Download BoNT_A Presentation UMass Dartmouth 081029

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.,
CEO
Worcester, 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


Cell based



permeabilized bacterial replication screen
whole-cell target-based luciferase reporter screens
Biofilm


purified enzymes essential for replication
(e.g., polymerase, gyrase, topoisomerase, helicase)
HTS for identification of biofilm inhibitors
Types of readouts

UV/Vis absorbancy, fluorescence, FRET,
time-resolved FRET, luminescence, radioisotopic
Lodish et al. 2003. Molecular
Medicinal Chemistry:
Cell Biology, 5th ed.
 Fully integrated medicinal chemistry drug discovery unit
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
Lead Identification
•
•
•
•
•
•
•
• Biochemical Screens
• Cell-Based Screens
• Re-tested in quadruplicate
Confirmed
Hits
Validated
Hits
• Secondary Assays
• IC50 & MIC criteria
• In vitro therapeutic index criteria
• QC & stability
Hit Validation
In Licensing
•MBX 1107 (USAMRIID)
•MBX 400 (Wayne St. U.)
October 29, 2008
Medicinal Chemistry
IC50 & MIC criteria
Serum effect
In vitro therapeutic index criteria
Confirmed SAR
MOA confirmation
Freedom to operate
Lead Compounds
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
Microbiotix Drug Discovery Portfolio
PROJECT
THERAPEUTIC TARGET
MOLECULAR TARGET
STATUS
MBX-500
Gram +; MRSA/VRE
Polymerase;
Gyrase/Topoisomerase
Pre-clinical
IND enabling
MBX-1162
Broad Spectrum Antibiotic
(biodefense)
DNA/Helicase
Pre-clinical
IND enabling
MBX-1131
C. botulinum (biodefense)
BoNT /A LC
SAR
MBX-400
Anti-beta/gamma Herpes;
(HCMV/HHV6/HHV8)
Polymerase
Preclinical
IND-enabling
MBX-222
EboV (biodefense)
Fusion
Hit/lead
MBX-1325
HCV
Polymerase
Hit/lead
ANTI-BACTERIAL
ANTI-VIRAL
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
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
1. Binding
2. Internaliz
ation
3. Translocat
ion
(LC
release)
4. Proteolyti
c
Cleavage
SNARE
complex
October 29, 2008
Therapeutic Approaches to BoNT Inhibition
Therapeutic
Anti-BoNT MAbs
Receptor decoys
Efficacy
Effective in mice (in vivo toxin
neutralization when premixed
with BoNT/A prior to injection)
Limitations
3 MAbs required (oligoclonal) for
adequate potency; limited postexposure utility
Effective in nerve assays when Co-administration of gangliosides
premixed prior to contact
required; limited post-exposure
utility
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
HC inhibitors
October 29, 2008
BoNT Current Treatment

