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