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Probing Nature for Antibiotics
Irosha Nayanthika NawarathneMichigan State University
04/30/08
health.howstuffworks.com
Struggle for living
dansaper.blogspot.com, www.photos-screensaver-maker.com, tecnocientista.info.com, www.creswell-crags.org.uk
“History of humankind can be regarded from a medicinal point of view as a struggle against infectious diseases”
Yoneyama, H., Katsumata, R., Biosci. Biotechnol. Biochem., 2006, 70,1060
Survival against infectious diseases
dodd.cmcvellore.ac.in, www.ayurvedicmedicine4u.com, www.rootsweb.com
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
How do the antibiotics act against bacteria?
Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19www.jacksofscience.com
Cell Wall Biosynthesis
β-lactams,Cyclosporins,Glycopeptides
How do the antibiotics act against bacteria?
Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.istockphoto.com
Protein BiosynthesisAminoglycosides,Macrolides,Tetracyclines,Oxazolidinones
How do the antibiotics act against bacteria?
Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 publications.nigms.nih.gov, www.istockphoto.com
DNA BiosynthesisQuinolones
RNA BiosynthesisRifampicin
How do the antibiotics act against bacteria?
Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.istockphoto.com
Metabolic pathways
Folic Acid MetabolismTrimethoprim, Sulfonamides
Fatty Acid BiosynthesisTriclosan, Isoniazid, Ethionamide
Why do we need more antibiotics?
- Developing antimicrobial resistance
Bacterial species Common types of Antimicrobial Types of Infections ResistanceStreptococcus 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
What should be targeted?
The compounds with,
Novel structures
New modes of action
Fernandes, P., Nature Biotechnology, 2006, 24, 1497
Where do the antibiotics come from?
NATURE
Where do the antibiotics come from?
NATURE
NP SS TS
Where do the antibiotics come from?
NATURE
NP SS TS
Helps in designing the molecules
Natural products as antibiotics Naturally occurring compounds that are end products
of secondary metabolism. Mostly extracted from plants, marine organisms, or
microorganisms.
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Natural products as antibiotics Naturally occurring compounds that are end products
of secondary metabolism. Mostly extracted from plants, marine organisms, or
microorganisms. Eg:
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Pal, S., Tetrahedron, 2006, 62, 3171
O
O
O
Me
OHO
O
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
Erythromycin
Isolation - Streptomyces erythreus in 1952Uses - Respiratory tract diseases, genital infectionsMOA - Inhibition of protein synthesis
Antibiotics which are semi-synthesized
Synthetically modified chemical compounds which are originated
from natural products.
Walsh, C., Antibiotics Actions origins and Resistance, 2003, 4
Erythromycin is
Acid unstableO
O
O
Me
OHO
O
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
O
O
Me
OO
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
HO
O
O
Me
OHO
O
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
O
O
Me
OO
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
O
O
Me
OO
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
69
12
O
O
OO
HO
O
-H2O
H2O
Pal, S., Tetrahedron, 2006, 62, 3171
Antibiotics which are semi-synthesized
O
O
O
Me
OMeO
O
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
96
12
N
O
O
Me
OMeO
O
Me2N
MeHO
Me
Me
N
O
96
12
N
O
O
OHO
O
Me2N
MeHO
Me
MeO O Me
OHOMeMe
HO
HO
96
12
O
O
Me
OMeO
O
Me2N
MeHO
Me
Me
N
O
96
12
Me
OO O
O
N
NN
Me
Clarithromycin Azithromycin
HMR3647TE802
Pal, S., Tetrahedron, 2006, 62, 3171
Antibiotics which are totally from synthesis
