november 7, 2008 microbiotix a product-focused, small molecule, anti-infective drug discovery...

November 7, 2008

MICROBIOTIX

A product-focused, small molecule, anti-infective drug discovery company

CONFIDENTIAL

November 7, 2008

The development of novel broad-spectrum anti-bacterials for

intracellular BW threats

November 7, 2008

AIMS Aim 1. Demonstrate potent, selective inhibitory activity of one or more bis-

(imidazolinylindole) compounds in animal models of infection (year 1). Milestone: Identify an inhibitor exhibiting in vivo efficacy (ED50<30 mg/kg) against >2 category A or B pathogens and minimum toxicity (MTD>300 mg/kg).

Aim 2. Establish the mechanism of action of the bis-(imidazolinylindole) class of compounds (year 1). Milestone: Defined mechanism of action and target which are common to multiple bacterial BW species but distinctly different in mammalian cells

Aim 3. Demonstrate structure-activity relationships for the potency and selectivity of the bis-(imidazolinylindole) class of compounds (year 2). Milestone: Identify key structural features for potency and selectivity; provide back-up compounds with MIC in serum <1 µg/ml with a selectivity index (CC50/MIC) >100.

Aim 4. Conduct IND-enabling pharmacokinetic, toxicology and safety pharmacology studies (year 2). Milestone: Complete two species GLP toxicology & safety pharmacology studies for the optimal bis-(imidazolinylindole) compound suitable for IND submission.

Aim 5. Prepare and file an IND application for a broad spectrum anti-bacterial active against intracellular BW threats (end of year 2). Milestone: IND approval for clinical Phase I human safety evaluation.

November 7, 2008

Aim 1 Demonstrate potent, selective inhibitory activity of one or more bis-(imidazolinylindole) compounds in animal models of infection (year 1).

November 7, 2008

Lead and Back-up Compounds

NH

NH

NH

NH

N

NH HN

N

N

NH HN

N

MBX 1066

MBX 1162

November 7, 2008

MBX 1066 Analogs

NHNC CN

Hetero

BrCN

Hetero

NH

B(OH)2NC

Suzuki

conditions

+

NH

HeteroN

NH HN

N

n n

P2S5diamine120 °C

Hetero

HN

HN

N

N

N N

N=

November 7, 2008

Current Work

NEtOOC COOEt

NOHC CHO

N

NO2

O2N

NC

CN

NHNC N

H CNNNH

NH

N

HN

NN

NHn n

1) LAH THF

2) DMP CH3CN

NO2NC

piperidine130 °C

P(OEt)3reflux

P2S5diamine

120 °C

November 7, 2008

Future Possibilities

NH

NH

N

NH HN

N

n n

NH

N

NHn HN

N

n

NH

SNH HN

NNH HN

NO O

nn

X

X

X

X

N

NHn

X

X

X

X

HN

N

nX = N, O, S

NH

N

NHn

HN

N

n

Tether

NH

NH

XX

NH N

H

N

NH HN

N

n

N

NH HN

N

HN

NN

NH

X = O, S, NH

n n n

nn

November 7, 2008

Exploration of Scale-up Conditions

NHNC N

H CN

NH

NH

NH

NH

NH

NH

NH

NH

NH

EtO

NH

OEt

N

NH2

N

NH2

R R

H2N

S

NH2

S

HN

SMe

NH

SMe

NHNC N

H CN

HCl (g)EtOH

R-NH2EtOH

P2S5

pyridne120 °C

MeIDMF

R-NH2solvents

November 7, 2008

Large Scale and Radio Syntheses

NH

NH

N

NH HN

N

NH

NH

N

NH HN

N

NH

NH

N

NH HN

N

MBX 1066

MBX 1162

MBX 1066*45 mCi (~1 mCi/mmol)

100 g (non-GMP)

120 g (non-GMP)

**

November 7, 2008

Average MIC (g/mL)

Bacterial Strain Test Site MBX 1066 MBX 1090 MBX 1113 MBX 1128 MBX 1162Burkholderia pseudomallei 1026b Calgary 0.65 3.2 >8 >8 0.375

