Discovery and Development of Novel Small Molecule Inhibitors of Botulinum Neurotoxin A

1. Discovery and Development of Novel Small Molecule Inhibitors of Botulinum Neurotoxin A Terry Bowlin, Ph.D. Microbiotix, Inc. Worcester, MA

2. BoNT Inhibitor Discovery MBX Overview BoNT Background BoNT Drug Discovery BoNT Assays

3. MICROBIOTIX A small molecule, anti-infective drug discovery company Terry L. Bowlin, Ph.D., CEO Worcester, MA

4. Microbiotix Corporate Overview • Launched in January 2000 with offices and laboratories in Worcester, Massachusetts • Core antibiotics technology based on scientific founders’ research at U Mass on inhibition of bacterial DNA replication • 10,739 sq. ft. of fully equipped office and microbiology and medicinal chemistry laboratory space • Fully integrated infectious disease microbiology and medicinal chemistry drug discovery capability • 25 employees with extensive experience in drug discovery and development • Active biodefense program for the discovery and development of novel antibacterial, antiviral and antivirulence factor therapeutics • Current preclinical pipeline of novel anti-bacterial and anti-herpes inhibitor

5. Microbiotix Discovery Platform Proprietary Screens: • Enzyme based • purified enzymes essential for replication (e.g., polymerase, gyrase, topoisomerase, helicase) • Cell based • permeabilized bacterial replication screen • whole-cell target-based luciferase reporter screens • Biofilm • HTS for identification of biofilm inhibitors • Types of readouts • UV/Vis absorbancy, fluorescence, FRET, time-resolved FRET, luminescence, radioisotopic Medicinal Chemistry: • Fully integrated medicinal chemistry drug discovery unit Compound Library: • Greater than 100K compounds with greater than 200 druglike chemotypes Lodish et al. 2003. Molecular Cell Biology, 5th ed.

6. Microbiotix Anti-Infective Drug Discovery • Compound Libraries • (Drug-like compounds • & natural products) • MBX 500 • MBX 222 Lead Identification • Medicinal Chemistry • IC50 & MIC criteria • Serum effect • In vitro therapeutic index criteria • Confirmed SAR • MOA confirmation • Freedom to operate • Biochemical Screens • Cell-Based Screens • Re-tested in quadruplicate HTS & Confirmation Confirmed Hits Validated Hits Lead Compounds • Secondary Assays • IC50 & MIC criteria • In vitro therapeutic index criteria • QC & stability • Ranked by criteria • Low resistance freq. • Passed acute tox • Effective in animals • Scalable synthesis • Patentable • Satisfactory market Lead Optimization Hit Validation • In Licensing • MBX 1107 (USAMRIID) • MBX 400 (Wayne St. U.) • Preclinical Candidates • MBX 500 • MBX 400

7. Microbiotix Drug Discovery Portfolio

8. BoNT Inhibitor Discovery MBX Overview BoNT Background BoNT Drug Discovery BoNT Assays

10. BoNT Medical Uses Cosmetic (Wrinkles, etc.) Dystonia (Muscle Contraction) Hyperhidrosis(Excess Sweating) Strabismus(Crossed Eyed) Blepharospasm(Excessive Blinking) Back Pain Migraine (Tension Headaches) Incontinence

12. Botulinum neurotoxins (BoNTs) are the most potent of • the biological toxins • Of the botulinum neurotoxins, BoNT/A is the most • potent (lethal dose 1ng/kg) • Due to their lethality, BoNTs are listed as category A • (highest priority) biothreat agents by the CDC • BoNTs are easily produced and may be delivered by • aerosol route • Consequently, these toxins represent a serious threat • to both military personnel and civilians The BoNT Threat

13. BoNT Serotypes • BoNT secreted by the anaerobic spore-forming bacterial • Clostridia species • Seven BoNT serotypes exists (A-G), which differ • significantly in amino acid sequence, protein substrates, • and substrate cleavage sites • Significant differences in the duration of the paralysis • caused by each

14. BoNT Mediated Paralysis • Significant differences in the duration of the paralysis • caused by each serotype: • BoNT/A paralysis lasts the longest, typically 4-6 • months, and this is a primary reason why it has • become popular for both medicinal and cosmetic • applications • The duration of paralysis from BoNT/A coupled with its • potency and the fact that several high resolution • crystal structures are available have made it possibly • the most tractable and relevant for immediate drug • discovery efforts

16. BoNT Substrate • Once inhaled into the lung, BoNTs are taken up by the • blood stream, target the peripheral cholinergic nerve • endings, and cause death by interrupting autonomic • nerve function • The zinc-dependent endopeptidase light chain (LC) • portion of BoNTs impair neuronal exocytosis through • proteolysis of essential SNARE (soluble NSF- • ethylmaleimide-sensitive factor attachment protein • receptor) components of neurotransmission

