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The development of novel broad-spectrum anti-bacterials for intracellular BW threats. Terry L. Bowlin, Ph.D. CEO, Microbiotix , Inc. Worcester, MA. The development of novel broad-spectrum anti-bacterials for intracellular BW threats. Microbiology Mechanism Animal studies. - PowerPoint PPT Presentation
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November 2007
The development of novel broad-spectrum anti-bacterials for
intracellular BW threats
Terry L. Bowlin, Ph.D.CEO, Microbiotix , Inc.Worcester, MA
November 2007
The development of novel broad-spectrum anti-bacterials for
intracellular BW threats
•Microbiology
•Mechanism
•Animal studies
November 2007
The development of novel broad-spectrum anti-bacterials
for intracellular BW threats
•Broad-spectrum anti-bacterials discovered by Sina Bavari, Ph.D.
November 2007
Microbiology Studies
• MIC’s against standard Gram-positive and Gram-negative laboratory strains
• MIC’s against category A and B bioterrorism pathogens
• Bactericidal activity of compounds
• Cytotoxicity (CC50) of compounds
November 2007
Average MIC (µg/mL)
Bacterial Strain MBX 1066 MBX 1090 MBX 1113 MBX 1128
Bacillus subtilis BD54 0.117 0.156 0.156 0.068
B. cereus ATCC 4342 0.078 0.156 0.156 0.521
B. thuringiensis ATCC 10792 0.078 0.313 0.235 0.182
B. anthracis Sterne 0.235 0.313 0.156 1.25
B. anthracis Ames ANR (pXO1-, pXO2-) 0.098 0.313 0.313 36.3
B. megaterium ATCC 12872 0.078 0.156 0.078 0.176
B. licheniformis ATCC 14580 0.059 0.313 0.156 0.117
Staphylococcus aureus ATCC 25923 0.117 0.625 0.313 0.283
S. aureus (Smith) ATCC 13709 0.078 0.313 0.156 0.078
Meth-res S. aureus (MRSA) 1094, clinical 0.137 0.625 0.313 0.508
S. aureus MT23142 NorA++ 0.039 0.313 0.235 0.088
Enterococcus faecalis ATCC 29212 0.137 0.313 0.313 0.107
Vanc-res E. faecalis (VRE) ATCC 51575 0.117 0.625 0.469 0.107
E. faecium ATCC 19434 0.059 0.156 0.274 0.088
VRE faecium B42762, clinical 0.039 0.313 0.156 0.068
MBX Gram Positive MIC Data (BSL 2)
November 2007
Average MIC (µg/mL)
Bacterial Strain MBX 1066 MBX 1090 MBX 1113 MBX 1128
Escherichia coli J53, lab strain 0.391 0.625 0.313 53.3
E. coli XL1Blue, lab strain 0.078 0.156 0.156 1.8
E. coli 700 TolC+ 1.25 0.625 0.313 80
E. coli 701 TolC- 0.156 0.156 0.156 21.3
Klebsiella pneumoniae 5657, clinical 0.235 0.580 0.352 16.3
Pseudomonas aeruginosa PAO1 7.5 25 25 >80
P. aeruginosa PAO1 ΔmexAB-oprM 1.15 >20 ND ND
P. aeruginosa 27853 2.5 12.5 1.09 >80
Burkholderia thailandensis E264 6.25 >80 35 >80
Stenotrophomonas maltophilia ATCC 13637 0.176 0.625 0.313 11.3
MBX Gram Negative MIC Data (BSL 2)
November 2007
Average MIC (g/mL)
Bacterial Strain Test Site MBX 1066 MBX 1090 MBX 1113 MBX 1128
P. aeruginosa PAO1 (control) Calgary 8 5.3 >8 >8*
S. aureus (Smith) ATCC 13709 (control)
Calgary 1.125 2 0.75 >8*
Burkholderia pseudomallei 1026b Calgary 0.65 3.2 >8 >8*
Burkholderia mallei GB3 Calgary 1 2 0.67 >8*
Bacillus anthracis Ames USAMRIID 0.067 0.099 0.11 0.145
Burkholderia mallei ATCC 23344 USAMRIID 0.42 1.6 1.8 >9.7
Burkholderia pseudomallei DD503 USAMRIID 1.7 3.1 1.8 >9.7
Francisella tularensis Schu4 USAMRIID ND 1.56 0.92 4.9
Yersinia pestis CO92 USAMRIID 3.4 >12.5 >7.4 >9.7
USAMRIID and U. Calgary MIC Data (BSL 3)
*Value determined only once.