Burkholderia mallei GB3 Calgary 1 2 0.7 >8 0.125

Burkholderia mallei ATCC 23344 USAMRIID 0.42 1.6 1.8 >9.7 0.6

Burkholderia pseudomallei DD503 USAMRIID 1.7 3.1 1.8 >9.7 0.9

Francisella tularensis Schu4 USAMRIID 1.7 1.6 0.9 4.9 1.8

Yersinia pestis CO92 USAMRIID 3.4 >12.5 >7.4 >9.7 3.5

Bacillus anthracis Ames USAMRIID 0.07 0.10 0.11 0.15 0.4

Bacillus anthracis Ames 105-6 (Cipro MIC > 100) USAMRIID 0.20 0.37 0.22 4.8 0.07

MBX Compounds Have Potent in vitro Activities Against Category A & B Biowarfare Agents

November 7, 2008

MIC90 Values for 20 Strains each of B. pseudomallei and B. mallei

Strain (n) Compound MIC90 (µg/mL) MIC50 (µg/mL) RangeBurkholderia

pseudomallei (20) Tetracycline 1 0.5 0.25 - 2

MBX 1066 2 1 0.5 - >8

MBX 1090 >8 8 1 - >8

MBX 1162 1 0.5 0.25 - 1

Burkholderia mallei (20) Tetracycline 0.125 0.06 0.03 – 0.25

MBX 1066 0.125 0.125 0.06 – 0.25

MBX 1090 0.25 0.25 0.125 - 1

MBX 1162 0.125 0.06 0.06 – 0.25

November 7, 2008

Demonstrate in vivo Potency in Various Murine Efficacy Models

November 7, 2008

No efficacy was demonstrated in a F. tularensis infection model

F . T u la r e n s is m o d e l

0

2 0

4 0

6 0

8 0

1 0 0

0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0

h o u r s p o s t c h a l la n g e

% m

ice

su

rviv

al

c o n t r o lM B X 1 0 9 0M B X 1 1 1 3M B X 1 1 4 2M B X 1 1 6 2

Infection: F. tularensis (Schu4 strain) given i.pTreatment: Compound (1.0, 1.5, 1.5, 0.5 mg/kg/injection for MBX 1090, 1142, 1162 and 1113, respectively) given i.p. qid starting at 6 hours post-infection and ending 5 days post-infection

November 7, 2008

MBX 1066 and 1142 were efficacious in a Yersinia pestis infection model

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 Infection: Y. pestis (100 cfu, CO92 strain) given i.pTreatment: Compound (1.5mg/kg/injection in 1.5 % DMSO in water) given i.p. qid starting at 6 hours post-infection and ending 5 days post-infection

Conclusion: MBX 1066 and 1142 were efficacious while MBX 1162 was not. Studies with IV compound administration are under way.

November 7, 2008

MBX 1066 is efficacious in a Yersinia pestis infection model when given by 2 different routes of administration

Infection: Y. pestis (100 cfu, CO92 strain) given i.pTreatment: Compound (2 mg/kg /injection in 1.5 % DMSO in water) given i.p. or i.m. qid starting at 6 hours post-infection and ending 5 days post-infection

Conclusion: MBX 1066, at 2 mg/kg dosing, was 60% and 20% protective when given i.p. or i.m., respectively. MBX 1162 was not efficacious by either route.

0

20

40

60

80

100

0 3 6 9 12 15 18

% s

urvi

val

Days post infection

Y.Pestis modelcontrol (n=10)

MBX 1066 IP (n=10)

MBX 1066 IM (n=10)

MBX 1162 IP (n=10)

MBX 1162 IM (n=10)

November 7, 2008

MBX 1162 demonstrated efficacy in a Burkholderia pseudomallei infection model

0

20

40

60

80

100

120

0 1 2 3 4 5

% S

urvi

val

Days Post-challenge

Burkholderia pseudomallei IV Treatment

Control

Tetracycline

MBX 1066

MBX 1090

MBX 1162

Infection: B. pseudomallei (1x 104 cfu, 1026b strain) given by the intranasal route (n = 5)Treatment: Compound (10 mg/kg/injection in 10% DMSO/PBS) given IV once at 1 hour post-infection

Conclusion: MBX 1162 was more efficacious than the control antibiotic, tetracycline, while MBX 1090 was equipotent to tetracycline. MBX 1066 caused immediate deaths in 2 mice and was not dosed to the remaining mice.

