October 29, 2008

October 29, 2008

October 29, 2008
A small molecule, anti-infective drug discovery company
Terry L. Bowlin, Ph.D.,
CEO
Worcester, MA

Microbiotix Corporate Overview
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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 unit
Lodish et al. 2003. Molecular
Cell Biology, 5th ed.
Compound Library:
 Greater than 100K compounds with greater than 200 druglike chemotypes
October 29, 2008

Microbiotix Anti-Infective Drug Discovery
Compound Libraries
(Drug-like compounds
& natural products)
• MBX 500
• MBX 222
HTS &
Confirmation
• Biochemical Screens
• Cell-Based Screens
• Re-tested in quadruplica te
Lead Identification
• Medicinal Chemistry
• IC
50
& MIC criteria
• Serum effect
• In vitro therapeutic index criteria
• Confirmed SAR
• MOA confirmation
• Freedom to operate
Confirmed
Hits
Validated
Hits
Lead Compounds
• Secondary Assays
• IC
50
& MIC criteria
• In vitro therapeutic index criteria
• QC & stability
Hit Validation
In Licensing
• MBX 1107 (USAMRIID)
• MBX 400 (Wayne St. U.)
Lead
Optimization
Preclinical Candidates
• MBX 500
• MBX 400
• Ranked by criteria
• Low resistance freq.
• Passed acute tox
• Effective in animals
• Scalable synthesis
• Patentable
• Satisfactory market
October 29, 2008

Microbiotix Drug Discovery Portfolio
PROJECT THERAPEUTIC TARGET MOLECULAR TARGET STATUS
ANTI-BACTERIAL
MBX-500
MBX-1162
MBX-1131
Gram +; MRSA/VRE
Broad Spectrum Antibiotic
(biodefense)
C. botulinum (biodefense)
Polymerase;
Gyrase/Topoisomerase
DNA/Helicase
BoNT /A LC
Pre-clinical
IND enabling
Pre-clinical
IND enabling
SAR
ANTI-VIRAL
MBX-400
MBX-222
MBX-1325
October 29, 2008
Anti-beta/gamma Herpes;
(HCMV/HHV6/HHV8)
EboV (biodefense)
HCV
Polymerase
Fusion
Polymerase
Preclinical
IND-enabling
Hit/lead
Hit/lead

October 29, 2008

October 29, 2008

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October 29, 2008

October 29, 2008

The BoNT Threat
 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
October 29, 2008

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
October 29, 2008

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
October 29, 2008

October 29, 2008

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 NSFethylmaleimide-sensitive factor attachment protein receptor) components of neurotransmission
October 29, 2008

October 29, 2008
1.Binding
2.Internaliz
ation
3.Translocat
ion
(LC release)
4.Proteolyti
c
Cleavage
SNARE complex

Therapeutic Approaches to BoNT Inhibition
Therapeutic
Anti-BoNT MAbs
Efficacy Limitations
Effective in mice ( in vivo toxin neutralization when premixed with BoNT/A prior to injection)
3 MAbs required (oligoclonal) for adequate potency; limited postexposure 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
LC inhibitors – peptides Efficacy demonstrated in vitro only
LC inhibitors – small molecules
Efficacy in vitro , & in neuronal cell culture or synaptosomes
Mechanism unclear; associated cytotoxicity of anti-malarials; no post-exposure protection
Non drug-like molecules with poor
ADME features
Higher potency with suitable
ADME properties needed
October 29, 2008

BoNT Current Treatment
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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 longterm 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 postexposure ‘rescue’ therapeutics
October 29, 2008

October 29, 2008

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 cellbased primary neuronal assays.
October 29, 2008

Compound Evaluation Flow Chart
Aim 1
MBX & NERCE cpd libraries
NSC240898
Deriv ativ es
Primary Screen (Identify & confirm backup hit series)
NSC240898
Aim 2
(A) In vitro Potency
• HPLC-based assay
• IC50 ≤ 100 nM
(B) Specificity
• Test of Zn
++ chelation
• Human MMP’s
• BoNT/B, BoNT/F, AT-LF
• IC
50 endo/IC
50
BoNT/A >10
(C) Cytotoxicity
• CC
50 vs. human cells
• Damage to neurons
• CC
50
/IC
50
>100
(D) In vivo Potency
• Inhibition of SNAP-25 cleavage
• Rescue of axon length loss
• IC
50
< 1 μM
Compounds suitable for Aim 3
FIG. 9. Compound Evaluation Flow Chart
October 29, 2008