November 2007
Average MIC (µg/mL)
Bacterial Strain MBX 1066 MBX 1142 MBX 1143 MBX 1162
Bacillus subtilis BD54 0.117 0.068 0.034 0.063
B. cereus ATCC 4342 0.078 0.107 0.039 0.054
B. thuringiensis ATCC 10792 0.078 0.156 0.078 0.117
B. anthracis Sterne 0.235 0.215 0.078 0.088
B. anthracis Ames ANR (pXO1-, pXO2-) 0.098 0.156 0.039 0.063
B. megaterium ATCC 12872 0.078 0.098 0.034 0.037
B. licheniformis ATCC 14580 0.059 0.156 0.049 0.088
Staphylococcus aureus ATCC 25923 0.117 0.274 0.117 0.156
S. aureus (Smith) ATCC 13709 0.078 0.215 0.078 0.102
Methicillin-res. S. aureus (MRSA) 1094, clinical 0.137 0.293 0.156 0.127
S. aureus MT23142 NorA ++ 0.039 0.178 0.078 0.166
Enterococcus faecalis ATCC 29212 0.137 0.176 0.078 0.156
Vanc-resistant E. faecalis (VRE) ATCC 51575 0.117 0.182 0.156 0.137
E. faecium ATCC 19434 0.059 0.235 0.137 0.186
VRE faecium B42762, clinical 0.039 0.235 0.156 0.137
MBX Gram Positive MIC Data for selected MBX 1066 Analogs
November 2007
Average MIC (µg/mL)
Bacterial Strain MBX 1066 MBX 1142 MBX 1143 MBX 1162
Escherichia coli J53, lab strain 0.391 0.43 0.195 0.274
E. coli XL1Blue, lab strain 0.078 0.215 0.098 0.254
E. coli 701 TolC- 0.156 0.254 0.156 0.137
Klebsiella pneumoniae 5657, clinical 0.235 0.254 0.137 0.146
Pseudomonas aeruginosa PAO1 7.5 0.938 0.235 0.293
P. aeruginosa PAO1 ΔmexAB-oprM 1.15 0.313 0.156 0.254
P. aeruginosa 27853 2.5 0.781 0.215 0.254
Burkholderia thailandensis E264 6.25 22.5 0.352 0.352
Stenotrophomonas maltophilia ATCC 13637
0.176 0.156 0.078 0.156
MBX Gram Negative MIC Data for selected MBX 1066 Analogs
November 2007
Average MIC (g/mL)
Bacterial Strain Test Site MBX 1066 MBX 1142 MBX 1143 MBX 1162
Burkholderia pseudomallei 1026b Calgary 0.65 1 1 0.375
Burkholderia mallei GB3 Calgary 1 ND ND ND
Burkholderia mallei ATCC 23344 USAMRIID 0.42 1.8 1.8 0.6
Burkholderia pseudomallei DD503 USAMRIID 1.7 1.8 0.6 ND
Francisella tularensis Schu4 USAMRIID ND 1.8 ND 1.8
Yersinia pestis CO92 USAMRIID 3.4 3.5 ND 3.5
USAMRIID and U. Calgary MIC Data for Selected MBX 1066 Analogs (BSL 3)
November 2007
Compound MBX 1090 MBX 1066 MBX 1142 MBX 1162
Time to reach cidal effect (hours) for S. aureus
4 2 1.5 1
Time to reach cidal effect (hours) for Y. pestis
≤1 ≤1 ≤1 ≤1
0
2
4
6
8
10
12
0 10 20 30
Lo
g C
FU
/mL
Time (hours)
MBX Compounds vs. S. aureus in a Time Kill Assay at 4x MIC
Control
MBX 1066
MBX 1090
MBX 1142
MBX 1162 0
2
4
6
8
10
12
0 10 20 30
Lo
g C
FU
/mL
Time (hours)
MBX Compounds vs. Y. pestis in a Time Kill Assay at 4x MIC
Control
MBX 1066
MBX 1090
MBX 1142
MBX 1162
Time kill assay for four MBX compounds represented at 4× their respective MIC values and tested against S. aureus ATCC strain 25923, panel A, or Y. pestis strain Kim Δpgm, CDI-, panel B. The threshold for determining bactericidal activity is at ~103 CFU/mL (a 3 log reduction in the original colony count).
