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Inhibiting the World’s Deadliest Toxin: Botulinum Neurotoxin (BoNT) Teresa Beary Blackwell Group October 15, 2009.

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Presentation on theme: "Inhibiting the World’s Deadliest Toxin: Botulinum Neurotoxin (BoNT) Teresa Beary Blackwell Group October 15, 2009."— Presentation transcript:

1 Inhibiting the World’s Deadliest Toxin: Botulinum Neurotoxin (BoNT) Teresa Beary Blackwell Group October 15, 2009

2 Natural sources of BoNT Clostridium botulinum: anaerobic, spore-forming, bacteria, lives in soil – Produces 7 immunologically distinct toxins, A-G – Type A is most common & most potent – Estimated LD 50 (for 150 lb human)  Intramuscular: 90 – 150 ng  Inhalation: 700 – 900 ng  Orally: 70 μg 2 Arnon, S.; et al. JAMA 2001, 285, ; Natural vectors of botulism: foodborne, wound, infant

3 The deadliest toxin in the world Class A bioterrorism agent: 275 g would kill the entire US population Arnon, S.; et al. JAMA 2001, 285, ; 3 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

4 Equine botulinum antitoxin Paralysis persists until reinnervation Arnon, S.; et al. JAMA 2001, 285, ; ; Schantz, E.; Johnson, E. Perspect. Biol. Med. 1997, 40, Epidemiology and therapeutics Onset and symptoms 6 hrs – 10 days (foodborne) 12 hrs – 4 days (inhalation) Treatment 4 vision speech swallowing Lab diagnostic: mouse bioassay

5 Worldwide therapeutic Truong, D.; Jost, W. Parkinsonism Relat D 2006, 12, ; Brin, M. Toxicon 2009, 54, Mass immunization is undesirable Edward Schantz (UW-Madison)

6 Normal neurotransmitter release Willis, B.; et al. Angew. Chem. Int. Ed. 2008, 47, Neuromuscular synapse muscle cell acetylcholine receptors SNARE proteinsmusclecontraction Neurotransmitter vesicle ( acetylcholine) complexation muscle cell

7 BoNT/A mechanism of action 7 Breidenbach, M.; Brunger, A. Nature 2004, 432, Neuromuscular synapse BoNT/A – zinc metalloprotease Heavy chain Light chain endocytosis No release of acetylcholinemuscleparalysis muscle cell translocation SNAP-25 cellular receptors

8 Glu His Tyr Gln Arg Mechanism of SNAP-25 cleavage Zuniga, J.; et al. Structure 2008, 16, Glu His Tyr Gln Arg Glu His Tyr Gln Arg Glu His Tyr Gln Arg BoNT/A SNAP-25

9 Approaches for inhibitor development 1. SNAP-25 peptide mimics 2. Pharmacophore screening 3. Hydroxamate-based structures 9 Glu His Tyr Gln Arg Glu

10 Enzymatic assays and definitions Boldt, G.: et al. J. Comb. Chem. 2006, 8, Fluorometric assay inhibitorBoNT/A LC + SNAPtide Relative fluorescence IC 50 : concentration that results in 50% inhibition K i : inhibition constant HPLC-based assay + SNAP-25 cleaved intact

11 Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu SNAP-25 (residues ) Zinc-chelating peptide inhibitors 11 Schmidt, J.; et al. FEBS Lett. 1998, 435, ; Schmidt, J.; Stafford, R. FEBS Lett. 2002, 532, Cys Arg Ala Thr Lys Met Leu Inhibitor CRATKML, K i = 1.9 μM Asp Glu Ser Ala K i (μM) 600 2,500 1,400 2,500 mpp-RATKML K i = 0.33 μM

12 Tetrapeptide inhibitors Kumaran, D.; et al. J. Biol. Chem. 2008, 283, K i (μM) Arg Arg Gly Cys0.157 Arg Arg Gly Leu0.660 Arg Arg Gly Ile0.786 Arg Arg Gly Met0.845 Gln Arg Ala Thr

13 I1 SNAP-25 Zn +2 Non zinc-chelating peptides Zuniga, J.; et al. Structure 2008, 16, Gln Arg Ala Thr Lys Met Leu SNAP-25 Inhibitor I1 – K i = μM Met Leu Arg Trp Thr DNP-DABDAB Future cyclic peptide inhibitors

