Superbug challenger from nature - Michigan State …...“Superbug Challenger” from Nature Irosha Nayanthika Nawarathne Michigan State University 02/14/07 O O N H O OH OH HO O FS06_SS07\

Platensimycin “Superbug

Challenger”

from Nature

Irosha

Nayanthika

NawarathneMichigan State University02/14/07

O

O

NH

OOH

OH

HO

O

“Infectious diseases are now the world's biggest killer of children and young adults. They account for more than 13 million deaths a year -

one in two deaths

in developing countries.

Over the next hour alone, 1 500 people will die from an infectious disease -

over half of them children

under five.”-

WHO Report on Infectious Diseases 2000

What are antibiotics?

Molecules that stop the microbial growth (both bacteria and fungi) or kill them outright

Walsh, C., Antibiotics Actions origins and Resistance, 2003, 4

Classification –

According to Mode of Action

GTP GHP DHF THF

dUMP dTMP

DNA

DNA or RNA synthesis

Protein synthesis

Cell wall synthesis

Folic acid metabolism

Fatty acid synthesis

Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19

Classification –

According to Mode of Action

Quinolones,Rifampin

Aminoglycosides,Macrolides,Tetracyclines,Oxazolidinones

β-lactams,Cyclosporins,Glycopeptides

Triclosan, Isoniazid, Ethionamide

Trimethoprim,Sulfonamides

GTP GHP DHF THF

dUMP dTMP

DNA

Walsh, C., Antibiotics Actions origins and Resistance, 2003, 13-18

Superbugs-:

Resistant to multiple classes of antibiotics

Bacterial species

Common types of Antimicrobial

Types of

Infections

Resistance

Streptococcus pneumoniae β-lactams, cephalosporins, macrolides

Otitis media, pneumonia,Tetracyclines

sinusitis, meningitis Staphylococcus aureusCommunity-associated Meticillin, cephalosporins, macrolides Skin, soft tissue, sepsis

pneumonia

Healthcare-associated Meticillin, cephalosporins, quinolones,

Endocarditis, pneumonia,aminoglycosides, macrolides

sepsis

Enterococcus spp.

Ampicillin, vancomycin, aminoglycosides Sepsis, urinary tract

Furuya, E.Y., Lowy, F.D., Nature, 2006, 4, 36

How to address the problem of resistance

•

Chemical synthesis of analogs of known natural products

Walsh, C., Antibiotics Actions origins and Resistance, 2003, 113

N

S

COOHO

O N

S

COOHO

O

N

S

COOHO

OPenicillin

Ampicillin

Carbenicillin

H2N

HOOC

β-lactamase

N

O OH

OCO2H

H

How to address the problem of resistance

•

Development of inhibitors of resistance mechanisms

N

S

OCO2H

HN

O

R

N

S

OCO2H

HN

O

R

Clavulanate

β-lactamaseHydrolysis of the antiboitic

Stop / minimize Hydrolysis of the antiboitic

Walsh, C., Antibiotics Actions origins and Resistance, 2003, 113

How to address the problem of resistance

•

Discovery of completely novel antibiotics with new modes of action

Inhibitors

of Fatty Acid Biosynthesis (Fab)by targeting Fab

enzymes

Platensimycin

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916

By systematic screening of 250,000 natural product extracts, using FabF-target based RNA-mediated gene silencing technique

Antisense RNA

mRNA

5` ………ATGGCCTGGACTTCA…………3` 3` ………TACCGGACCTGAAGT…………5`

Sense DNAAntisense

DNA

Transcription

5` ………AUGGCCUGGACUUCA…………3`

Met - Ala - Trp - Thr - Ser -

Translation

Peptide

Discovery of Platensimycin

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916Forsyth, R.A., Molecular Biology, 2002, 43, 1387

Wang, J., et al, Antimicrob. Agents Chemother., 2006, 50, 519

Reduced or No FabF

expression

5`………

AUGGCCUGGACUUCA………3`3`………

UACCGGACCTGTTGU ………5`ds

RNA

Degradation of fabF

mRNA or inhibition of translation

In Prokaryotes-

Discovery of Platensimycin

From a strain of Streptomyces platensis recovered from soil samples

Higher sensitivity towards FabF

inhibitors

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916Forsyth, R.A., Molecular Biology, 2002, 43, 1387