November 7, 2008

MBX 1162 again demonstrated efficacy in a Burkholderia pseudomallei infection model

Infection: B. pseudomallei (1x 106 cfu, 1026b strain) given by the i.p.l route (n = 5)Treatment: Compound (10 mg/kg/injection in 10% DMSO/PBS) given i.p. once at 1 hour post-infection

Conclusion: MBX 1162 and 1090 were equivalent to the control antibiotic, tetracycline, while MBX 1066 displayed no potency.

0

20

40

60

80

100

120

0 1 2 3 4

% S

urvi

val

Days Post-infection

B. pseudomallei i.p. Treatment

Vehicle control

Tetracycline

MBX 1090

MBX 1066

MBX 1162

November 7, 2008

MBX 1090 and 1162 demonstrated efficacy in a Burkholderia mallei infection model

0

20

40

60

80

100

120

0 1 2 3 4 5

% S

urvi

val

Days Post-challenge

Burkholderia mallei IV Treatment

Control

Tetracycline

MBX 1090

MBX 1162

Infection: B. mallei (1x 106 cfu, GB5 strain) given by the intranasal route (n = 5)Treatment: Compound (10 mg/kg/injection in 10% DMSO/PBS) given IV once at 1 hour post-infection

Conclusion: MBX 1090 and 1162 were 60% and 20% protective when given in a single IV dose where the control antibiotic, tetracycline, provided 100% protection. MBX 1066 was not tested due to the deaths observed in the B. pseudomallei model.

November 7, 2008

Demonstrated efficacy in a Bacillus anthracis infection model

Infection: B. anthracis (860 cfu, Ames strain) given by the i.p. route (n = 10)Treatment: Compound (10 mg/kg for MBX 1066 and 1162, 5 mg/kg for MBX 1090 in 10% DMA/D5W) given IV at 6 hours post-infection; 5 mg/kg for MBX 1066 and 1162, 2 mg/kg for MBX 1090 given IV at 18 and 42 hours post-infection—a total of 3 treatments

Conclusion: MBX 1090 was 40% protective when given in three IV doses. MBX 1066 and 1162 did not protect when administered in three doses.

0

25

50

75

100

0 5 10 15 20

% s

urvi

val

Days post challenge

B. anthracis Triple IV treatment

control

MBX 1066

MBX 1090

MBX 1162

November 7, 2008

Demonstrated efficacy in a Bacillus anthracis infection model

Infection: B. anthracis (860 cfu, Ames strain) given by the i.p. route (n = 10)Treatment: Compound (10 mg/kg for MBX 1066 and 1162, 5 mg/kg for MBX 1090 in 10% DMA/D5W) given IV at 6 hours post-infection; only 1 treatment

Conclusion: MBX 1162 was 60% protective when given in one IV dose. MBX 1066 and 1090 were not protective when administered in one IV dose.

0

25

50

75

100

0 5 10 15

% s

urvi

val

Days post challenge

B. anthracis Single IV treatment

control

MBX 1066

MBX 1090

MBX 1162

November 7, 2008

Survivors

Group n IV treatment Dose, mg/kg 8 hr 18 hr 24 hr 48 hr % survival

1 10 DMA/D5W - 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

7 10 MBX 1162 10 10 10 10 10 100

8 10 MBX 1162 1 9 6 6 6 60

Demonstrated efficacies of MBX compounds in a murine S. aureus infection model

Infection: S.aureus (4X108 cfu, Smith strain) injected i.p. Treatment: Compound (10 or 1 mg/kg in 10% DMA/D5W) given IV 15 min. post-infection

Conclusion: MBX 1162 was 100 and 60% protective when given in one IV dose at 10 and 1 mg/kg, respectively. MBX 1066 and 1090 were 80 and 90% protective, respectively, when administered in one IV dose. MBX 1113 and 1128 killed mice at the 10 mg/kg dose and were not protective at the 1 mg/kg dose.