October 29, 2008
BoNT Biological Assays
 FRET Assays
 HPLC Assay
 Neuronal Cell Assays

Enzyme Based Assays
Fluoresence Resonance Energy Transfer (FRET)
Standard Assay For Recombinant BoNT LcA (DACIA SUBSTRATE)
For Characterization of MBX Compounds
REAGENT
DMSO or Compound
[STOCK]
100 %
QUANTITY (

L)
1
[FINAL]
1 %
Sterile Water
HEPES pH 7.4
55M
200 mM
44
25
N/A
50 mM
Tween 20
BonT LcA
DACIA Substrate
0.5%
1
 g/mL
200

M
10
10
10
0.05%
10 ng in rxn
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 BoNT LcA (FITC SUBSTRATE)
For Characterization of MBX Compounds
REAGENT
DMSO or Compound
Sterile Water
HEPES pH 8.2
[STOCK]
100 %
55M
200 mM
QUANTITY (
1
34
25

L) [FINAL]
1 %
N/A
50 mM
Tween 20
BonT LcA
FITC Substrate
0.5%
1
 g/mL
100

M
20
10
10
0.1%
10 ng in rxn
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.
October 29, 2008

October 29, 2008
H
N
H
N

Detection Method for BoNT LcA
FRET (DACIA) Assay
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0 100 200 300 400
Vmax Points = 41
Well
Vmax Per Second
R^2
D1
-0.001
0.154
E1
0.787
0.989
500 600 700 800 900 1000 1100 1200 1300 1400
Time (secs)
1500 1600 1700 1800 1900 2000 2100 2200 2300 2400
D1
E1
HEAT DENATURED BoNT LcA CONTROL
10 ng BoNT LcA
October 29, 2008

Enzyme Based Assay-HPLC
REAGENT
DMSO or Compound
Sterile Water
HEPES pH 7.4
NP-40
BoNT LcA
Substrate (DACIA)
[STOCK]
100%
55M
200 mM
0.5%
1
 g/mL
2 mM
QUANTITY (

L) [FINAL]
1.5
46
38
15
45
4.5
1%
N/A
50 mM
0.05%
45 ng in rxn
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
October 29, 2008

Detection Method for BoNT LcA HPLC Assay
Heat Denatured BoNT LcA Reaction
October 29, 2008
Native BoNT LcA Reaction

Compound Library
20,000 cpds
Maybridge & Microsource
Discovery
50,000 cpds
Chembridge DIVERSet TM
HTS Screening
Library
100,000 Cpds
~200 chemotypes
3,770 cpds, natural products
& derivatives
AnalytiCon Discovery
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
30,000 cpds
GLSynthesis, MBX, & other sources
October 29, 2008

Ongoing HTS at Microbiotix
Libraries Screened:
 Tim Tec Natural Products
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Chembridge 50K
Chem Div 2
Number of Compounds
Screened
Number of
Primary Hits
Number
Confirmed via
FRET Assay
70,400 330 114
Typical Z’ Score=0.69
% Final Confirmed Hit
Rate: FRET + Ongoing
HPLC Secondary
Assays
0.16
October 29, 2008

Examples of Select Screening Hits
ID
CB 6346186
CB 6352178
CB 6696465
CB 6698977
IC
50
(µM)
FRET
16
5.92
33
19
CB 7774777
CB 7781727
CB 7785416
CB 7836164
39
38
18
13
IC
50
(µM)
HPLC
58
35
78
78
ND
ND
61
59
ID
CB 7620237
CB 7662532
CB 7725216
CB 7738585
IC
50
(µM)
FRET
24
31
10
16
CB 7869065
CB 7853216
CB 7898734
CB 7924532
10
7.15
8.22
15
IC
50
(µM)
HPLC
88
72
27
>100
14
10
13
12
October 29, 2008