Totally synthesized molecules which are potent as antibiotics. Three main types. 1. Sulfa drugs 2. Quinolones 3. Oxazolidinones
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis
SO
O HN
H2N
ON
Sulfa drugs (Sulphonamides)
Uses - Urinary tract infections, pneumonia etc.MOA - Inhibition of folate synthesis
Harold, P.L., O’Grady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272
Sulfamethoxazole
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis
SO
O HN
H2N
ON
Sulfa drugs (Sulphonamides) Naturally occurring
Uses - Urinary tract infections, pneumonia etc.MOA - Inhibition of folic acid biosynthesis
Harold, P.L., O’Grady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272
Sulfamethoxazole
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82
p-aminobenzoic acid
H2N
COOH
Antibiotics which are totally from synthesis
HNN N
OCO2HF
Quinolones
Ciprofloxacin
Uses - Urinary tract infections, Lower respiratory infections, Gastrointestinal infectionsMOA - Inhibition of DNA synthesis
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis
HNN N
OCO2HF
NH
O
Quinolones Naturally occurring
N
O
OHCiprofloxacin
Uses - Urinary tract infections, Lower respiratory infections, Gastrointestinal infectionsMOA - Inhibition of DNA synthesis
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Kunze, B., Hofle, G., Reichenbach, H., J. Antibiotics, 1987, 40, 258
Aurachin C
Aurachin D
Antibiotics which are totally from synthesis
NO
F N O
O
H
HN
O
CH3
Oxazolidinones
Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current Drug Targets-Infectious Disorders, 2001, 1,181
Linezolid
Uses - Soft tissue infections, skin infections, Tuberculosis etc.MOA - Inhibition of protein synthesis
Antibiotics which are totally from synthesis
NO
F N O
O
H
HN
O
CH3
MeO
OHN
O
OH
Oxazolidinones Naturally occurring
Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current Drug Targets-Infectious Disorders, 2001, 1,181Zappia, G., et al., Mini-Reviews in Medicinal Chemistry, 2007, 7, 389
Linezolid
Uses - Soft tissue infections, skin infections, Tuberculosis etc.MOA - Inhibition of protein synthesis
(-)-Cytoxazone
NO
OHH
O
H
(+)-Sreptazolin
Sources of antibacterial drugs from 1981 to 2002
10%
68%
21% 1%NP
SS
TS
NM
Newman, D.J., Cragg, G.M., Snader, K.M., J. Nat. Prod., 2003, 66, 1022
Ways of probing nature for antibiotics
NATURE
Approach ABy exploring the novel
Natural Products
Approach BGenerating
the Nature Mimics
Ways of probing nature for antibiotics
NATURE
Approach ABy exploring the novel
Natural Products
Approach BGenerating
the Nature Mimics
New antibiotics New architectural scaffolds
Approach AConventional way of NP discovery
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006www.spc.int, www.oceanexplorer.noaa.gov, www.nature.com, www.textbookofbacteriology.net
Extraction to the solvents
Isolation and Structure Elucidation
Natural materials
Bioassay guided fractionation
Approach AConventional way of NP discoveryWhy isn’t it successful?
Problems associated with the growth or the availability of the source
Replication of the hits Do not distinguish novel from old Mostly miss the novel compounds due to the lack of
sensitivity No hints about MOA Cannot reveal potency at screening stage
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541
Approach AWhat are the new strategies to explore
nature for NPs
Molecular Biology based Techniques
Novel culturing techniques
Heterologous expression of biosynthetic genes & Metagenomics
Genomics and Combinatorial biosynthesis
Precursor directed biosynthesis & Mutasynthesis
Differential sensitivity screening approach
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541
Donadio, S., Chemistry & Biology, 2006, 13, 560
