October 29, 2008

Discovery and Development of Novel Small Molecule Inhibitors of

Botulinum Neurotoxin A

Terry Bowlin, Ph.D.Microbiotix, Inc.Worcester, MA

October 29, 2008

BoNT Inhibitor Discovery

MBX Overview

BoNT Background

BoNT Drug Discovery

BoNT Assays

October 29, 2008

MICROBIOTIX

A small molecule, anti-infective drug discovery company

Terry L. Bowlin, Ph.D., CEOWorcester, MA

October 29, 2008

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

October 29, 2008

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 unitCompound Library: Greater than 100K compounds with greater than 200 druglike chemotypes

Lodish et al. 2003. Molecular Cell Biology, 5th ed.

October 29, 2008

Microbiotix Anti-Infective Drug Discovery

Confirmed Hits

Validated Hits

Lead Compounds

Preclinical Candidates•MBX 500•MBX 400

• Biochemical Screens• Cell-Based Screens• Re-tested in quadruplicate

Compound Libraries(Drug-like compounds

& natural products)•MBX 500•MBX 222

• Secondary Assays•IC50 & MIC criteria•In vitro therapeutic index criteria

• QC & stability

• Medicinal Chemistry• IC50 & MIC criteria• Serum effect• In vitro therapeutic index criteria• Confirmed SAR• MOA confirmation• Freedom to operate

• Ranked by criteria• Low resistance freq.• Passed acute tox• Effective in animals• Scalable synthesis• Patentable• Satisfactory market

HTS & Confirmation

Hit Validation

Lead Identification

Lead Optimization

In Licensing•MBX 1107 (USAMRIID)•MBX 400 (Wayne St. U.)

October 29, 2008

Microbiotix Drug Discovery Portfolio

PROJECT THERAPEUTIC TARGET MOLECULAR TARGET STATUS

ANTI-BACTERIAL

MBX-500 Gram +; MRSA/VRE Polymerase; Gyrase/Topoisomerase

Pre-clinical IND enabling

MBX-1162 Broad Spectrum Antibiotic (biodefense)

DNA/Helicase Pre-clinicalIND enabling

MBX-1131 C. botulinum (biodefense) BoNT /A LC SAR

ANTI-VIRAL

MBX-400 Anti-beta/gamma Herpes; (HCMV/HHV6/HHV8)

Polymerase Preclinical IND-enabling

MBX-222

MBX-1325

EboV (biodefense)

HCV

Fusion

Polymerase

Hit/lead

Hit/lead

October 29, 2008

BoNT Inhibitor Discovery

MBX Overview

BoNT Background

BoNT Drug Discovery

BoNT Assays

October 29, 2008

October 29, 2008

BoNT Medical Uses

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

October 29, 2008

October 29, 2008

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

October 29, 2008

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

BoNT Serotypes

October 29, 2008

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

BoNT Mediated Paralysis

October 29, 2008

October 29, 2008

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

BoNT Substrate

October 29, 2008

1. Binding2. Internalizat

ion3. Translocati

on(LC release)

4. ProteolyticCleavageSNAREcomplex

October 29, 2008

Therapeutic Efficacy Limitations

Anti-BoNT MAbs Effective in mice (in vivo toxin neutralization when premixed with BoNT/A prior to injection)

3 MAbs required (oligoclonal) for adequate potency; limited post-exposure utility

Receptor decoys Effective in nerve assays when premixed prior to contact

Co-administration of gangliosides required; limited post-exposure utility

HC inhibitors Effective in isolated mouse diaphragm muscle twitch model

Mechanism unclear; associated cytotoxicity of anti-malarials; no post-exposure protection

LC inhibitors – peptides Efficacy demonstrated in vitro only

Non drug-like molecules with poor ADME features

LC inhibitors – small molecules

Efficacy in vitro, & in neuronal cell culture or synaptosomes

Higher potency with suitable ADME properties needed

Therapeutic Approaches to BoNT Inhibition

October 29, 2008

The currently available BoNT toxoid 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

BoNT Current Treatment

October 29, 2008

BoNT Inhibitor Discovery

MBX Overview

BoNT Background

BoNT/A Inhibitor Drug Discovery

Assays/Results

October 29, 2008

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.

