inhibiting the world’s deadliest toxin: botulinum neurotoxin (bont) teresa beary blackwell group...

Inhibiting the World’s Deadliest Inhibiting the World’s Deadliest Toxin:Toxin:

Botulinum Neurotoxin (BoNT)Botulinum Neurotoxin (BoNT)

Teresa BearyTeresa Beary

Blackwell GroupBlackwell Group

October 15, 2009October 15, 2009

Natural sources of BoNTNatural sources of BoNTClostridium botulinum: anaerobic, spore-forming,

bacteria, lives in soil

– Produces 7 immunologically distinct toxins, A-G

– Type A is most common & most potent

– Estimated LD50 (for 150 lb human)

Intramuscular: 90 – 150 ng Inhalation: 700 – 900 ng Orally: 70 μg

2Arnon, S.; et al. JAMA 2001, 285, 1059-1070. ; http://www.cdc.gov

Natural vectors of botulism: foodborne, wound, infant

The deadliest toxin in the worldThe deadliest toxin in the worldClass A bioterrorism agent:

275 g would kill the entire US population

Arnon, S.; et al. JAMA 2001, 285, 1059-1070. ; http://www.cdc.gov3

BoNT/A is:2,000,000 x more toxic than Sarin

10,000 x ‘ ‘ ‘ VX

In 1991, Sadam Hussein reported

the production of 19,000 L of

BoNT/A to UN officials

3 x the lethal dose for the world’s population

Equine botulinum antitoxin

Paralysis persists until reinnervation

Arnon, S.; et al. JAMA 2001, 285, 1059-1070. ; http://www.cdc.gov ; Schantz, E.; Johnson, E. Perspect. Biol. Med. 1997, 40, 317-327.

Epidemiology and therapeuticsEpidemiology and therapeutics

Onset and symptoms

6 hrs – 10 days (foodborne)12 hrs – 4 days (inhalation)

Treatment

4

vision

speech

swallowing

Lab diagnostic: mouse bioassay

Worldwide therapeuticWorldwide therapeutic

Truong, D.; Jost, W. Parkinsonism Relat D 2006, 12, 331-355. ; Brin, M. Toxicon 2009, 54, 676-682. 5

Mass immunization is undesirable

Edward Schantz (UW-Madison)

Normal neurotransmitter releaseNormal neurotransmitter release

Willis, B.; et al. Angew. Chem. Int. Ed. 2008, 47, 8360-8379. 6

Neuromuscular synapse

muscle cell

acetylcholine receptors

SNARE proteins

musclemusclecontractioncontraction

Neurotransmitter vesicle( acetylcholine)

complexation

muscle cell

BoNT/A mechanism of actionBoNT/A mechanism of action

7Breidenbach, M.; Brunger, A. Nature 2004, 432, 925-929.

Neuromuscular synapse

BoNT/A – zinc metalloprotease

Heavy chain

Light chain

endocytosis No release ofacetylcholine

musclemuscleparalysisparalysis

muscle cell muscle cell

translocationSNAP-25

cellular receptors

Glu

OH

O

OZn+2

OO

H

NHH

Glu

His

His

Tyr GlnArg

Mechanism of SNAP-25 cleavageMechanism of SNAP-25 cleavage

Zuniga, J.; et al. Structure 2008, 16, 1588-1597. 8

Glu

OH

NH

O

O

O

OH

HZn+2

Glu

His

His

Tyr

GlnArg

Glu

OH

NH

OO

O

OH

HZn+2

Glu

His

His

Tyr GlnArg

Glu

OH

O

OZn+2

OO

NHH

H

Glu

His

His

Tyr GlnArg

BoNT/A

SNAP-25

Approaches for inhibitor Approaches for inhibitor developmentdevelopment

1. SNAP-25 peptide mimics

2. Pharmacophore screening

3. Hydroxamate-based structures

9

OH

NH

O

O

O

OH

HZn+2

Glu

His

His

Tyr

GlnArg

Glu

Enzymatic assays and definitionsEnzymatic assays and definitions

Boldt, G.: et al. J. Comb. Chem. 2006, 8, 513-521. 10

Fluorometric assay

inhibitor BoNT/A LC

+SNAPtide

Relative fluorescence

IC50: concentration that results in 50% inhibition

Ki: inhibition constant

HPLC-based assay

+SNAP-25

cleaved

intact

Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu

SNAP-25 (residues 187-203)

Zinc-chelating peptide inhibitorsZinc-chelating peptide inhibitors

11Schmidt, J.; et al. FEBS Lett. 1998, 435, 61-64. ; Schmidt, J.; Stafford, R. FEBS Lett. 2002, 532, 423-426.

