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