Approach AWhat are the new strategies to explore
nature for NPs
Molecular Biology based Techniques
Novel culturing techniques
Heterologous expression of biosynthetic genes & Metagenomics
Genomics and Combinatorial biosynthesis
Precursor directed biosynthesis & Mutasynthesis
Differential sensitivity screening approach
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541
Donadio, S., Chemistry & Biology, 2006, 13, 560
Approach A
Extraction
to the Solvents
Producing organisms
found in nature
Pathogen
Precursor Directed Biosynthesis & Mutasynthesis
Wild type
Mutant type
Approach APrecursor Directed Biosynthesis & Mutasynthesis
HO
O OH
O
OH
OH
O
O
OH
O
O
O OH
Natural Biosynthetic pathway
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
Wild type
Approach APrecursor Directed Biosynthesis and Mutasynthesis
HO
O OH
O
OH
OH
O
O
OH
O
O
O OH
HO
O
N
O
OH
O
O
O
N
Precursor-Directed Biosynthesis
Wild type
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
Approach APrecursor Directed Biosynthesis and Mutasynthesis
HO
O OH
O
OH
OH
OHO
O
NO
OH
O
O
O
N
Mutasynthesis
Mutant
Mutasynthon
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
Approach AMutasynthesis
Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901Galm, U., et al, Chemistry & Biology, 2004, 11, 173
Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
OH
HN
O
O O
OH
MeO
OMe
MeMeO
O OHO
NH2
Ring A Ring B Ring C
Novobiocin (Albamycin)
OH
HN
O
O O
OH
ClO
OMe
MeMeO
O OHO
NH
Me
Ring A Ring B Ring C
Clorobiocin
Approach AMutasynthesis
OH
HN
O
O O
OH
ClO
OMe
MeMeO
O OHO
NH
Me
CloQ- mutants
Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901Galm, U., et al, Chemistry & Biology, 2004, 11, 173
Eustảquio, A.S., et al, Arch. Microbiol., 2003, 180, 25
Approach AMutasynthesis
Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901
CloQ-mutantClorobiocin
Approach AMutasynthesis
CloQ-mutantClorobiocin
OH
O
HO
Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901
Approach AMutasynthesis
CloQ-mutantAnalogs of Clorobiocin
HN
O O
OH
ClO
OMe
MeMeO
O OHO
NH
Me
O
RCl
OH
O
HO Br
OH
O
HO
HN
OH
O
HO
OH
O
HO
HN
OH
O
HO
OH
O
HO
OH
O
HO
HN
OH
O
HO
CH3
NH2
O
HO
O
O O O
R1
R2
O
HO
R3
Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Galm, U., et al, Antimicrob. Agents Chemother., 2004, 48, 1307
Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901
Approach AWhat are the new strategies to explore
nature for NPs
Molecular Biology based Techniques
Novel culturing techniques
Heterologous expression of biosynthetic genes & Metagenomics
Genomics and Combinatorial biosynthesis
Precursor directed biosynthesis & Mutasynthesis
Differential sensitivity screening approach
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541
Donadio, S., Chemistry & Biology, 2006, 13, 560
Approach ADifferential sensitivity screening
approach
Extraction
to the solvents
Couzin, J., Nature, 2006, 314, 34, ForsythR.A., Molecular Biology, 2002, 43, 1387
Wang, J., et al, Antimicrob. Agents Chemother., 2006, 50, 519
Producingorganism from nature
Disabled type
Wild type
Increased sensitivity
Low
Normal
Pathogen Expression of certain protein/s
Target the pathway
Approach ADifferential sensitivity screening approachFatty Acid Biosynthesis… 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
HO
O O
SCoA O
O O
S ACP
O O
S ACP
OH O
S ACP
O
S ACP
O
S ACP
CoASH
FabD
C02 + CoASHFabH
CoA
O
NADPH NADP
FabG
H2O
FabAFabZ
FabIFabKFabL
NADPH NADP
Biosynthesis of Saturated Fatty Acids
Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305
HO
O O
SCoA O
O O
S ACP
O O
S ACP
ACPSH CoASH
FabD
C02 + CoASHFabF
NADPH NADP
FabG
H2O
FabAFabZ
FabIFabKFabL
NADPH NADP
O
S
OH O
S ACP
O
S ACP
O
S ACP
ACP
Biosynthesis of Saturated Fatty Acids
Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305