Wang, J., et al, Antimicrob. Agents Chemother., 2006, 50, 519

Structure Elucidation of Platensimycin

Through Isolation followed by Determination of Structure and Stereochemistry

O

O

NH

OOH

OH

HO

O

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916

HR-ESI-FTMSC24

H27

NO7

UV/IR/DEPT/13C, 1H, HMQC2 methyls5 methylenes7 methines

4-Aromatic/Olefinic1-Oxy

10 non protonated

C’s 1-Ketone2-Carbonyls1-Oxygen bearing sp3 C2-Oxygen bearing sp2

C’s

Structure Elucidation of Platensimycin

Singh, S.B., et al, J. AM. CHEM. SOC., 2006, 128, 11916

Structure Elucidation of Platensimycin

TOCSY/COSY

HMBC

2

3

OH

H

6

7

HH

7̀6̀ 11 13

9

Singh, S.B., et al, J. AM. CHEM. SOC., 2006, 128, 11916

O

O

NH

OOH

OH

HO

O

Structure Elucidation of Platensimycin

O

OOH

OHH

HO

O OHNH

1

Combining aminobenzoic

acid unit to the ketolideHMBC ESIMS

O

O0

m/z

-

273

Singh, S.B., et al, J. AM. CHEM. SOC., 2006, 128, 11916

Stereochemistry of Platensimycin

Relative : NOESY and Scalar Couplings

Absolute : Anomalous Scattering

Synthesis of 6’-bromoplatensimycin

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916

O

O

NHOH

1. NBS, acetone, THF

rt, 2.5 h

OOH

O

O

NHOH

OOH

HO2C

Br

HO2C

S S

R

S

S

S

Stereochemistry of Platensimycin

Absolute and RelativeCrystal structure of 6’-Bromoplatensimycin

Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916

O

OO

NH

COOHOH

OH

1 4

715

10

12

Br

Structural Similarity of Platensimycin

Similar to known terpenoid structures

O

H

O

O

O

H

HO

HRO

17 Carbons 20 Carbons 20 Carbons

ent-KaurenePierisformoside

GPlatensimycin

Habich, D., Nussbaum, F.V., ChemMedChem, 2006, 1, 951Wang, L.Q., Qin, G.W., Chen, S., Li, C., Fitoterapia, 2001, 72, 779

O-glucoside

from Pieris

formosaIn plants and fungi

Microbiological profiles and Toxicity of Platensimycin

Potent, Broad Spectrum Gram positive activity in vitroNo cross resistance to major superbugsOrganism and genotype Platensimycin Linezolid

Antibacterial activity (MIC, µg/ml)

S. aureus (MSSA) 0.5 4S. aureus (MRSA) 0.5 2S. aureus (MRSA,

macrolideR) 0.5 2S. aureus (MRSA, linezolidR) 1 32S. aureus (VISA) 0.5 2Enterococcus faecalis (macrolideR) 1 1Enterococcus faecium (VRE) 0.1 2S. pneumoniae 1 1

Wang, J., et al, Nature, 2006, 441, 358

MIC

–

Concentration of inhibitor used to result no visible growth of the pathogens

Microbiological profiles and Toxicity of Platensimycin

Efflux mechanism limits the activity

Organism and genotype Platensimycin Linezolid

Antibacterial activity (MIC, µg/ml)

E. coli (wild-type) >64 >64E. coli (tolC) 16 32

Wang, J., et al, Nature, 2006, 441, 358

MIC

–

Concentration of inhibitor used to result no visible growth of the pathogens

Microbiological profiles and Toxicity of Platensimycin

Acts selectively

Organism and genotype Platensimycin Linezolid

Toxicity (µg/ml)