USAMRIID HTS BoNT/A LC Inhibitors
Structure Compound ID
%
Inhibition
NSC 661,755 62%
Structure Compound ID
%
Inhibition
Q2-61 50%
NSC 357,756 57%
NSC 119,889 56%
Q2-15 60%
Q2-43 52%
October 29, 2008

October 29, 2008

The BoNT/A LC pseudo-peptide inhibitor Mpp-RATKML (K i
=330nM) docked within the BoNT/A LC substrate binding cleft (Burnett et al, JBC, 2007, 282: 5004-14)
October 29, 2008

Refined Pharmacophore for BoNT/A LC
Inhibition
A
Planar
Components: A&B
Hydrophobic
Components: C&D
Positive Ionizable
Component: E
October 29, 2008

New BoNT/A LC Inhibitors: Potencies, Search
Query Fits and Distances Between Components
October 29, 2008

Chick Neuronal Cell Assay
1) Embryonic chicken spinal motor neuron cells were isolated utilizing methods described 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 lysed
5) Lysates were run on a 12% gel and transferred to nitrocellulose
6) 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)
Burnett et al. (2007) J. Biol. Chem. 282, 5004-5014
Kuhn, T.B. (2003) Methods Cell Biol. 71,67-87
October 29, 2008

Chick Neuronal Cell Morphological Analysis
Green=staining for tubulin
Red=staining for actin filaments
Blue=staining for DNA
October 29, 2008
NSC 240898 is well tolerated by neurons and is an effective inhibitor of BoNT/A
LC-mediated cleavage of SNAP-25 in cells

SNAP-25 Western Blot Analysis
Chick Primary Neuronal Cells
October 29, 2008

Analysis of Hit NSC240898
HN
N
H
NH
2
NSC240898
MBX-1131
O
H
2
N
NH
Optimization
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Neuron uptake
BoNT /A LC inhibition: 61%@20 µM
CC
50
> 40 µM
O
R
X Y
X = NH, S
Y = CH, N
R'
R = CN, CONH
2
, C(=NH)NH
2
October 29, 2008
Type I analogs
Three-ring scaffold
R'
R
X Y
X = NH, S
Y = CH, N
R = CN, CONH
2
, C(=NH)NH
2
Type II analogs
Two-ring scaffold

October 29, 2008
Cl
Docking Analysis
O
N
H
OH
N
N
H
NH
Cl
2,4-dichlorocinnamic hydroxamate
MBX-1107
O
HN
N

BoNT SAR: Summary
R
X Y
O R''
R'
 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
October 29, 2008

Structures of BoNT/A Inhibitors
O
N
NH
N
H
MBX 1107
HN
N
MBX 1131 (NSC240898)
MBX 1130
MBX 1140
MBX 1340
October 29, 2008
MBX 1195
MBX 1341

MBX
ID
Enzyme Specificity of Select BoNT LcA Inhibitors
IC
50
(µM)
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
1140
1195
15
1.35
IND
8.9
0.84
2.7
26
8.1
4.4
5.5
0.83
3.9
>100
>100
ND
< 25
35
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 IC
50 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 6well 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 antirabbit 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
MBX Compounds (80

M)
Control BoNT/A 1130 1107 1131 1195 1340 1341
Uncleaved
Cleaved
120
100
80
60
40
20
0
Control BoNT/A 1130 1107 1131
MBX compounds (80 µM)
1195 1340 1341
October 29, 2008

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
October 29, 2008

120.000
100.000
80.000
60.000
40.000
20.000
0.000
1
Cytotoxic effects of 1131 & 1140 on N2a cells
1131 1140
10
Comopunds (

M)
100
October 29, 2008
1000

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
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All 7 compounds exhibited potency in the enzyme assays of 117 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 (IC
50
=
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., CSO
Michelle M. Butler, Ph.D., Steven Cardinale, MS
Arnab Basu, Ph.D., Joselynn Wallace, BS
Microbiotix - Medicinal Chemistry:
Norton P. Peet, Ph.D., Director of Chemistry
John D. Williams, Ph.D.
Bing Li, Ph.D., Ramdas Pai, MS
Shen Gu, Ph.D.
NIAID – 5U01 AI070430-02
October 29, 2008

October 29, 2008