HO
O O
SCoA O
O O
S ACP
O O
S ACP
ACPSH CoASH
FabD
C02 + CoASHFabF
NADPH NADP
FabG
H2O
FabAFabZ
FabIFabKFabL
NADPH NADP
O
S
OH O
S ACP
O
S ACP
O
S ACP
ACP
Biosynthesis of Saturated Fatty Acids
Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305
Antisense RNA
mRNA
5` ………ATGGCCTGGACTTCA…………3` 3` ………TACCGGACCTGAAGT…………5`
Sense DNA Antisense DNA
Transcription
5` ………AUGGCCUGGACUUCA…………3`
Met - Ala - Trp - Thr - Ser -
Translation
Peptide
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
Approach A
RNA-mediated gene silencing techniqueDifferential sensitivity screening approach
Reduced or No FabF expression
5`……… AUGGCCUGGACUUCA………3`3`……… UACCGGACCTGTTGU ………5`
ds RNA
Degradation of fabF mRNA or inhibition of translation
In Prokaryotes-
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
RNA-mediated gene silencing techniqueDifferential sensitivity screening approachApproach A
Approach ADifferential sensitivity screening
approachResults - RNA-mediated gene silencing technique
Wild type
fabF Anti-sense
Wang, J., et al, Nature, 2006, 441, 358
Inhibitor (μg)
Approach ADifferential sensitivity screening
approachResults - RNA-mediated gene silencing technique
Wild type
fabF Anti-sense
Wild type
fabF Anti-sense
200 times more potent than Cerulenin
Wang, J., et al, Nature, 2006, 441, 358Price, 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
Inhibitor (μg)
Approach ADifferential sensitivity screening
approach
O
O
NH
OOH
OH
HO
O
Platensimycinfrom a strain of Streptomyces platensis
Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916
Discovery of Platensimycin
Approach ADifferential sensitivity screening
approachPotency of PlatensimycinOrganism and genotype Platensimycin Linezolid
Antibacterial activity (MIC, µg/ml) S. aureus (MSSA) 0.5 4 S. aureus (MRSA) 0.5 2 S. aureus (MRSA, macrolideR) 0.5 2 S. aureus (MRSA, linezolidR) 1 32 Enterococcus faecium (VRE) 0.1 2
MIC – Concentration of inhibitor used to result no visible growth of the pathogens
Wang, J., et al, Nature, 2006, 441, 358
Toxicity (µg/ml) HeLa MTT (IC50) >1,000 >100
IC50 – Concentration of the inhibitor used to kill 50% population of the living cells
Cell - free gel - elongation assay
Wang, J., et al, Nature, 2006, 441, 358
Malonyl-ACPC4:1(Δ2)-ACP
C4:0-ACP
>6C-ACP
Approach ADifferential sensitivity screening approachHigh FabF selectivity
Heath, R.J., Nat.Prod.Rep., 2002, 19, 581
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
CoASH
FabD
C02 +CoASHFabH
CoA
O
NADPH
NADP
FabG
H2O
FabAFabZ
FabIFabKFabL
FabF
NADPH
NADP
Ways of probing nature for antibiotics
NATURE
Approach ABy exploring the novel
Natural Products
Approach BGenerating
the Nature Mimics
New antibiotics New architectural scaffolds
Approach B
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541
Generating Nature Mimics
Biosynthetic pathway
Designing theoretical chemical space that fits the active site or
docking the database structures
Translate to a real structure by synthesis
Enzyme purification &
3D structural determination
Approach B
Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Essential for the bacterial growth
Does not exist in mammals
Generating Nature MimicsBiosynthesis of lysine… A good target
isoleucine
threonine
methionine
Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Biosynthesis of lysine
HO
O
H2N CO2H
O
O
H2N CO2H
PO
HOO
H
O
H2N CO2H
NHO2C
OH
CO2H
NHO2C CO2H
NHO2C CO2H
HO2C
O
CO2H
NHR
HO2C
NH2
CO2H
NHR
HO2C
NH2
CO2H
NH2
HO2C
NH2
CO2H
NH2
H2N CO2H
NH2
aspartokinase
ASA-DH
ASA
DHDPSpyruvate
HTPA
DHDP
THDP
DHDPR
acyl-CoA THDP N-acyltransferase
R = succinyl / acetyl
DAP-ATDAP deacylase
LL-DAP
DAP-epimerase
meso-DAP
DAP decarboxylase
lysine
R = succinyl / acetyl
isoleucine