Rapid Bactericidal Activity of MBX 1066, 1090, 1142 and 1162
Summary of time kill results
A B
November 2007
StrainMBC (µg/mL)
MBX 1090MIC (µg/mL)MBX 1090
MBC/MIC ratio (1090)
MBC (µg/mL)MBX 1066
MIC (µg/mL)MBX 1066
MBC/MIC ratio (1066)
B. anthracis Ames ciproR 1.168 0.584 2 1.292 1.292 1
B. anthracis 1024 2.336 1.168 2 0.646 0.162 4
B. anthracis vollum 1.168 0.584 2 0.324 0.324 1
Comparison of MBC and MIC values for infectious B. anthracis strains
November 2007
Compound Cytotoxicity on HeLa cells
CompoundsCC50
(µg/mL)MIC S. aureus 25923
(µg/mL)Selectivity Index
(in vitro)
MBX 1066 >20 0.117 >170
MBX 1090 10 0.625 16
MBX 1113 3 0.313 9.6
MBX 1128 17 0.283 60
MBX 1142 14 0.274 51
MBX 1143 13 0.117 111
MBX 1162 4 0.156 26
HB-EMAU 35 5 7
November 2007
MICROBIOLOGY SUMMARY
• The activity of the original lead compounds and analogs have been confirmed in our laboratories and our collaborators with similar anti-bacterial potency
• Our lead series displays favorable in vitro selectivity index with low mammalian cell cytotoxicity
• Analogs of our lead series have been tested and several maintain activity against the Gram-positive strains while displaying greater potency against Gram-negative strains
• Rapid bactericidal activity observed in time kill assays
• Future work:
• We will continue to acquire and test other relevant bacterial strains against the current compounds and new series as they are synthesized
November 2007
Mechanism Studies
•MMS•DNA Binding•Replix•Helicase•In Situ•Efflux•Resistance•Membrane Effects
November 2007
Macromolecular Synthesis Assays in S. aureus — MBX 1066
DNA synthesis is the most sensitive macromolecular pathway to MBX 1066 treatment – effects are observed at >10 μg/ml
MBX-1066-40xMBX-1066-20x
MBX-1066-10xMBX-1066-5x
0
20
40
60
80
100
120
DNA RNAProtein Cell
wallLipid
% o
f C
on
tro
l
Macromolecule
MBX-1066 (5x, 10x, 20x, 40x MIC)
CiprofloxacinRifampicin-10x
Chloram-10xVancomycin-10xIrgasan-2x
0
20
40
60
80
100
120
140
DN
A
RN
A
Pro
tein
Ce
ll w
all
Lip
id
% o
f C
on
tro
l
Macromolecule
Rifampicin (RNA), Chloramphenicol (protein), Ciprofloxacin (DNA), Vancomycin (cell wall) and Irgasan (lipid)
November 2007
Conclusion: Half-maximal DNA interaction by MBX 1066 occurs at about 0.4 μM (~0.3 μg/ml)
Fluorescence Enhancement of MBX 1066 in the Presence of DNA – Concentration Dependence
November 2007
MBX 1066, 1090 and 1113 are Potent Inhibitors of Replix™, a Permeable Cell DNA Replication Assay
IC50 µM (µg/mL) Against Permeable Bacteria
Compound B. subtilis B. anthracis
MBX 1066 2.2 (1.5) 4.1 (2.8)
MBX 1090 4.8 (3.0) 7.7 (4.8)
MBX 1113 2.6 (0.95) 6.1 (2.2)
HB-EMAU (pos. ctl.) 1.1 (0.35) 2.0 (0.63)
November 2007
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20 30 40 50 60 70 80
Concn (uM)
% I
nhib
itio
n
0.00
20.00
40.00
60.00
80.00
100.00
120.00
0 10 20 30 40 50 60 70 80
M02
E10
N18
F21
MBX1066
MBX1090
Log. (E10)
Log. (M02)
Log. (N18)
Log. (F21)
Log. (MBX1066)
Log. (MBX1090)Conclusion: MBX 1066 & 1090 are very potent B. anthracis helicase inhibitors with
IC50’s of <1 μM (<0.6 μg/ml)
Helicase Inhibition by MBX 1066 & 1090 as Measured by 32P-Based Unwinding Assay – Comparison to Other Helicase Inhibitors
November 2007
In situ Fluorescence of MBX 1066 in S. aureus cells is Consistent with Cell Penetration & DNA Binding
None 1 X MBX 1066 4 X MBX 1066 1 X MBX 1090 4 X MBX 1090
4 X MBX 1113
DIC
DAPI
DIC
DAPI
Intracellular fluorescence readily detected at 1X MICConsistent with DNA-dependent fluorescence enhancement
1 X MBX 1066
Contrast enhanced10X zoom
cytoplasmiclocalization
November 2007
