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