HeLa

MTT (IC50

) >1,000 >100Candida albicans (MIC) >64 >64

Wang, J., et al, Nature, 2006, 441, 358

IC50 –

Concentration of the inhibitor used to kill 50%

population of the living cells

Microbiological profiles and Toxicity of Platensimycin

In vivo Studies

In a mouse model of disseminated S. aureus

infection

Wang, J., et al, Nature, 2006, 441, 358

Fatty Acids in bacteria…

•

Synthesis of membrane lipids which regulates membrane fluidity

eg

-

Phospholipids and Glycolipids

•

Metabolic storage

eg

-

Triacylglycerols

•

Cell Signal Transduction

Nelson, D.L., Cox, M.M., Lehninger Principles of Biochemistry, 2005, 4, 787

FAB …….. is a good target

FAB Type I-

In mammals

FAB Type II-

In bacteria

Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305

SH

SH

SH

S

O

O S

OO

S

O

O O

SSH

OH O

SSH

O

SSH

O

S

SH

O

SSH

S CoAO

O

O

SCoA

O

CO2NADPH+H

NADP

H2O

NADPH+ H

NADP

CoASH

CoASH

Biosynthesis of Saturated Fatty Acids

FabD

FabH FabG

FabZ

FabI

/ K / L

Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305

ACP

SH

SH

SH

S

O

O S

OO

S

O

R

R

O O

SSH

R

OH O

SSH

R

O

SSH

R

O

S

SH

R

O

SSH

S CoAO

O

O

SCoA

O

CO2NADPH+H

NADP

H2O

NADPH+ H

NADP

CoASH

CoASH

Biosynthesis of Saturated Fatty Acids

FabD

FabF FabG

FabZ

FabI

/ K / L

Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305

ACP

SH

SH

SH

S

O

O S

OO

S

O

R

R

O O

SSH

R

OH O

SSH

R

O

SSH

R

O

S

SH

R

O

SSH

S CoAO

O

O

SCoA

O

CO2NADPH+H

NADP

H2O

NADPH+ H

NADP

CoASH

CoASH

Inhibitors of Fatty Acids Biosynthesis

Triclosan,Isoniazid,Ethionamide

PlatensimycinCerulenin,

Thiolactomycin

Campbell, J.W., Cronan, J.E.Jr., Annu.Rev.Microbiol., 2001, 55, 305Price, A.C., et al, The Journal of Biological Chemistry, 2001, 276, 6551

Heath, R.J., White, S.W., Rock, C.O., Progress in Lipid Research, 2001, 40, 467

Platensimycin- Inhibitor of Fatty Acid Synthesis

DNA,RNA, Protein and Cell wall Biosynthesis

Wang, J., et al, Nature, 2006, 441, 358Kodali, S., et al, J.Biol.Chem., 2005, 280, 1669

Mode of Action of Platensimycin

High FabF selectivityCell - free gel - elongation assay

HO

O O

SCoA HO

O O

S ACP

O O

S ACP

OH O

S ACP

O

S ACP

O

S ACP

HO

O O

S ACP

CO2 +ACPSH

ACPSH CoASH

FabD

C02 +CoASHFabH

CoA

O

NADPH

NADP

FabG

H2O

FabAFabZ

FabIFabKFabL

FabF

NADPH

NADP

Wang, J., et al, Nature, 2006, 441, 358Heath, R.J., Nat.Prod.Rep., 2002, 19, 581

Malonyl-ACPC4:1(Δ2)-ACP

C4:0-ACP

>6C-ACP

Mode of Action of Platensimycin

Inhibits FabF by direct targetingDirect binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

Synthesis of 3H-dihydroplatensimycin

O

O

NH

OH

OOH

O

O

NH

OH

OOH

1. 5%Pd/ C , Isoproponol

Introduce Tritiumgasfromuraniumtritide bed(2.5 ml at 740mmHg)(5 Ci,2 Ci/ml)

2. 0.2NNaOH/ MeOH Specific activityof 15.8 Ci/mol

HO2C HO2C

T

Wang, J., et al, Nature, 2006, 441, 358

Mode of Action of Platensimycin

Inhibits FabF by direct targetingDirect binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

Scintillation Proximity Assay

Wang, J., et al, Nature, 2006, 441, 358

Cu2+

SPA

His6Tag

FabF

Mode of Action of Platensimycin

Inhibits FabF by direct targetingDirect binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

Scintillation Proximity Assay

Wang, J., et al, Nature, 2006, 441, 358

SPACu2+

His6Tag

FabFDPMN

Mode of Action of Platensimycin

Inhibits FabF by direct targetingDirect binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