14 Initial pharmacophore proposal stericelectronic Pharmacophore: an ensemble of steric and electronic features that is necessary to ensure the optimal interactions with a specific biological target Burnett, J.; et al. Biochem. Bioph. Res. Co. 2003, 310, 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

15 4-amino-7-chloroquinoline (ACQ) derivatives Burnett, J.; et al. Biochem. Bioph. Res. Co. 2003, 310, Q2-15: 60% inhibition at 20 μM Possible dual activity: inhibit protease and prevent translocation translocation Chloroquine: 20% at 50 μM

16 Peptide contributions to pharmacophore Burnett, J.; et al. J. Biol. Chem. 2007, 282, Molecular docking analysis Account for SAR studies Arg Ala Thr Lys Met Leu mpp-RATKML K i = 0.33 μM Fits A - E F & G F F: Potential new H-bonds G G: hydrophobic anchor Leu: optimal length ~24 Å

17 New 4-amino-7-chloroquinoline derivatives Burnett, J.; et al. J. Med. Chem. 2007, 50, IC 50 = 7.0 μM IC 50 = 3.2 μM

18 New scaffolds and cell-based assays Burnett, J.; et al. J. Biol. Chem. 2007, 282, Became concentrated within cells in 30 minutes Well tolerated up to 40 μM Dose-dependent protection of SNAP-25 Chicken motor neuron assay NSC K i = 10 μM, IC 50 = 3.0 μM untreated10 nM BoNT/ABoNT/A + 40 uM

19 Continued optimization Hermone, A.; et al. ChemMedChem 2008, 3, NSC Q2-15 NSC

20 Synthesizing a chimera Burnett, J.; et al. Bioorg. Med. Chem. Lett. 2009, 19, Mixture K i = 0.6 μM 1)NSC PhSH, EtOH 2)RP-HPLC MeOH: H 2 O 0.1% TFA  4 TFA 4 : 1 A : B A B

21 Tested Gln and Arg hydroxamates, 50 μM Hydroxamate-based inhibitors Boldt, G.; et al. J. Comb. Chem. 2006, 8, % inhibition75% inhibition K i = 60 μM R = 82% 68% 82% No improvement Gln Arg Ala Thr SNAP-25

22 More robust development of hydroxamates Boldt, G.; et al. Org. Lett. 2006, 8, p-chloro-cinnamic hydroxamate IC 50 = 15 μM derivatives N 2 CH 2 NH 2 OH cat. KCN 150 compounds IC 50 = 0.41 μM K i = 300 nM 2,4-dichloro-cinnamic hydroxamate

23 Cell-based and in vivo assays Eubanks, L.; et al. P. Natl. Acad. Sci. USA 2007, 104, Mouse cholinergic neuron assays Toxic at concentrations ≥ 5 μM Mouse toxicity bioassay IC 50 = 0.41 μM K i = 300 nM Discrepancy between in vitro and in vivo 1) BoNT/A 2) 1 mM inhibitor 16% survival No toxicity observed

24 Crystal structures reveal active site flexibility Silvaggi, N.; et al. Chem. Biol. 2007, 14, ,4-dichlorocinnamic hydroxamate L -arginine hydroxamate aspartic acid phenylalanine Zn 2+

25 2 nd generation hydroxamates Čapková, K.; et al. Bioorg. Med. Chem. Lett. 2007, 17, Fused ring derivatives: IC 50 = 21 – 71 μM R = Br, Me, CF 3 IC 50 = 0.6 – 0.8 μM Equivalent to R = Cl (IC 50 = 0.9 μM) X = Cl, Br Y = C, S, NH, NMe

26 Summary 26 K i = 41 nM Mixture K i = 600 nM K i = 300 nM Met Leu Arg Trp Thr DNP-DABDAB Pseudopeptide Pharmacophore design Hydroxamate  4 TFA

27 Future 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, IC 50 = 2.1 μM

28 Acknowledgements Helen Blackwell Blackwell Group Practice talk attendees Aaron McCoy Kelsey Mayer Paul White Joey Stringer Reto Frei Margie Mattmann Aaron Crapster Tony Breitbach Joe Grim Drew Palmer Christie McInnis J. P. Gerdt Knick Praneenararat 28


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