threonine
methionine
HO
O
H2N CO2H
O
O
H2N CO2H
PO
HOO
H
O
H2N CO2H
NHO2C
OH
CO2H
NHO2C CO2H
NHO2C CO2H
HO2C
O
CO2H
NHR
HO2C
NH2
CO2H
NHR
HO2C
NH2
CO2H
NH2
HO2C
NH2
CO2H
NH2
H2N CO2H
NH2
aspartokinase
ASA-DH
ASA
DHDPSpyruvate
HTPA
DHDP
THDP
DHDPR
acyl-CoA THDP N-acyltransferase
R = succinyl / acetyl
DAP-AT DAP deacylase
LL-DAP
DAP-epimerase
meso-DAP
DAP decarboxylase
lysine
R = succinyl / acetyl
Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Biosynthesis of lysine
Approach BGenerating Nature Mimics
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Proposed mechanism
Approach BGenerating Nature Mimics
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
Supportive data
Acyl-enzyme intermediate(Streptococcus pneumoniae)
Faehnle, C.R., Coq, J.L., Liu, X., Viola, R.E., Journal of Biological Chemistry, 2006, 281, 31031 Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Approach BGenerating Nature Mimics
Cox, R.J., Gibson, J.S., Martín, M.B.M., Chem. Commun., 2001, 1710Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
Inhibitors of lysine biosynthesis
Approach BGenerating Nature Mimics
Cox, R.J., Gibson, J.S., Martín, M.B.M., Chem. Commun., 2001, 1710Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
Inhibitors of lysine biosynthesis
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Approach BGenerating Nature Mimics
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255
Reverse Biosynthesis
OH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH
In vitro assays
Approach B
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Generating Nature Mimics
KI (ASA) KI (Phosphate)
- -
750 μM 2130μM
214 μM 92μM
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Direct assay
OH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH
Competitive assays
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Approach BGenerating Nature Mimics
Direct assay
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Competitive assaysOH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Approach BGenerating Nature Mimics
Direct assay
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Competitive assaysOH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Approach B
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Generating Nature Mimics
Direct assay
- - 4.2-5.0
750 μM 2130μM 6.1
214 μM 92μM 6.2-6.4
KI (ASA) KI (Phosphate) 2nd pKa
OH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH
Competitive assays
Approach B
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Generating Nature Mimics
Pre-incubation assay
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
95μM
-
-
KI (ASA)
OH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH + inhibitor
Time-dependent inhibition assays
Approach B
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
OH
O
NH2
X
OP
HOHO
O
Generating Nature Mimics
Pre-incubation assay
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
X
O
H3N CO2
PO
HOO
Cys-EnzS
X
H3N CO2
PO
HOO
O SCys-Enz
ASA-DH
Time-dependent inhibition assaysOH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
ASA-DH + inhibitor
Ways of probing nature for antibiotics
NATURE
Approach ABy exploring the novel
Natural Products
Approach BGenerating
the Nature Mimics
New antibiotics New architectural scaffolds
Please, Don’t flush!
Average american receives more than 11 prescriptions a year.
About 3.3 billion a total.
Nonprescription drugs !
Halford, B., C & EN News, 2008, 86, 13Halford, B., C & EN News, 2008, 86, 16
Acknowledgement
Dr. WalkerDr. HausingerDr. ArnostiDr. StoltzfusDr. Stephen Soisson, Dr. Jun Wang (Merck)
Labmates - Behnaz, Danielle, Joshua, Mark, Washington, Yemane
Friends - Samantha, Sue, Tharanga, Xiaofei
Thank you all !Thank you all !
