Probing Nature for Antibiotics

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

Kevin D Walker

How ancient people survived? Using nature.

Survival against infectious diseases

dodd.cmcvellore.ac.in, www.ayurvedicmedicine4u.com, www.rootsweb.com

Kevin D Walker

They used natural resources to fight the infectious diseases. Nature was the solution at all times.

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

Owner

Commonest infectious diseases like Lower respiratory tract diseases, Meningitis, Diarrhea can be cured using Antibiotics.

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


Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.istockphoto.com

Protein BiosynthesisAminoglycosides,Macrolides,Tetracyclines,Oxazolidinones


Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 publications.nigms.nih.gov, www.istockphoto.com

DNA BiosynthesisQuinolones

RNA BiosynthesisRifampicin


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

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

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


NATURE

NP SS TS

Kevin D Walker

Either the treatment for Infectious disease is a Natural product, semisynthetic analog or a total synthetic molecule nature supplies all of them


NATURE

NP SS TS

Helps in designing the molecules

Kevin D Walker

Nature helps in designing the total synthetic 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

Me

O O Me

OHOMeMe

HO

HO

Erythromycin

Isolation - Streptomyces erythreus in 1952

Uses - Respiratory tract diseases, genital infections

MOA - 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 unstable

O

O

O

Me

OHO

O

Me2N

MeHO

Me

Me

O O Me

OHOMeMe

HO

HO

O

O

Me

OO

Me2N

MeHO

Me

Me

O O Me

OHOMeMe

HO

HO

HO

O

O

Me

OHO

O

Me2N

MeHO

Me

Me

O O Me

OHOMeMe

HO

O

O

Me

OO

Me2N

MeHO

Me

Me

O O Me

OHOMeMe

HO

HO

O

O

Me

OO

Me2N

MeHO

Me

Me

O O Me

OHOMeMe

HO

6

9

12

O

O

O

O

HO

O

-H2O

H2O

Pal, S., Tetrahedron, 2006, 62, 3171

Antibiotics which are semi-synthesized

O

O

O

Me

OMeO

O

Me2N

MeHO

Me

Me

O 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

Me

O 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



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



SO

O HN

H2N

ON

Sulfa drugs (Sulphonamides) Naturally occurring


MOA - Inhibition of folic acid biosynthesis


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


HN

N N

O

CO2HF

Quinolones

Ciprofloxacin

Uses - Urinary tract infections, Lower respiratory infections, Gastrointestinal infectionsMOA - Inhibition of DNA synthesis



HN

N N

O

CO2HFNH

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


N

O

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


N

O

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

Kevin D Walker

Almost every drug so far has derived from nature. For novel drugs why wouldn't we rely on nature. So best way to find new drugs is by searching them in nature.

Ways of probing nature for antibiotics

NATURE

Approach ABy exploring the novel

Natural Products

Approach BGenerating

the Nature Mimics


NATURE


Natural Products


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


Donadio, S., Chemistry & Biology, 2006, 13, 560

Kevin D Walker

Due to the time issues the last two methods will be discussed using several examples.


nature for NPs









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


Kevin D Walker

Synthetic analog of one of the precursors is supplied to the culture of producing organism and it get incorporate into a new product.

Approach APrecursor Directed Biosynthesis and Mutasynthesis

HO

O OH

O

OH

OH

O

HO

O

NO

OH

O

O

O

N

Mutasynthesis

Mutant

Mutasynthon


Kevin D Walker

The biosynthetic pathway of producing that precursor is interrupted. So the organism has to use the supplied analog.This method will lead to produce new compounds which are possibly biologically active.

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

Me

OO

Me

Me

MeO

O OHO

NH2

Ring A Ring B Ring C

Novobiocin (Albamycin)

OH

HN

O

O O

OH

Cl

OO

Me

Me

MeO

O OHO

NH

Me


Clorobiocin

Kevin D Walker

Biologically active compounds but not so useful as they are toxic to human cells. Use of above method led to synthesise compounds in this category with low or no toxicity but still potent antibiotics.


OH

HN

O

O O

OH

Cl

OO

Me

Me

MeO

O OHO

NH

Me

CloQ- mutants


Eustảquio, A.S., et al, Arch. Microbiol., 2003, 180, 25


Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901

CloQ-mutant

Clorobiocin


CloQ-mutant

ClorobiocinOH

O

HO

Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901


CloQ-mutant

Analogs of Clorobiocin

HN

O O

OH

Cl

OO

Me

Me

MeO

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


nature for NPs









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


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



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



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


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


RNA-mediated gene silencing techniqueDifferential sensitivity screening approachApproach A

Owner

The low FabF expressing S.aureus cells were made using described technique and the natural products were screened against them. They found only one hit which led to the discovery of Platensimycin.

Kevin D Walker

The organism is modified such a way that it's not capable of normal expression of a useful protein to it's survival. So it is indeed disable. So more prone to be affected by even relatively weaker inhibitor then.That gives the organism to show higher sensitivity towards the inhibitors of that specific enzyme.


approachResults - RNA-mediated gene silencing technique

Wild type

fabF Anti-sense

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

Inhibitor (μg)


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

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


approachPotency of Platensimycin

Organism 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


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


Malonyl-ACPC4:1(Δ2)-ACP

C4:0-ACP

>6C-ACP

Approach ADifferential sensitivity screening approach

High 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

Owner

The assay was performed using radioactive malonyl coA. Scintillation film was taken after the protein gel electorphoresis. PMN shows the accumulation of butroyl ACP inhibiting the elongation condensation step which is facilitated by FabF enzyme.