Cys Arg Ala Thr Lys Met Leu

Inhibitor CRATKML, Ki = 1.9 μM

Asp

Glu

Ser

Ala

Ki (μM)

600

2,500

1,400

2,500

O

O

OH

O

O

Me

mpp-RATKML Ki = 0.33 μM

SH

O

Tetrapeptide inhibitorsTetrapeptide inhibitors

Kumaran, D.; et al. J. Biol. Chem. 2008, 283, 18883-18891. 12

Ki (μM)

Arg Arg Gly Cys 0.157

Arg Arg Gly Leu 0.660

Arg Arg Gly Ile 0.786

Arg Arg Gly Met 0.845

Gln Arg Ala Thr

SH

S

I1

SNAP-25

Zn+2

Non zinc-chelating peptidesNon zinc-chelating peptides

Zuniga, J.; et al. Structure 2008, 16, 1588-1597. 13

Gln Arg Ala Thr Lys Met Leu

SNAP-25

Inhibitor I1 – Ki = 0.041 μM

Met LeuArg Trp Thr

O2N NO2

NH

NH2

O

NH

NH2

O

DNP-DAB DAB

Future cyclic peptide inhibitors

Initial pharmacophore proposalInitial pharmacophore proposalPharmacophore: an ensemble of stericsteric and electronicelectronic features that is

necessary to ensure the optimal interactions with a specific biological target

Burnett, J.; et al. Biochem. Bioph. Res. Co. 2003, 310, 84-93. 14

• A & B: planar components➙ biaryl/triaryl systems

• E: positive ionizable substituent➙ salt bridges / solubility

• C & D: hydrophobic moieties➙ fit hydrophobic pockets

• A contains heteroatom➙ zinc chelators / displace water

N

HN

Cl

N

Me

4-amino-7-chloroquinoline (ACQ) 4-amino-7-chloroquinoline (ACQ) derivativesderivatives

Burnett, J.; et al. Biochem. Bioph. Res. Co. 2003, 310, 84-93. 15

N

HN

Cl

NH

NH

N Cl

Q2-15: 60% inhibition at 20 μMQ2-15: 60% inhibition at 20 μM

Possible dual activity: inhibit protease and prevent translocation

translocation

Chloroquine: 20% at 50 μMChloroquine: 20% at 50 μM

Peptide contributions to Peptide contributions to pharmacophorepharmacophore

Burnett, J.; et al. J. Biol. Chem. 2007, 282, 5004-5014. 16

Molecular docking analysis

• Account for SAR studies

SH

O

Arg Ala Thr Lys Met Leu

mpp-RATKML Ki = 0.33 μM

Fits A - E F & GF & G

• FF: Potential new H-bonds

• GG: hydrophobic anchor

• Leu: optimal length ~24 Å

OAc

OAcH

HN

NCl

HN

OMe

O

New 4-amino-7-chloroquinoline New 4-amino-7-chloroquinoline derivatives derivatives