17. Binding • Internalization • Translocation • (LC release) • Proteolytic • Cleavage • SNARE • complex

18. Therapeutic Approaches to BoNT Inhibition

19. BoNT Current Treatment • The currently available BoNTtoxoid vaccine, as well as experimental preventative antibodies, cannot counter these toxins after they penetrate neurons • Critical care mechanical ventilation is the only treatment option once neurons have been intoxicated and diaphragm muscles cease to function • The effects of internalized BoNTs can last for months (6), and long-term mechanical ventilation would be impractical if even a limited number of individuals were simultaneously intoxicated • Therefore, there is an urgent need to identify and develop low molecular weight non-peptidic inhibitors that will serve as both prophylactics and post-exposure ‘rescue’ therapeutics

20. BoNT Inhibitor Discovery MBX Overview BoNT Background BoNT/A Inhibitor Drug Discovery Assays/Results

21. BoNT Drug Discovery • Due to the lethality and difficulty of treating • intoxication with BoNTs, new small-molecule • inhibitors of these toxins are critically needed. • We have identified a new series of BoNT/A • inhibitors with potency in both enzyme and cell- • based primary neuronal assays.

22. Compound Evaluation Flow Chart

23. BoNT Biological Assays • FRET Assays • HPLC Assay • Neuronal Cell Assays

24. Enzyme Based Assays Fluoresence Resonance Energy Transfer (FRET) Standard Assay For Recombinant BoNTLcA (DACIA SUBSTRATE) For Characterization of MBX Compounds REAGENT [STOCK] QUANTITY (L) [FINAL] DMSO or Compound 100 % 1 1 % Sterile Water 55M 44 N/A HEPES pH 7.4 200 mM 25 50 mM Tween 20 0.5% 10 0.05% BonTLcA 1 g/mL 10 10 ng in rxn DACIA Substrate 200 M 10 20 M Incubate at 37°C for 40 minutes. Monitor Ex 398 nm Em 485 nm every minute for kinetic measurement. At the end of 40 minutes, stop reactions with 10 µL 5% Acetic Acid. Read Ex 398 nm Em 485 nm in endpoint mode. Alternative Assay For Recombinant BoNTLcA (FITC SUBSTRATE) For Characterization of MBX Compounds REAGENT [STOCK] QUANTITY (L) [FINAL] DMSO or Compound 100 % 1 1 % Sterile Water 55M 34 N/A HEPES pH 8.2 200 mM 25 50 mM Tween 20 0.5% 20 0.1% BonTLcA 1 g/mL 10 10 ng in rxn FITC Substrate 100 M 10 10 M Incubate at 37°C for 60 minutes. Monitor Ex 490 nm Em 523 nm every minute for kinetic measurement. At the end of 60 minutes, stop reactions with 10 µL 500 mM EDTA pH 8.0. Read Ex 490 nm Em 523 nm in endpoint mode.

26. Detection Method for BoNTLcA FRET (DACIA) Assay

27. Enzyme Based Assay-HPLC REAGENT [STOCK] QUANTITY (L) [FINAL] DMSO or Compound 100% 1.5 1% Sterile Water 55M 46 N/A HEPES pH 7.4 200 mM 38 50 mM NP-40 0.5% 15 0.05% BoNTLcA 1 g/mL 45 45 ng in rxn Substrate (DACIA) 2 mM 4.5 60 M Incubate at 37°C for 40 minutes. At the end of 40 minutes, stop reactions with 15 µL 5% Acetic Acid. Read Ex 398 nm Em 485 nm in endpoint mode. HPLC Conditions Solvent A: 0.1% TFA Solvent B: 0.1% TFA in 70% Acetonitrile Inject 100 µl sample Gradient: 35% B to 40% B over 21 min, 100% for 10 min Monitor effluent at 365 nm

28. Detection Method for BoNTLcA HPLC Assay Heat Denatured BoNTLcA Reaction Native BoNTLcA Reaction

29. Compound Library 20,000 cpds Maybridge & Microsource Discovery 50,000 cpds Chembridge DIVERSetTM • Chemical Filters: • To include: • ~200-500 Da • Lipinski “rule of 5” • To exclude: • Cytotoxic fragments • Metal complexes • Highly conjugated ring systems • Oxime esters • Nitroso groups • Strong Michaelson acceptors HTS Screening Library 100,000 Cpds ~200 chemotypes 3,770 cpds, natural products & derivatives AnalytiCon Discovery 30,000 cpds GLSynthesis, MBX, & other sources

30. Ongoing HTS at Microbiotix • Libraries Screened: • Tim Tec Natural Products • Chembridge 50K • Chem Div 2 Typical Z’ Score=0.69