Back-up slides
Approach ADifferential sensitivity screening approachIn vivo studies of Platensimycin
In a mouse model of disseminated S. aureus infection
Wang, J., et al, Nature, 2006, 441, 358
Timeline of discovery of novel classes of antibiotics and introduction in clinic
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Approach BGenerating the Nature Mimics
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
Faehnle, C.R., Coq, J.L., Liu, X., Viola, R.E., Journal of Biological Chemistry, 2006, 281, 31031 Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458
Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115
Antibiotics which are totally from synthesis
SO
O HN
H2N
ON
GTP DHP DHF THF
Sulfa drugs (Sulphonamides) Naturally occurring
Uses - Urinary tract infections, pneumonia etc.MOA - Inhibition of folate synthesis
Harold, P.L., O’Grady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272
Sulfamethoxazole
Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82
HN
N NH
NO
O PPH2N
COOH
H2N
p-aminobenzoic acid
Approach APrecursor Directed Biosynthesis
Nayer, J.H.C., Trends. Biochem. Sci., 1991, 16, 195Nayer, J.H.C., Trends. Biochem.Sci., 1991, 16, 234
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25
Penicillium Chrysogenum
(Penicillium notatum)
N
S
HHO
OO
H HH2N
6-APA
Approach APrecursor Directed Biosynthesis
Penicillium Chrysogenum
(Penicillium notatum)
N
S
HHO
OO
H HHN
O
N
S
HHO
OO
H HHN
O
OOO
OH
O
OH
Penicillin G
Penicillin V
Nayer, J.H.C., Trends. Biochem. Sci., 1991, 16, 195Nayer, J.H.C., Trends. Biochem.Sci., 1991, 16, 234
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25
Approach AMutasynthesis
Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901Galm, U., et al, Chemistry & Biology, 2004, 11, 173
Eustảquio, A.S., et al, Arch. Microbiol., 2003, 180, 25
OH
HN
O
O O
OH
MeO
OMe
MeMeO
O OHO
NH2
Ring A Ring B Ring C
Novobiocin (Albamycin)
OH
HN
O
O O
OH
ClO
OMe
MeMeO
O OHO
NH
Me
Ring A Ring B Ring C
Clorobiocin
Nov L Clo L
Where do the antibiotics come from?
NATURE
NP SS TS
Kekule stucture of benzene
www.boomeria.org
Approach APrecursor Directed BiosynthesisDrawbacks
Involves complex purification procedures
Require high concentrations of synthetic precursor
Only few intermediates will incorporate into the product
Kennedy, J., Nat. Prod. Rep., 2008, 25, 25Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
O S
OO
S
O
O O
SSH
OH O
SSH
O
SSH
O
S
SH
O
SSH
O
O
SCoA
O
CO2 NADPH + HNADP
H2O
NADPH + H
NADP
CoASH
A
O
S
SH
Continues...
Cerulenin,Thiolactomycin
Triclosan,Isoniazid,
Ethionamide
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
Inhibitors of Fatty Acids Biosynthesis
Where do the antibiotics come from?
NATURE
Approach B
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Generating the Nature Mimics
OH
O
NH2OHP
HOHO
O
F F
OH
O
NH2OHP
HOHO
O
H H
Cox, R.J., Gibson, J.S., Martín, M.B.M., Chem. Commun., 2001, 1710Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
O
O
H3N CO2
PO
HOO
Cys-EnzS
O
H3N CO2
PO
HOO
O SCys-Enz
OS
Cys-Enz
CO2H3N
HOPO
O
OS
Cys-Enz
CO2H3N
OH
CO2H3N
H
NADPHNADP
Cys-EnzS
ASA-DH
acyl-enzymeintermediate
ASA
O
OH
O
NH2OP
F FHO
HOO
OH
O
NH2OP
HOHO
O
OH
O
NH2
HN
OP
HOHO
O
H H
Approach B
OH
O
NH2
O
OP
HOHO
OOH
O
NH2O
NADP NADPH
H2PO4
Generating the Nature Mimics
Direct assay
Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
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
A
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
O S
OO
S
O
O O
SSH
OH O
SSH
O
SSH
O
S
SH
O
SSH
O
O
SCoA
O
CO2 NADPH + HNADP
H2O
NADPH + H
NADP
CoASH
A
O
S
SH
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
Continues...