No signal of binding

1. Loss of olefin may result no binding

2. It may require co-substratesfor the binding

Wang, J., et al, Nature, 2006, 441, 358Olesen, J.G., Structure, 2001, 9, 233

Apo FabF/FabB

Acyl Enzyme Intermediate

Mode of Action of Platensimycin

Inhibits FabF by direct targetingDirect binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

•

Platensimycin inhibits Acyl-Enzyme Intermediate

•

Stable

Acyl-Enzyme Intermediatethrough Mutation C163Q

Wang, J., et al, Nature, 2006, 441, 358Olesen, J.G., Structure, 2001, 9, 233

McGuire, K.A., Biochemistry, 2001, 40, 9836

Mode of Action of Platensimycin

Inhibits FabF by direct targeting

Wang, J., et al, Nature, 2006, 441, 358Olesen, J.G., Structure, 2001, 9, 233

Apo FabF/FabB

A.E.IntermediateMutated FabF/

FabB

A.E.Intermediate

Mode of Action of Platensimycin

Inhibits FabF by direct targeting

Wang, J., et al, Nature, 2006, 441, 358Olesen, J.G., Structure, 2001, 9, 233

Mutated FabF/

FabB

A.E.Intermediate Mutated FabF

Mode of Action of Platensimycin

Inhibits FabF by direct targetingProposed mechanism

“

ACYL BINDING SITE ”

“

MALONYL BINDING SITE ”

ACYL-ENZYME INTERMEDIATE

Brown, E.D., Nature, 2006, 441, 293Habich, D., Nussbaum, F.V., ChemMedChem, 2006, 1, 951

O

O

S

O

ACP

R S

O

ACP

S

O

OH

O

ACP

R

O

S

O

ACP

CO2

H SFabF

CLOSED

R S

O

OPEN

FabF

H SFabF

CLOSED

Mode of Action of Platensimycin

Inhibits FabF by direct targetingProposed mechanism

R S

O

ACP

S

O

OH

O

ACP

R

O

S

O

ACP

CO2

H SFabF

CLOSED

R S

O

OPEN

O

O

S

O

ACP FabF

H SFabF

CLOSED

“

ACYL BINDING SITE ”

“

MALONYL BINDING SITE ”

ACYL-ENZYME INTERMEDIATE

Brown, E.D., Nature, 2006, 441, 293Habich, D., Nussbaum, F.V., ChemMedChem, 2006, 1, 951

PMN

N

N

N

N His303His298

H H

O S

OO

pantetheine

Active site tunnel

Phe229

Mode of Action of Platensimycin

Inhibits FabF by direct targeting

Wang, J., et al, Nature, 2006, 441, 358Heath, R.J., Nat.Prod.Rep., 2002, 19, 581

McGuire, K.A., Biochemistry, 2001, 40, 9836

Mutated FabF FabB

A.E.IntermediateO

O

NH

OOH

OH

HO

O

Mode of Action of Platensimycin

Inhibits FabF by direct targeting

Direct binding assay employing 3H-dihydroplatensimycin and purified recombinant enzyme

Wang, J., et al, Nature, 2006, 441, 358

• Developing resistance through mutations at active site

would be hard

O

O

NH

OOH

OH

HO

O

O

OO

NH

COOH

OH

HO O

OO

HO

OMOM

OMOMNH2MeO2C

1 2 3

O

OO

o

O

O

O

TBSO

4567

Retrosynthesis

of Platensimycin

Amide bond formation

Double alkylation

Ketyl radical cyclizationAnd Etherification

Cycloisomerization

Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

O

EtO

O

EtO

OTBS

Br

OTBS1. LDA

2. LDABr

3. DIBAL-H, -78oCto-20oCthenHCl

4. TBSCl

O

TBSO

O

TBSO

CpRu(MeCN)3 PF6

cat.

6. LiHMDS, TMSCl

5.

7. 1.1eq. Pd(OAc)222oC

8. HCl aq

O

O

8 10 11

1213

O

TBSO

9

6

92%

97% 84%twosteps

92%1: 1d.r.