November 7, 2008

Initial compound IV toxicity studies in mice

Compound MBX 1066 MBX 1162Route/vehicle IV; 20% DMSO/D5W IV; 10% DMA/D5W

LD50 (mg/kg) 20 40-50

Maximum tolerated dose (mg/kg) 10-20 ≈40

No adverse effect dose (mg/kg) 10 10

ED50 (mg/kg, IV) < 10, IV< 1, i.p.

1-3, IV< 1, i.p.

Conclusion: MBX 1162 and 1066 were both non-toxic at the 10 mg/kg IV dose but displayed signs of toxicity at higher concentrations.

November 7, 2008

Aim 3 Demonstrate structure-activity relationships for the potency and selectivity of the bis-(imidazolinylindole) class of compounds (year 2).

November 7, 2008

Task 4.3.1 Perform molecular modeling studies

November 7, 2008

Task 4.3.2 Synthesis of bis-(imidazolinylindole) analogs4.3.2.1 Synthesis of NSC 317,880 (MBX 1090) and analogs

November 7, 2008

4.3.2.2 Synthesis of NSC 317,881 (MBX 1066) and analogs

November 7, 2008

4.3.2.3 Synthesis of NSC 330,687 (MBX 1113) and analogs

November 7, 2008

Evaluation of bis-(imidazolinylindole) analogs

Potency measurement of MBC/MIC values on intact cells

November 7, 2008

MBX compounds act with a bactericidal mechanism of action with rapid killing kinetics observed

0

2

4

6

8

10

12

0 10 20 30

Log

CFU

/mL

Time (hours)

MBX Compounds vs. Y. pestis in a Time Kill Assay at 4x MIC

Control

MBX 1066MBX 1090MBX 1142

MBX 1162 0123456789

0 10 20 30

Log

CFU

/mL

Time (hours)

MBX Compounds vs. B. anthracis in a Time Kill Assay at 4x MIC

Control

MBX 1066MBX 1090MBX 1142

MBX 1162

Time (hr) to cidal effect

Species / Compound MBX-1066 MBX-1090 MBX-1142 MBX-1162

Y. pestis ≤1 ≤1 ≤1 ≤1

B. anthracis 6 ≤1 4 ≤1

November 7, 2008

Mammalian cytotoxicity values provide good selectivity indices

CompoundsHeLa Cell CC50

(µg/mL)MIC S. aureus 25923

(µg/mL)Selectivity Index

(in vitro)

MBX 1066 32.5 0.12 270

MBX 1090 10 0.63 16

MBX 1113 3 0.31 9.6

MBX 1128 17 0.28 60

MBX 1142 14 0.27 51

MBX 1143 13 0.12 111

MBX 1162 4 0.16 26

MBX 1195 15 45 0.33

MBX 1196 15 0.16 96

HB-EMAU 35 5 7

Method: Human HeLa cells were exposed for 72 hours to serial dilutions of compounds, then assessed for cell viability using an MTT assay

Conclusion: MBX 1066 displayed the highest selectivity index, with 4 other compounds displaying indices >50