Average MIC (µg/mL)
Bacterial Strain MBX 1066 MBX 1142 MBX 1143 MBX 1162
Pseudomonas aeruginosa PAO1 7.5 0.938 0.235 0.293
P. aeruginosa PAO1 ΔmexAB-oprM 1.15 0.313 0.156 0.254
MBX MIC Data for MBX 1066 & AnalogsIsogenic P. aeruginosa Strains +/- a Major Efflux Pump
Conclusion: MIC of MBX 1066 is significantly improved by loss of major efflux pump; analogs may be better at escaping
efflux
November 2007
Mutation to Resistance to MBX 1066 is Rare in S. aureus NCTC-8325 Serial Passage
A B C D E F G H
Hig
he
st S
ub
leth
al
Co
nce
ntr
ati
on
(F
old
MIC
)
128643216
8421
0.50.25
0.125MBX 1066
1 5 10 15 20
Time (days)
MBX 1090
128643216
8421
0.50.25
0.125
1 5 10 15 20
Time (days)
MBX 1113
1 5 10 15 20
Time (days)
128643216
8421
0.50.25
0.125
S. aureus NCTC 8325
Resistant mutants-16X MIC
November 2007
MBX 1090 Resistant Mutants are not Cross-Resistant to MBX 1066
Clone MBX-1066 MBX-1090 MBX-1113A1 1 32 4A2 1 64 4A3 1 32 4C1 2 32 4C2 2 32 4C3 2 32 4C4 2 32 4G1 1 32 4WT 2 2 2
RESISTANCE (FOLD MIC)
MICs vs MBX 1090, MBX 1066, and MBX 1113
No cross resistance to MBX 1066, suggesting different MOAs for MBX 1090 and MBX 1066
November 2007
Bacterial membrane perturbation assay using DiSC3(5)
DiSC3(5)
Ex-622Em-670
e- transportQUENCHQUENCH
2H+
2H+
Ex-622Em-670
Membrane disrupter
Membrane potential
perturbation
No membrane potential No membrane potential perturbation by compoundperturbation by compound
Membrane potential perturbation by compound
November 2007
0
100
200
300
400
500
600
700
800
No
cm
pd
CC
CP
Va
n-3
2X
11
62-
0.2
5X
11
62-
1X
11
62-
4X
11
62-
32
X
10
66-
0.2
5X
10
66-
1X
10
66-
4X
10
66-
32
X
RF
U
DiSC3(5) Membrane Perturbation Assay of MBX 1066 & Analog MBX 1162
Results of DiSC3(5) assay 10 min after compound addition
Conclusion: MBX 1066 & 1162 do not perturb membrane potential at concentrations near the MIC
November 2007
MBX 1066 does not disrupt HeLa cell membranes
0
5
10
15
20
64X
MIC
16X
MIC
1X
MIC
64X
MIC
16X
MIC
1X
MIC
No
an
tib
ioti
c
To
talL
ysi
s
RF
U x
103
MBX-1066 VAN
0
5
10
15
20
64X
MIC
16X
MIC
1X
MIC
64X
MIC
16X
MIC
1X
MIC
No
an
tib
ioti
c
To
talL
ysi
s
RF
U x
103
MBX-1066 VAN
• Monolayers of HeLa cells were exposed to MBX 1066 and a control antibiotic (vancomycin) for 1 h.
• Activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) released into the media was measured after 30 min.
• Similar results obtained with MBX 1090 and MBX 1113
November 2007
Favorable Features of Lead Series Antibacterial Mechanism
• In vitro therapeutic index (CC50/MIC >170)
• Rapidly bactericidal • DNA synthesis is the most sensitive macromolecular pathway at higher
concentrations• Interacts with DNA
• Fluorescence increase in the presence of DNA (Max1/2~0.4 μM)
• Inhibits ReplixTM (IC50 ~2 μM) & replicative helicase (IC50~1 μM)
• ~2x preference for AT-rich B. anthracis DNA vs. calf thymus DNA• Target appears to be intracellular
• Fluorescence enhancement observed within bacterial cells • MIC is significantly lower in efflux mutant of P. aeruginosa
• Resistance seen with some analogs and others exhibit very low frequency of mutation to resistance
• Minimal effects on cell membranes
November 2007
Future Mechanism Studies Perform genetic expression profile analysis. Expression profiling in the presence of various concentrations of bis(imidazolinylindole) compounds to identify genes up- and down-regulated in response to compound treatment
Perform target under-expression hypersensitivity and over-expression resistance assays. For implicated single gene targets, construct and test strains over- and under-expressing those putative targets to confirm MOA in the cell