BoNT Drug Discovery

October 29, 2008

MBX & NERCE cpd libraries

(A) In vitro Potency• HPLC-based assay

NSC240898Derivatives

• IC50 ≤ 100 nM

FIG. 9. Compound Evaluation Flow Chart

Primary Screen (Identify & confirm backup hit series)

(B) Specificity• Test of Zn++ chelation

• Human MMP’s• BoNT/B, BoNT/F, AT-LF

(C) Cytotoxicity• CC50 vs. human cells• Damage to neurons

(D) In vivo Potency• Inhibition of SNAP-25 cleavage

• Rescue of axon length loss

• CC50/IC50 >100

• IC50endo/IC50BoNT/A >10

Compounds suitable for Aim 3

Feed

bac

k to

SA

R

• IC50 <1 μM

Aim 1

Aim 2

NSC240898

S B

D D

Compound Evaluation Flow Chart

October 29, 2008

BoNT Biological Assays

FRET Assays

HPLC Assay

Neuronal Cell Assays

October 29, 2008

Standard Assay For Recombinant BoNT LcA (DACIA SUBSTRATE)For Characterization of MBX Compounds

REAGENT [STOCK] QUANTITY (L) [FINAL]DMSO or Compound 100 % 1 1 %Sterile Water 55M 44 N/AHEPES pH 7.4 200 mM 25 50 mM Tween 20 0.5% 10 0.05%BonT LcA 1 g/mL 10 10 ng in rxn DACIA Substrate 200 M 10 20 MIncubate 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 BoNT LcA (FITC SUBSTRATE)For Characterization of MBX Compounds

REAGENT [STOCK] QUANTITY (L) [FINAL]DMSO or Compound 100 % 1 1 %Sterile Water 55M 34 N/AHEPES pH 8.2 200 mM 25 50 mM Tween 20 0.5% 20 0.1%BonT LcA 1 g/mL 10 10 ng in rxn FITC Substrate 100 M 10 10 MIncubate 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.

Enzyme Based AssaysFluoresence Resonance Energy Transfer (FRET)

October 29, 2008

NH2-S-N-R-T-R-I-D-E-A-N-K-R-A-C-R-M-L-COOH

NH2-S-N-R-T-R-I-D-E-A-N-K-COOH

NH2-R-A-C-R-M-L-COOH +

NHNH

NO

O

NO O

BoNT/A LC

O

CH3

NO

NH

CH2

O

S

CH3

CH3

NHNH

NO

O

NO O

O

CH3

NO

NH

CH2

O

S

CH3

CH3

398 nm

398 nm

485 nm

FIG. 2. FRET Assay for BoNT/A LC Peptidase Activity

485 nm

485 nm

485 nm

NH2-S-N-R-T-R-I-D-E-A-N-K-R-A-C-R-M-L-COOH

NH2-S-N-R-T-R-I-D-E-A-N-K-COOH

NH2-R-A-C-R-M-L-COOH +

NHNH

NO

O

NO O

BoNT/A LC

O

CH3

NO

NH

CH2

O

S

CH3

CH3

NHNH

NO

O

NO O

O

CH3

NO

NH

CH2

O

S

CH3

CH3

398 nm

398 nm

485 nm

FIG. 2. FRET Assay for BoNT/A LC Peptidase Activity

485 nm

485 nm

485 nm

October 29, 2008

D1 HEAT DENATURED BoNT LcA CONTROLE1 10 ng BoNT LcA

Time (secs)

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 24000

200

400

600

800

1000

1200

1400

1600

1800

2000

Well D1 E1

Vmax Per Second -0.001 0.787

R^2 0.154 0.989

Vmax Points = 41

Detection Method for BoNT LcA FRET (DACIA) Assay

October 29, 2008

REAGENT [STOCK] QUANTITY (L) [FINAL]DMSO or Compound 100% 1.5 1%Sterile Water 55M 46 N/AHEPES pH 7.4 200 mM 38 50 mM NP-40 0.5% 15 0.05%BoNT LcA 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 ConditionsSolvent A: 0.1% TFA Solvent B: 0.1% TFA in 70% AcetonitrileInject 100 µl sampleGradient: 35% B to 40% B over 21 min, 100% for 10 minMonitor effluent at 365 nm