November 7, 2008

MBX 1066 and 1162 Maintain Excellent Potencies Against Multiple Isolates of

Gram-positive and Gram-negative Species

November 7, 2008

Gram-Positive (Staphylococci) Laboratory/Clinical Strains

Gr+ Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

Staphylococcus aureus MSSA 27 MBX 1066 0.25 0.12 0.004-0.5

MBX 1162 0.5 0.12 0.008-0.5

Linezolid 4 2 2-4

Vancomycin 1 0.5 0.5-2

Daptomycin 0.5 0.5 0.25-1

S. aureus MRSA 12 MBX 1066 0.12 0.06 0.06-0.12

MBX 1162 0.12 0.06 0.03-0.12

Linezolid 4 2 2-4


Daptomycin 0.25 0.25 0.12-0.5

Staphylococcus epidermidis MSSE 27 MBX 1066 0.03 0.008 0.004-0.06

MBX 1162 0.06 0.03 0.008-0.06

Linezolid 2 1 0.5-2

Vancomycin 2 1 1-4

Daptomycin 1 0.5 0.5-1

S. epidermidis MRSE 12 MBX 1066 0.03 0.015 0.004-0.03

MBX 1162 0.06 0.015 0.008-0.06

Linezolid 2 1 1-2

Vancomycin 2 2 1-2


November 7, 2008

Gram-Positive (Enterococci) Laboratory/Clinical Strains


Enterococcus faecalis VSE 27 MBX 1066 0.06 0.06 0.004-0.12

MBX 1162 0.06 0.06 0.004-0.25

Linezolid 2 2 0.5-2

Vancomycin 2 1 0.5-2

Daptomycin 2 1 0.03-4

E. faecalis VRE 12 MBX 1066 0.06 0.03 0.015-0.06

MBX 1162 0.03 0.015 0.008-0.03

Linezolid 1 1 0.5-2

Vancomycin >64 >64 >64


Enterococcus faecium VSE 27 MBX 1066 0.015 0.004 0.002-0.03

MBX 1162 0.015 0.004 0.002-0.03

Linezolid 4 2 2-4


Daptomycin 4 4 1-8

E. faecium VRE 12 MBX 1066 0.004 0.004 0.002-0.008

MBX 1162 0.004 0.004 0.004-0.008

Linezolid 2 2 1-2

Vancomycin >64 >64 64->64

Daptomycin 4 2 1-4

November 7, 2008

Gram-Positive (Streptococci) Laboratory/Clinical Strains


Streptococcus pneumoniae PSSP 27 MBX 1066 0.03 0.015 0.008-0.12

MBX 1162 0.03 0.03 0.015-0.03

Linezolid 2 1 0.5-2

Vancomycin 0.25 0.25 0.12-0.25

Daptomycin 0.25 0.06 <0.03-0.5

S. pneumoniae PRSP 12 MBX 1066 0.06 0.03 0.03-0.06

MBX 1162 0.06 0.03 0.015-0.06

Linezolid 1 1 0.5-1

Vancomycin 0.25 0.25 0.25-0.5

Daptomycin 0.12 0.06 <0.03-0.12

Streptococcus agalactiae 12 MBX 1066 0.06 0.06 0.03-0.12

MBX 1162 0.06 0.06 0.06-0.12

Linezolid 2 2 1-2

Vancomycin 0.5 0.5 0.5-1


Streptococcus pyogenes 12 MBX 1066 0.03 0.03 0.03

MBX 1162 0.03 0.03 0.03

Linezolid 2 1 1-2

Vancomycin 1 1 0.5-1


November 7, 2008

Gram-Negative (Nonfermentors) Laboratory/Clinical Strains

Gr- Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

A. baumannii 27 MBX 1066 >16 2 0.06->16

MBX 1162 0.5 0.25 0.12-4

Imipenem 1 0.12 0.06-8

Tigecycline 1 0.5 0.06-4

Ciprofloxacin 2 0.5 0.015->8

A. baumannii MDR 13 MBX 1066 >16 >16 1->16

MBX 1162 4 2 0.12-4

Imipenem >32 4 0.06->32

Tigecycline 4 2 0.25->32

Ciprofloxacin >8 >8 0.12->8

P. aeruginosa 27 MBX 1066 >16 >16 0.06->16

MBX 1162 1 0.25 0.03->16

Imipenem >8 1 0.5->8

Tigecycline >8 8 1->8

Ciprofloxacin >2 0.25 0.12->2

B. cepacia 11 MBX 1066 0.06 <0.015 <0.015-4

MBX 1162 0.12 0.06 0.03-0.25

Imipenem >8 4 4->8

Tigecycline 4 2 1-4

Ciprofloxacin 2 2 0.5-2

November 7, 2008

Gram-Negative (Enterobacteriaceae) Laboratory/Clinical Strains


Escherichia coli 27 MBX 1066 0.5 0.12 0.03-0.12

MBX 1162 0.25 0.12 0.06-0.25

Imipenem 0.25 0.25 0.06-0.5

Tigecycline 0.25 0.12 0.12-0.25


Klebsiella pneumoniae 27 MBX 1066 8 2 0.25->16

MBX 1162 0.5 0.25 0.12-1

Imipenem 16 0.25 0.06-32



K. pneumoniae ESBL 12 MBX 1066 >16 1 0.5->16

MBX 1162 0.5 0.12 0.06-0.5

Imipenem 1 0.25 0.12-2


Ciprofloxacin >8 >8 0.06->8

Serratia marcescens 12 MBX 1066 2 1 0.06-2

MBX 1162 0.25 0.12 0.12-0.5

Imipenem >8 4 2->8

Tigecycline 1 1 0.5-2

Ciprofloxacin 1 0.25 0.06->2

November 7, 2008

Gram-Negative (Atypical) and Gram-positive (Anaerobe) Laboratory/Clinical Strains