Map loci responsible for resistance. Select resistant strains and map resulting mutations to identify genes which can confer resistance
Identification of site specificity for DNA interaction. Determine the nucleotide sequence preferential for binding
November 2007
ANIMAL STUDIES
B. Anthracis
Y.Pestis
B.Pseudomallei
S. Aureus
November 2007
In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis
0
20
40
60
80
100
0 10 20 30
Days post infection
% s
urv
iva
lcontrol (n=10)
PW 317881 (n=10)
MBX 1090 (n=10)
MBX 1113
In vivo testing in a murine B. anthracis infection model
MBX1066
November 2007
In Vivo Testing in Y. Pestis Murine Model (USAMRIID)
Y.Pestis survival study
0
20
40
60
80
100
0 5 10 15 20 25 30
Days post-infection
% m
ice
surv
ival
control
MBX 1066
MBX 1142
MBX 1162
November 2007
Efficacy of MBX 1162 in a murine IP/IP B. pseudomallei infection model
Three groups of 5 Balb/C mice (female, 20-22g) were inoculated intraperitoneally with 106 cells of Burkholderia pseudomallei strain
1026b. Mice were treated intraperitoneally ten minutes post infection with tetracycline (10 mg/kg), MBX 1162, or vehicle alone
Survivors
Group, n Treatment Dose, mg/kg 24 hours 48 hours 72 hours % survival
1, 10 Vehicle control -- 10 6 2 20
2, 10 Tetracycline 10 10 10 9 90
3, 10 MBX 1090 10 10 10 8 80
4, 10 MBX 1066 10 10 6 3 30
5, 15 Vehicle control -- 15 3 ND 20
6, 15 Tetracycline 10 15 15 ND 100
7, 15 MBX 1162 10 15 15 ND 100
November 2007
survivors
Group n treatment Dose, mg/kg 8 hr 18 hr 24 hr 48 hr%
survival
1 10DMA/D5W,
pH 3.52- 2 2 2 2 20
2 10 Dapto 10 10 10 10 10 100
3 10 MBX 1066 10 9 8 8 8 80
4 10 MBX 1090 10 10 9 9 9 90
5 2 MBX 1113 10 2 mice died immediately after injection
5’ 8 MBX 1113 1 2 1 1 1 12.5
6 1 MBX 1128 10 1 mouse died immediately after injection
6' 9 MBX 1128 1 5 2 0 0 0
Efficacies of MBX compounds in a murine IP/IV S. aureus infection model
November 2007
Survivors
Treatment Route nDose, mg/kg
8 hours 18 hours 24 hours 48 hours % survival
Vehicle control (D/PO)
i.p. 6 -- 1 0 0 0 0
Daptomycin i.p. 6 10 6 6 6 6 100
MBX 1162 i.p. 10 1 10 10 10 10 100
MBX 1162 i.p. 10 10 10 10 10 10 100
Vehicle control(DMA/D5W)
IV 6 -- 1 1 1 1 17
Daptomycin IV 6 10 6 6 6 6 100
MBX 1162 IV 10 1 9 6 6 6 60
MBX 1162 IV 10 10 10 10 10 10 100
In vivo testing in a S. aureus murine infection model with i.p. or IV treatment
November 2007
NCI ID Dose (mg/kg/ injection)
Sched. Route #Mice #Surviving on Day 5
100 Q04DX003 i.p. 6 6MBX 1090 200 Q04DX003 i.p. 6 6
400 Q04DX003 i.p. 6 6100 Q04DX003 i.p. 6 6
MBX 1066 200 Q04DX003 i.p. 6 6400 Q04DX003 i.p. 6 625 Q04DX003 i.p. 6 6
MBX 1113 50 Q04DX003 i.p. 6 6100 Q04DX003 i.p. 6 5200 Q04DX003 i.p. 6 5400 Q04DX003 i.p. 6 250 Q01DX005 i.p. 6 6
MBX 1128 100 Q01DX005 i.p. 6 6200 Q01DX005 i.p. 6 6
Toxicity Determination in Mice
November 2007
LEAD SERIES SUMMARY Very potent broad spectrum agent active against Gram-
positive and -negative biodefense bacterial pathogens
Rapidly bactericidal
MOA consistent with DNA interaction/helicase inhibition
Variable resistance pattern (some compounds/not all)
Effective in murine models against Gram-positive and Gram-negative bacteria, with ED50<10mg/kg
Well tolerated, with murine MTD >400mg/kg
Easy and inexpensive to synthesize
Next step: IND enabling GLP toxicology/parmacology
November 2007
ACKNOWLEDGMENTS
• USAMRIID: Sina Bavari, Ph.D., Rekha Panchal, Ph.D.
• University of Calgary: Donald Woods, Ph.D.
• Defense Threat Reduction Agency (DTRA)
November 2007
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