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 post-exposure ‘rescue’ therapeutics
October 29, 2008
BoNT Inhibitor Discovery
MBX Overview
BoNT Background
BoNT/A Inhibitor Drug Discovery
Assays/Results
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
MBX & NERCE
cpd libraries
NSC240898
Derivatives
SBDD
Aim 1
NSC240898
Primary Screen (Identify &
confirm backup hit series)
Aim 2
(A) In vitro Potency
• HPLC-based assay
(B) Specificity
• Test of Zn++ chelation
• Human MMP’s
• BoNT/B, BoNT/F, AT-LF
• IC 50endo/IC 50BoNT/A >10
(C) Cytotoxicity
• CC50 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
Feedback to SAR
• IC50 ≤ 100 nM
BoNT Biological Assays
 FRET Assays
 HPLC Assay
 Neuronal Cell Assays
October 29, 2008
Enzyme Based Assays
Fluoresence Resonance Energy Transfer (FRET)
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/A
HEPES 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 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
[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%
BonT LcA
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.
October 29, 2008
BoNT/A LC
NH2-S-N-R-T-R-I-D-E-A-N-K-R-A-C-R-M-L-COOH
O
N
O
398 nm
S
NH
CH2
NH
O
CH3
H
N
O N
O
O
N CH3
O
CH3
+
NH2-R-A-C-R-M-L-COOH 398 nm
NH2-S-N-R-T-R-I-D-E-A-N-K-COOH
O
N
S
CH2
O
NH
O
H
N
CH3
NH
O
N CH3
O
CH3
O N
O
485 nm
485 nm
485 nm
FIG. 2. FRET Assay for BoNT/A LC Peptidase Activity
October 29, 2008
485 nm
Detection Method for BoNT LcA
FRET (DACIA) Assay
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0
Vmax Points = 41
Well
Vmax Per Second
R^2
100
200
300
400
500
600
700
900
1000
1100
1200
1300
Time (secs)
D1
E1
-0.001
0.154
0.787
0.989
D1
E1
October 29, 2008
800
HEAT DENATURED BoNT LcA CONTROL
10 ng BoNT LcA
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
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)
1.5
46
38
15
45
4.5
[FINAL]
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
Native BoNT LcA Reaction
October 29, 2008
Compound Library
20,000 cpds
50,000 cpds
Maybridge & Microsource
Discovery
Chembridge DIVERSetTM
HTS Screening
Library
100,000 Cpds
~200 chemotypes
3,770 cpds, natural products
& derivatives
AnalytiCon Discovery
October 29, 2008
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
Ongoing HTS at Microbiotix
Libraries Screened:
 Tim Tec Natural Products
 Chembridge 50K
 Chem Div 2
Number of Compounds
Screened
Number of
Number
Primary Hits Confirmed via
FRET Assay
% Final Confirmed Hit
Rate: FRET + Ongoing
HPLC Secondary
Assays
70,400
330
0.16
114
Typical Z’ Score=0.69
October 29, 2008
Examples of Select Screening Hits
ID
IC50 (µM)
FRET
IC50 (µM)
HPLC
ID
IC50 (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
October 29, 2008
Compound ID
%
Inhibition
Structure
Compound ID
%
Inhibition
NSC 661,755
62%
Q2-61
50%
NSC 357,756
57%
Q2-15
60%
NSC 119,889
56%
Q2-43
52%
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
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
N
H
NH2
NSC240898
MBX-1131
Neuron uptake
 BoNT/A LC inhibition: 61%@20 µM
 CC50 > 40 µM

O
HN
NH
H2N
Optimization
O
X
R
Y
X = NH, S
R'
Y = CH, N
R = CN, CONH2, C(=NH)NH2
Type I analogs
Three-ring scaffold
October 29, 2008
R'
R
X
Y
X = NH, S
Y = CH, N
R = CN, CONH2, C(=NH)NH2
Type II analogs
Two-ring scaffold
Docking Analysis
O
Cl
N
H
OH
O
N
NH
N
H
Cl
2,4-dichlorocinnamic
hydroxamate
October 29, 2008
N
HN
MBX-1107
BoNT SAR: Summary
O
R
X
R''
Y
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
MBX 1131 (NSC240898)
N
HN
MBX 1130
MBX 1140
MBX 1195
MBX 1340
MBX 1341
October 29, 2008
Enzyme Specificity of Select BoNT LcA Inhibitors
IC50 (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 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
Control BoNT/A
1130
1107
1131
1195
1340
1341
Uncleaved
Cleaved
120
Rel. Int. SNAP25 (%)
100
80
60
40
20
0
1131
MBX compounds (80 µM)
October 29, 2008
1195
1340
1341
Dose-dependent Inhibition of BoNT/A Activity in
Primary Rat Neurons by MBX 1131
MBX 1131 (µM)
Control
Uncleaved
Cleaved
October 29, 2008
BoNT/A
100
50
25
12.5
Cytotoxic effects of 1131 & 1140 on N2a cells
1131
1140
Viability (%)
120.000
100.000
80.000
60.000
40.000
20.000
0.000
1
10
100
Comopunds (M)
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



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., 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