H. influenzae 12 MBX 1066 >16 4 1->16

MBX 1162 4 1 0.5-4

Levofloxacin 0.06 0.015 0.008-1

Azithromycin 2 1 0.5-2

Cefotaxime >4 1 0.03->4

Amox/Clav 8/4 1/0.5 0.5/0.25-16/8

Clostridium difficile 16 MBX 1066 0.12 0.06 0.03-0.25

MBX 1162 0.06 0.06 0.03-0.12

Clindamycin >8 4 0.25->8

Imipenem 4 4 0.5->8

Metronidazole 0.5 0.12 0.06->8

November 7, 2008

Average MIC (µg/mL)Bacterial Strain MBX 1066 MBX 1090 MBX 1113 MBX 1128 MBX 1162E. coli 700 TolC+ (efflux proficient) 1.3 0.63 0.31 80 0.16E. coli 701 TolC- (efflux deficient) 0.16 0.16 0.16 21 0.14Pseudomonas aeruginosa PAO1 (efflux proficient) 7.5 3.1 25 >80 0.29

P. aeruginosa PAO1 ΔmexAB-oprM (efflux deficient) 1.15 3.1 1.3 >80 0.25

The Effect of Efflux on Activity of Compounds

November 7, 2008

Aims 1 and 3 Research SummaryChemistry– Original 4 compounds plus MBX 1066 analogs and saltsSmall scale of > XX analogs in SAR programScale-up synthesis of XX compounds at > g(kg)

Microbiology--Potent in vitro activity against Gram-pos. and Gram-neg. pathogens, especially category A or B bioterrorism pathogens

Potency maintained even when looking at >10 isolates/species (MIC90 values)

Rapidly bactericidal mechanism of actionPotent in vivo efficacy against several murine infection models

Low 3-day cytotoxicity (CC50) of compounds

November 7, 2008

Aim 2 Establish the mechanism of action of the bis-(imidazolinylindole) class of compounds (year 1).

November 7, 2008

Aim 2 – Mechanism of ActionNSC 317880, NSC 317881, NSC 330687, NSC

369718 & New analogs

1. Macromolecular Synthesis Assays & cidal/static determination(DNA, RNA, protein, cell wall, & lipid biosynthesis)

2. Cell Membrane Integrity Assays-- Fluorescent dye retention – membrane potential-- HeLa cell lysis – membrane lysis

3.a. Genes Up-Regulated in Resistant Strains-- Identify over-expressed E. coli genes which confer resistance

3.b. Mapping Mutations to Resistance-- Select resistant mutants; then,-- map by comparative genome sequencing (CGS)

4. Expression Profiling-- Identify genes or patterns of genes up- or down-

regulated in response to treatment with compound

5. Target Confirmation -- Demonstrate MIC alterations in response to up- or down-

regulation of the putative target-- Demonstrate plasmid-mediated transfer of resistance in

>1 species

Inhibits 0 or 1 pathwayInhibits >1 pathway

DNA+RNA @ ≥10X MIC

NONo

Membrane is target

Completed

Completed

Completed Completed

6. Experiments not in original plan-- DNA binding studies (Eric Long, IUPUI)-- Phenotypic Macroarray (Biolog)

Not Done

Not Done (no discreet targets ID)

November 7, 2008

Macromolecular Synthesis Assays

•None of the MMS pathways affected at killing dose (5x MIC)•Unknown target•DNA synthesis is inhibited at >10X MIC (secondary effect)

MBX-1066-40xMBX-1066-20x

MBX-1066-10xMBX-1066-5x

0

20

40

60

80

100

120

DNA RNA Protein Cell wall Lipid

% o

f Con

trol

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

Prot

ein

Cel

l wal

l

Lipi

d

% o

f Con

trol

Macromolecule

Rifampicin (RNA), Chloramphenicol (protein), Ciprofloxacin (DNA), Vancomycin (cell wall) and Irgasan (lipid)