31. Examples of Select Screening Hits

32. USAMRIID HTS BoNT/A LC Inhibitors

34. The BoNT/A LC pseudo-peptide inhibitor Mpp-RATKML (Ki=330nM) docked within the BoNT/A LC substrate binding cleft (Burnett et al, JBC, 2007, 282: 5004-14)

35. A Refined Pharmacophore for BoNT/A LC Inhibition Planar Components: A&B Hydrophobic Components: C&D Positive Ionizable Component: E

36. New BoNT/A LC Inhibitors: Potencies, Search Query Fits and Distances Between Components

37. Chick Neuronal Cell Assay • Embryonic chicken spinal motor neuron cells were isolated utilizing methods • described by Kuhn • Neuronal cell cultures were incubated overnight at 37°C prior to BoNT/A • intoxication • Cells were pre-incubated with inhibitor for 45 min, followed by 3.5 hour • incubation with 10 nMBoNT/A and inhibitor • Cells were then lysed • Lysates were run on a 12% gel and transferred to nitrocellulose • Blots were probed with SMI 81 mouse anti-SNAP-25 primary antibody, • followed by probing with horseradish peroxidase-conjugated goat anti-mouse • secondary antibody in combination with ECL Western blotting detection system • Developed blot is analyzed via densitometry (UN-SCAN-IT gel automated • digitizing system) Burnett et al. (2007) J. Biol. Chem. 282, 5004-5014 Kuhn, T.B. (2003) Methods Cell Biol. 71,67-87

38. Chick Neuronal Cell Morphological Analysis Green=staining for tubulin Red=staining for actin filaments Blue=staining for DNA NSC 240898 is well tolerated by neurons and is an effective inhibitor of BoNT/A LC-mediated cleavage of SNAP-25 in cells

39. SNAP-25 Western Blot Analysis Chick Primary Neuronal Cells

40. Analysis of Hit NSC240898 • Neuron uptake • BoNT/A LC inhibition: 61%@20 µM • CC50 > 40 µM NSC240898 MBX-1131 Optimization Type I analogs Three-ring scaffold Type II analogs Two-ring scaffold

41. Docking Analysis 2,4-dichlorocinnamic hydroxamate MBX-1107

42. BoNT SAR: Summary • Basic substituents at R are required for BoNTLcA inhibitory activity • Basic substituents at R’ increase activity further • Small substituents on indole N are tolerated • Heteroatoms Y decrease BoNT activity • Small substituents such as F, Cl at R’’ are tolerated • Substitution of the phenoxy group with indole maintains potency

43. Structures of BoNT/A Inhibitors MBX 1107 MBX 1131 (NSC240898) MBX 1130 MBX 1140 MBX 1195 MBX 1340 MBX 1341

45. BoNT LcA Enzymatic Activity • The original lead NSC 240898 was resynthesized (MBX 1131) and demonstrated to • be as potent as it was in the original screen, with an IC50 of 16.5 µM • MBX 1107, a structural analog of MBX 1131, is as potent as MBX 1131 in the • enzymatic (FRET and HPLC) assays • MBX 1107 shows greater specificity for BoNTLcA than does MBX 1131 • in assays for related metalloproteases (BoNTLcB, anthrax lethal factor and human • MMPs) • Compounds MBX 1130, 1140, 1196, 1340 and 1341, with related but • distinct bis-(indole) structures, are the most potent BoNTLcA enzyme inhibitors we • have synthesized to date, with MBX 1140 displaying a 10-fold increase in potency • over MBX 1131 and 1107

46. Rat Neuronal Cell Assay Cells are harvested from 7-8 day old rat cerebella, washed and cultured in 6-well plates (>7days) Once the cells have become networked, they are preincubated (15min.) with test compounds or diluent (DMSO) Cells are inoculated with BoNT/A and incubated for 3 hrs (37 °C) Cells are treated with 1 M NaOH, to inactivate the BoNT and lysed. Samples are run on SDS-PAGE gels and transferred to membranes for immunoblot analysis with rabbit anti-SNAP-25 and HRP-conjugated goat anti-rabbit IgG Band intensities are read and normalized using scanning densitometry

47. Inhibition of BoNT/A Activity in Primary Rat Neurons by MBX Compounds at 80 µM MBX Compounds (80 mM) Uncleaved Cleaved Control BoNT/A 1130 1107 1131 1195 1340 1341

48. Dose-dependent Inhibition of BoNT/A Activity in Primary Rat Neurons by MBX 1131 MBX 1131 (µM) Control BoNT/A 100 50 25 12.5 Uncleaved Cleaved

50. BoNT/A Inhibitor Cell-Based Results • MBX 1131 is the most potent of the Microbiotix BoNT/A inhibitors in the rat neuronal SNAP-25 cleavage assay, followed by MBX 1140. MBX 1107 has very little activity in this assay. • Compounds MBX 1195, 1340 and 1341 appear to have activity at a single concentration of 80 µM.