Enzyme Based Assay-HPLC

October 29, 2008

Detection Method for BoNT LcA HPLC Assay

Heat Denatured BoNT LcA Reaction

Native BoNT LcA Reaction

October 29, 2008

HTS Screening Library 100,000 Cpds ~200 chemotypes

20,000 cpds

Maybridge & Microsource Discovery

50,000 cpds

Chembridge DIVERSetTM

3,770 cpds, natural products & derivatives

AnalytiCon Discovery

30,000 cpds

GLSynthesis, MBX, & other sources

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

Compound Library

October 29, 2008

Number of Compounds Screened

Number of Primary Hits

Number Confirmed via FRET Assay

% Final Confirmed Hit Rate: FRET + Ongoing HPLC Secondary Assays

70,400 330 114 0.16

Libraries Screened:Tim Tec Natural ProductsChembridge 50KChem Div 2

Ongoing HTS at Microbiotix

Typical Z’ Score=0.69

October 29, 2008

Examples of Select Screening Hits

IDIC50 (µM) FRET

IC50 (µM) HPLC

IDIC50 (µM) FRET

IC50 (µM) HPLC

CB 6346186 16 58 CB 7620237 24 88

CB 6352178 5.92 35 CB 7662532 31 72

CB 6696465 33 78 CB 7725216 10 27

CB 6698977 19 78 CB 7738585 16 >100

CB 7774777 39 ND CB 7869065 10 14

CB 7781727 38 ND CB 7853216 7.15 10

CB 7785416 18 61 CB 7898734 8.22 13

CB 7836164 13 59 CB 7924532 15 12

October 29, 2008

USAMRIID HTS BoNT/A LC Inhibitors

Structure Compound ID%

InhibitionStructure Compound ID

% Inhibition

NSC 661,755 62% Q2-61 50%

NSC 357,756 57% Q2-15 60%

NSC 119,889 56% Q2-43 52%

MeO

HONH

HO OH

OMe

OHHN

OHHO

NH

NH2 O

N

NHHN

N

OHO OH

I

I

I

I

COOH

NH

NH

HN

N

NCl

Cl

NH

NH

N

Cl

NH

N

Cl

NH

N

N

Cl

NH

N

Cl

October 29, 2008

October 29, 2008

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)

October 29, 2008

A

Refined Pharmacophore for BoNT/A LC Inhibition

Planar Components: A&B

Hydrophobic Components: C&D

Positive IonizableComponent: E

October 29, 2008

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

October 29, 2008

1) Embryonic chicken spinal motor neuron cells were isolated utilizing methodsdescribed by Kuhn

2) Neuronal cell cultures were incubated overnight at 37°C prior to BoNT/A intoxication

3) Cells were pre-incubated with inhibitor for 45 min, followed by 3.5 hour incubation with 10 nM BoNT/A and inhibitor

4) Cells were then lysed5) Lysates were run on a 12% gel and transferred to nitrocellulose6) 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

7) Developed blot is analyzed via densitometry (UN-SCAN-IT gel automated digitizing system)

Chick Neuronal Cell Assay

Burnett et al. (2007) J. Biol. Chem. 282, 5004-5014Kuhn, T.B. (2003) Methods Cell Biol. 71,67-87

October 29, 2008

Chick Neuronal Cell Morphological Analysis

Green=staining for tubulinRed=staining for actin filamentsBlue=staining for DNA

NSC 240898 is well tolerated by neuronsand is an effective inhibitor of BoNT/ALC-mediated cleavage of SNAP-25 in cells

October 29, 2008

SNAP-25 Western Blot Analysis

Chick Primary Neuronal Cells

October 29, 2008

NSC240898MBX-1131

Neuron uptake BoNT/A LC inhibition: 61%@20 µM CC50 > 40 µM

NH

OHN

H2NNH

NH2

Type I analogsThree-ring scaffold

Type II analogsTwo-ring scaffold

Optimization

Analysis of Hit NSC240898

R X YO

R'X = NH, S

R = CN, CONH2, C(=NH)NH2

Y = CH, N

R X YR'

X = NH, S

R = CN, CONH2, C(=NH)NH2

Y = CH, N

October 29, 2008

Cl

Cl

O

NH

OH

2,4-dichlorocinnamic hydroxamate

NH

N

NH

O

HNN

MBX-1107

Docking Analysis

October 29, 2008

Basic substituents at R are required for BoNT LcA 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

BoNT SAR: Summary

R X YO

R'

R''

October 29, 2008

NH

H2N

NH

O

NH2HN

•2 HCl

MBX 1131 (NSC240898)MBX 1131 (NSC240898)

NH

ON

NH

HNN

MBX 1107MBX 1107

NH

NH

H2N

NH

NH2

NH•2 HClNH

N

NH2

N NH

N

NH2

N

• 4TFA

MBX 1140MBX 1140

MBX 1130MBX 1130

Structures of BoNT/A InhibitorsStructures of BoNT/A Inhibitors

OHNNH

N

NH HN

N•2 TFA

MBX 1195MBX 1195

NH

NH

N

NH

N

NH

HO OH

•2 TFA

MBX 1341MBX 1341

MBX 1340MBX 1340NH

NH

N

NH HN

N

HO

HO OH

OH•2 HCl

October 29, 2008

Enzyme Specificity of Select BoNT LcA InhibitorsIC50 (µM)