NATURE


Natural Products


the Nature Mimics


Approach B


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

Kevin D Walker

This is the way of how nature gives ideas in designing totally synthetic molecules with bioactivity.

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

Kevin D Walker

Lysine and few other amino acids (threonine, methionine, isoleucine) are supplied to human only through their diet. We cannot produce it in the body.

isoleucine

threonine

methionine


Biosynthesis of lysine

HO

O

H2N CO2H

O

O

H2N CO2H

P

O

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


Kevin D Walker

This pathway is important to bacteria as the highlighted compounds involve in cell signaling, methylations and cell wall biosynthesis.

isoleucine

threonine

methionine

HO

O

H2N CO2H

O

O

H2N CO2H

P

O

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


DAP-AT DAP deacylase

LL-DAP

DAP-epimerase

meso-DAP

DAP decarboxylase

lysine



Biosynthesis of lysine

Kevin D Walker

Focus will be given to the enzyme activity, mechanism and dynamics of ASA-DH (Aspartate semi aldehyde dehydrogenase).

Approach BGenerating Nature Mimics

O

O

H3N CO2

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH

acyl-enzymeintermediate

ASA

O


Proposed mechanism


O

O

H3N CO2

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH


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


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

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH


ASA

O

Inhibitors of lysine biosynthesis



O

O

H3N CO2

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH


ASA

O

Inhibitors of lysine biosynthesis

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

Kevin D Walker

Synthetic analogs of starting material to inhibit ASA-DH enzyme and then the whole pathway of producing lysine.


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

O

PHO

HOO

OH

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


KI (ASA) KI (Phosphate)

- -

750 μM 2130μM

214 μM 92μM

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

Direct assay

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH

Competitive assays

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H


Direct assay



Competitive assays

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H


Direct assay



Competitive assays

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH

Kevin D Walker

In order to bind to the active site very well and inhibit the reaction, phosphate needs to be singly charged.

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

Approach B




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

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH

Competitive assays

Kevin D Walker

The flouro groups make the phosphate group in the first analog to be doubly charged be changing the 2nd pKa to a low value. So it doesn't act as a very good inhibitor.

Approach B

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H


Pre-incubation assay



95μM

-

-

KI (ASA)

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH + inhibitor

Time-dependent inhibition assays

Approach B

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

OH

O

NH2

X

O

PHO

HOO


Pre-incubation assay



X

O

H3N CO2

P

O

HOO

Cys-EnzS

X

H3N CO2

P

O

HOO

O SCys-Enz

ASA-DH

Time-dependent inhibition assays

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

ASA-DH + inhibitor

Kevin D Walker

Here the increased electrophilicity of the carbonyl by fluoro groups led the first compound to be more potent, since nuclephile would favour attacking it more than attacking the natural substrate.


NATURE


Natural Products


the Nature Mimics


Kevin D Walker

Nature is the best source of antibiotics, forever.Looking at it in different ways and being creative will result in finding more and more drugs in future.

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 approach

In vivo studies of Platensimycin

In a mouse model of disseminated S. aureus infection


Timeline of discovery of novel classes of antibiotics and introduction in clinic


Approach BGenerating the Nature Mimics

O

O

H3N CO2

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH


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


SO

O HN

H2N

ON

GTP DHP DHF THF

Sulfa drugs (Sulphonamides) Naturally occurring


MOA - Inhibition of folate synthesis


Sulfamethoxazole

Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82

HN

N NH

N

O

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

OO

O

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



Eustảquio, A.S., et al, Arch. Microbiol., 2003, 180, 25

OH

HN

O

O O

OH

Me

OO

Me

Me

MeO

O OHO

NH2


Novobiocin (Albamycin)

OH

HN

O

O O

OH

Cl

OO

Me

Me

MeO

O OHO

NH

Me


Clorobiocin

Nov L Clo L


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


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

Owner

Fab F catalyses the elongation condensation step.


NATURE

Approach B

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

Generating the Nature Mimics

OH

O

NH2OH

PHO

HOO

F F

OH

O

NH2OH

PHO

HOO

H H


O

O

H3N CO2

P

O

HOO

Cys-EnzS

O

H3N CO2

P

O

HOO

O SCys-Enz

OS

Cys-Enz

CO2H3N

HOP

O

O

OS

Cys-Enz

CO2H3N

OH

CO2H3N

H

NADPHNADP

Cys-EnzS

ASA-DH


ASA

O

OH

O

NH2O

P

F FHO

HOO

OH

O

NH2O

PHO

HOO

OH

O

NH2

HN

O

PHO

HOO

H H

Approach B

OH

O

NH2

O

O

PHO

HOO

OH

O

NH2O

NADP NADPH

H2PO4

Generating the Nature Mimics

Direct assay



SH

SH

SH

S

O

O S

OO

S

O

O O

SSH

OH O

SSH

O

SSH

O

S

SH

O

SSH

SCoA

O

O

O

SCoA

O

CO2NADPH + H

NADP

H2O

NADPH + H

NADP

CoASH

CoASH

A


FabD

FabH FabG

FabZ

FabI / K / L


ACP

Owner

ACP- Acyl carrier proteinFabH catalyses the initial condensation step.

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


FabD

FabF FabG

FabZ

FabI / K / L


Continues...

Owner

Fab F catalyses the elongation condensation step.

Documents

Probing Nature for Antibiotics