Burnett, J.; et al. J. Med. Chem. 2007, 50, 2127-2136. 17

ICIC5050 = 7.0 μM = 7.0 μM ICIC5050 = 3.2 μM = 3.2 μM

HN

NCl

N

NH

NCl

HN

N

Cl

New scaffolds and cell-based New scaffolds and cell-based assaysassays

Burnett, J.; et al. J. Biol. Chem. 2007, 282, 5004-5014. 18

• Became concentrated within cells in 30 minutes

• Well tolerated up to 40 μM

• Dose-dependent protection of SNAP-25

Chicken motor neuron assay

O

NH

NH

NH2

HN

NH2

NSC 240898

Ki = 10 μM, IC50 = 3.0 μM

untreated 10 nM BoNT/A BoNT/A + 40 uM 240898

Continued optimizationContinued optimization

Hermone, A.; et al. ChemMedChem 2008, 3, 1905-1912. 19

O

NH

NH

NH2

HN

NH2

NSC 240898

N

HN

Cl

NH

NH

N Cl

Q2-15

N NH

N

N

HN N

NSC 328398

O

NH

N

NH2

HN

NH2HN N

Cl

O

NH

NH

NH2

N

NH2N

Cl

NH

Synthesizing a chimeraSynthesizing a chimera

Burnett, J.; et al. Bioorg. Med. Chem. Lett. 2009, 19, 5811-5813. 20

Mixture Ki = 0.6 μM

N Cl

Cl

H2N NH2

1) NSC 240898PhSH, EtOH

2) RP-HPLCMeOH: H2O0.1% TFA

4 TFA

4 TFA

4 : 1A : B

A

B

N NH

Cl

NH2

Tested Gln and Arg hydroxamates, 50 μM

Hydroxamate-based inhibitorsHydroxamate-based inhibitors

Boldt, G.; et al. J. Comb. Chem. 2006, 8, 513-521. 21

NH

HO

O

NH2

NH

NH2

HN

NH

HO

O

NH2

H2N O

5% inhibition 75% inhibition

Ki = 60 μM

NH

HO

OHN

NH

NH2

HN

R

R = SN

O OS

N

O O

82%68%

82%No improvement

Gln Arg Ala Thr

SNAP-25

More robust development of More robust development of hydroxamateshydroxamates

Boldt, G.; et al. Org. Lett. 2006, 8, 1729-1732. 22

p-chloro-cinnamic hydroxamate

Cl

O

NH

OH

IC50 = 15 μM

derivatives

R

O

OH R

O

OMe R

O

NH

OHN2CH2

NH2OH

cat. KCN

150compounds

Cl

O

NH

OH

Cl

IC50 = 0.41 μM

Ki = 300 nM

2,4-dichloro-cinnamic hydroxamate

Cell-based and Cell-based and in vivo in vivo assaysassays

Eubanks, L.; et al. P. Natl. Acad. Sci. USA 2007, 104, 2602-2607. 23

Mouse cholinergic neuron assays

Toxic at concentrations ≥ 5 μM

Mouse toxicity bioassay

Cl

O

NH

OH

Cl

IC50 = 0.41 μM

Ki = 300 nMDiscrepancy between in vitro and in vivo

1) BoNT/A

2) 1 mM inhibitor

16% survival

No toxicity observed

Crystal structures reveal active site Crystal structures reveal active site flexibilityflexibility

Silvaggi, N.; et al. Chem. Biol. 2007, 14, 533-542. 24

2,4-dichlorocinnamic hydroxamate L-arginine hydroxamate

aspartic acidaspartic acid

phenylalaninephenylalanine

Zn2+

22ndnd generation hydroxamates generation hydroxamates

Čapková, K.; et al. Bioorg. Med. Chem. Lett. 2007, 17, 6463-6466. 25

X

O

HN

OH

YCl

HN

O

OHFused ring derivatives:

IC50 = 21 – 71 μM

Cl

HN

OH

O

R R = Br, Me, CF3

IC50 = 0.6 – 0.8 μM

Equivalent to R = Cl (IC50 = 0.9 μM)

X = Cl, Br

Y = C, S, NH, NMe

SummarySummary

26

Cl

O

NH

OH

Cl

Ki = 41 nM

Mixture Ki = 600 nM

Ki = 300 nM

Met LeuArg Trp Thr

O2N NO2

NH

NH2

O

NH

NH2

O

DNP-DAB DAB

Pseudopeptide

Pharmacophore design

Hydroxamate

4 TFA

O

NH

NH

NH2

N

NH2N

Cl

NH

Future directionsFuture directions

27

• In vitro assays that better correspond to in vivo results

• Inhibitors designed to distort active site cavity

• Drug-candidate refinements

➙ potency

➙ absorption

➙ distribution

➙ metabolism

➙ toxicity

Roxas-Duncan, V.; et al. Antimicrob. Agents. Ch. 2009, 53, 3478-3486.

NH

N

N

O

OH

Cl

IC50 = 2.1 μM

AcknowledgementsAcknowledgementsHelen Blackwell

Blackwell Group

Practice talk attendeesAaron McCoyKelsey MayerPaul WhiteJoey StringerReto FreiMargie MattmannAaron CrapsterTony BreitbachJoe GrimDrew PalmerChristie McInnisJ. P. GerdtKnick Praneenararat

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