MBX ID

BoNT/A Fluorescence

Assay

BoNT/A HPLC Assay

BoNT/B HPLC Assay

AT LF MMP-1 MMP-9

1131 16.5 11 21 17 >100 11

1107 12.5 9.4 >100 43 >100 24

1130 15 8.9 26 5.5 >100 < 25

1140 1.35 0.84 8.1 0.83 >100 35

1195 IND 2.7 4.4 3.9 ND ND

1340 4.4 2.8 ND ND ND ND

1341 2.8 3.2 ND ND ND ND

AT LF, Bacillus anthracis lethal factor; MMP, human Matrix Metalloprotease; IND, Indeterminate due to autofluorescence or quenching of the compound; ND, Not determined

October 29, 2008

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 BoNT LcA than does MBX 1131 in assays for related metalloproteases (BoNT LcB, 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 BoNT LcA enzyme inhibitors we have synthesized to date, with MBX 1140 displaying a 10-fold increase in potency over MBX 1131 and 1107

October 29, 2008

Rat Neuronal Cell Assay

1) Cells are harvested from 7-8 day old rat cerebella, washed and cultured in 6-well plates (>7days)

2) Once the cells have become networked, they are preincubated (15min.) with test compounds or diluent (DMSO)

3) Cells are inoculated with BoNT/A and incubated for 3 hrs (37 °C)4) Cells are treated with 1 M NaOH, to inactivate the BoNT and lysed. 5) 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

6) Band intensities are read and normalized using scanning densitometry

October 29, 2008

Inhibition of BoNT/A Activity in Primary Rat Neurons by MBX Compounds at 80 µM

Inhibition of BoNT/A Activity in Primary Rat Neurons by MBX Compounds at 80 µM

MBX Compounds (80 M)MBX Compounds (80 M)

UncleavedCleaved

UncleavedCleaved

Control BoNT/A Control BoNT/A

1130 1107 1131 1195 1340 1341 1130 1107 1131 1195 1340 1341

October 29, 2008

Dose-dependent Inhibition of BoNT/A Activity in Primary Rat Neurons by MBX 1131

100 50 25 12.5

MBX 1131 (µM)

Control BoNT/A

UncleavedCleaved

October 29, 2008

Cytotoxic effects of 1131 & 1140 on N2a cells

0.000

20.000

40.000

60.000

80.000

100.000

120.000

1 10 100 1000

Comopunds (M)

Viab

ility

(%)

1131 1140

October 29, 2008

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.

October 29, 2008

BoNT/A LC Inhibitor Status Over 100 compounds have been made and tested

Established a BoNT/A LC fluorescent based assay for HTS (Z’ factor > 0.8)

Established a BoNT/A LC HPLC assay

Established MMP 1, 2, 3 and 9 assays; anthrax LF

Established cytotoxicity assays: HeLa, MRC-5, HFF

BoNT/B LC assay is being developed

Compound profiling in secondary assays in progress

Co-Crystallography Studies are under way

October 29, 2008

BoNT/A Inhibitor Summary

All 7 compounds exhibited potency in the enzyme assays of 1-17 µM, with varying degrees of specificity, when tested against other metalloproteases

MBX 1140 was the most potent compound in the series

In the cell-based assay, MBX 1131 (NSC240898) and 1140 displayed the greatest potencies (IC50 = 40 µM and 70 µM, respectively)

October 29, 2008

BoNT/A Inhibitor Conclusions

The new series of compounds, based on MBX 1131 (NSC240898), show promise for the

treatment of lethal BoNT/A intoxication.

October 29, 2008

October 29, 2008

Acknowledgements

USAMRIID: Sina Bavari, Ph.D. Rekha Panchal, Ph.D.

James Burnett, Ph.D.

NCI: Rick Gussio, Ph.D.

Tufts Vetinary School: John Beak-Park, Ph.D. Microbiotix – Biology:

Don Moir, Ph.D., CSOMichelle M. Butler, Ph.D., Steven Cardinale, MSArnab Basu, Ph.D., Joselynn Wallace, BS

Microbiotix - Medicinal Chemistry: Norton P. Peet, Ph.D., Director of ChemistryJohn D. Williams, Ph.D.Bing Li, Ph.D., Ramdas Pai, MSShen Gu, Ph.D.

NIAID – 5U01 AI070430-02

October 29, 2008