 µM

SHN HN

HN

HN

NN

ClCl

Ki = .302 µM

Staudinger  Aza  Wittig  (SAW)  

32  Adv.  Heterocycl.  Chem.  1995,  Vol.  64,  159-‐  249  Angew.  Chem.  Int.  Ed.  2005,  44,  5188  –  5240  J.  Org.  Chem.  2004,  69,  4299  

R1N

NN PR3

R= Alkyl, Aryl

R1NPR3

N2

H2OStaudingerReduction

R1NH2

R LG

OStaudingerLigation

R NH

OR1

O

R3R2

StaudingerAza Wittig

N

R3R2

R1 OPR RR

From  hydrazine  to  triazine  

33  

N

NHNPMBR

H2NPMBN N3 R

O

OH

O

O

O

H2NHN OH

THF reflux on O

O

N NH OH

63%

1) MsCl Et3N THFrt 20 min

2) NaN3 DMF 50COvernight

89% 2 steps

O

O

N NH N3

PMBBr, K2CO3, NaI

DMF rt 2dO

O

N NPMB N3

70%

O

O

N NPMB N3

NH2NH2 (HCl)2

THF H2O rt 2.5hH2N

PMBN N3

60-80%

NH

PMBN N3

R

O

OH

Coupling R

O

29-89%

N

NH

NPMBR

PR3

MW 180°C35-55%

N

NH

NPMBR 55°C 1hr

TFA N

NH

NHR

78%

Future  Goals  •  Finish  the  synthesis  of  both  targets.      

34  

Acknowledgements    •  Dr.  Wipf    •  Pete  Chambers,  Sage  Bowser  •  CollaboraKons  •  Dr.  Jane  Wang,  Dr.  Manuj  Tandon,  and  Dr.  John  Schmitz    PKD  •  Dr.  Sina  Bavari,  Dr.  Jonathan  Nuss,  Dr.  James  Burnel,  and  Dr.  Rick  Gussio    BoNT  

•  Funding:    SAIC/NIH  •  Wipf  group  past  and  present  

35  

PKD1  

36  

Tetazolium  reduction  mechanism  

37  

38