68%twosteps85%

Racemic Synthesis of PlatensimycinSynthesis of pentacyclic

carboxylic acid -

3

Cycloisomerization

Dehydrosilylation

Appel reaction

Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

O

O 6

O

OO

OH

8. 2.2 eq. SmI2 ,

HFIP, THF/HMPA-78oC

O

OO

HO

9. TFA

10. KHMDS,MeI

O

O

11. KHMDS

I

O

OBO

O

BO

O

12. Grubbs catalyst13. 5 eq. Me3NO

95%

14. 3 eq. NaClO2,2-methyl-2-buteneNa2PO4, tBuOH/H2O

15 16

1718193

46%, 2:1 d.r.

87%

88%

79%

6 eq.

85%, 6:1 E / Z95%

Racemic Synthesis of PlatensimycinSynthesis of pentacyclic

carboxylic acid -

3

Single electron reduction

Pinnick oxidation

Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

Racemic Synthesis of PlatensimycinSynthesis of aromatic amine -

2

OHNO2

OH

OMOMNH2

OMOM

OMOMNHR

OMOM2. H2,Pd/C cat.

1. NaH, MOMCl

OMOMNHR

OMOMCO2Me

OMOMNH2

OMOMCO2Me

3. Boc2O

MeO CN

O

R = BoC

5. 2050C, Microwave

5 min.

2

20 21 22

23

82%

99%

99%

4. 1 eq.nBuLi,1 eq. TMSCl

then 2.2 eq. nBuLi

54%

83%

Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

O

OO

HOOMOM

NH2OMOM

MeO2C

O

OONH

CO2MeOMOM

OMOM

1. 4 eq.HATU, Et3N

O

OO

NH

COOHOH

OH

2. LiOH

3. aq. HCl

2 3 24

1

85%

90% two steps

Racemic Synthesis of PlatensimycinCoupling of Carboxylic acid-3 with Aromatic amine-2

Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

HATU

–

O-(7-azabenzotriazol-1-yl)-N,N,N`,N`-tetramethyluronium hexafluorophosphate

O

OO

HOOMOM

NH2OMOM

MeO2C

O

OONH

CO2MeOMOM

OMOM

1. 4 eq.HATU, Et3N

O

OO

NH

COOHOH

OH

2. LiOH

3. aq. HCl

2 3 24

1

85%

90% two steps

Racemic Synthesis of PlatensimycinCoupling of Carboxylic acid-3 with Aromatic amine-2

Racemic PMN Nicolaou, K.C., Li, A., Edmonds, D.J., Angew. Chem. Int. Ed. 2006, 45, 7086

Summary and The Future Goals

O

O

NH

OOH

OH

HO

OPreclinical and

clinical trials

Explore the parts of PMN that are not involved in FabF binding

Designing analoguesFor the SAR studies

Enantioselective

synthesis

Proven potency as an antibacterial against superbugs

Developing resistance will be a slow process

No mammalian cell cytoxicity

Acknowledgement

Dr. Walker Dr. Borhan Dr. Jackson Dr. Wulf

Dr. Hausinger Dr. Arnosti

Dr. Stoltzfus

Labmates

-

Amanda, Danielle, Karin, Mark, Sanjit, Washington, Yemane

Friends

-

Aman

D., Calvin, Chandana, Samantha, Sanjukta,Sue,

Sulagna,Tharanga, Toyin, Xiaofei, Zhenjie

Dr.Sheo

B. Singh, Dr.Stephen

Soisson, Dr.Jun

Wang

Happy Valentine’s Day!

Happy Valentine’s Dayfilled with Love and Hope !

Few back-up slides

Discovery of Platensimycin Relative

Steriochemistry

NOESY

18-methyl and H-11(axial) -

β-faceH-9 and H-14 (axial) -

α-face

Discovery of Platensimycin Structure

Elucidation

HMBCAmino benzoic Acid Unit

HOHH

HO

O OHNH

6`

2`3`

7`

Discovery of Platensimycin Structure

Elucidation

HMBCKetolide

Unit

Cyclohexenone ring

OH

H

O1

4 6

8

18

Biosynthesis of Malonyl

CoA

Biotincarrierprotein

HNO

S

HNNH

O

RS

HNNH

O

S

HNN

O

BaseHO O

OPO

OHOH

HO O

O

HOP

ADP

O

OH

RS

HN

O

OH

O

H2C

O

SCoA

O

O O

SCoA

Microbiological profiles and Toxicity of Platensimycin

More potent than FabF inhibitor cerulenin