S. aureusMBX-1066 Controls

November 7, 2008

Membrane perturbation assays

0

1

2

3

4

5

6

7

Flou

resc

ence

rat

io (r

ed/g

reen

)

0

5

10

15

20

64X

MIC

16X

MIC

1X M

IC

64X

MIC

16X

MIC

1X M

IC

No a

ntib

iotic

Tota

lLys

is

RFU

x103

MBX-1066 VAN

0

5

10

15

20

64X

MIC

16X

MIC

1X M

IC

64X

MIC

16X

MIC

1X M

IC

No a

ntib

iotic

Tota

lLys

is

RFU

x103

MBX-1066 VAN

MBX-1066 does not perturb bacterial or mammalian cellular membranes at therapeutically relevant concentrations

Bacterial membrane perturbationDiOC(2)/FACS

Mammalian membrane lysisLDH release assay

November 7, 2008

Map loci responsible for bis-(imidazolinylindole) resistance

Hig

hest

Sub

leth

al C

once

ntra

tion

(Fol

d M

IC)

A B C D E F G H

128643216

8421

0.50.25

0.125 MBX 1066

1 5 10 15 20Time (days)

MBX 1090

128643216

8421

0.50.25

0.125

1 5 10 15 20Time (days)

MBX 1113

1 5 10 15 20Time (days)

128643216

8421

0.50.25

0.125

S. aureus NCTC 8325

Resistant mutants-16X MIC

MBX-1066 resistance is rare

Serial passage of S. aureus NCTC-8325 in subinhibitory compound concentrations to select resistance mutants

MBX-1113 resistance is rare

MBX-1162 resistance is rare (data not shown)

November 7, 2008

Map loci responsible for bis-(imidazolinylindole) resistance

328800 329600 330400 331200

SAOUHSC_00314mepR

MarR-like repressor

SAOUHSC_00315mepA

Multi Antimicrobial Extrusion (MATE)

drug/sodium antiporter

SAOUHSC_00316mepB

Unknown function

WT

1090R mutants (A1, B1, and C1)

mepR mepA mepB

X

OFF

ON

Compound NCTC 8325 (WT) 1090R d20A1MBX-1066 0.5 0.5MBX-1090 1 8MBX-1113 1 2MBX-1128 1 1MBX-1162 0.5 0.5MBX-1195 16 32MBX-1196 1 1MBX-1335 0.0625 0.0625distamycin 50 200

MIC (µg/ml)

No cross resistance vs. other bis-(imidazolinylindole) compounds

Model confirmed by extensive genetic and transcription profiling analyses (see poster)

•MBX-1090 is a MepA substrate•Other bis-(imidazolinylindole) compounds are NOT MepA substrates

November 7, 2008

Bottom line Analyses of MBX-1090 resistant mutants

have not identified MOA MBX-1090 resistant mutants have identified

a novel mechanism of resistance based on drug efflux

Resistance mechanism does not affect MBX-1066 and analogs

Additional experiments in progress to elucidate MOA….

November 7, 2008

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

MBX-1066 fluorescent enhancement from B. anthracis or calf thymus genomic

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

0.001 0.01 0.1 1 10 100

[uM] nt bp's

ratio

of 1

-(fre

edru

g/dr

ug+D

NA)

1066+B.anthracis DNA1066+Calf Thymus DNA

Affinity of both MBX 1066 for AT-rich B. anthracis DNA is ~2-fold stronger than for calf thymus DNA

DNA Interaction with MBX-1066 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracis Genomic DNA

Analysis of DNA binding activity of bis-(imidazolinylindole) compounds

Why?—structural similarity with DNA minor groove binders

November 7, 2008

In situ fluorescence of MBX-1066 in S. aureus cells is consistent with DNA binding at 1X MIC

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 7, 2008

5’-CGXXXXC3’-GCXXXXGA

A AAA

Fluorescent displacement assayMBX-1162

Preference for A/T rich sequencesHighest affinity for AATT

136 possible sequences

Relative affinity for AATTScatchard plot (Kapp)

Dr. Eric Long (IUPUI)

Detailed analyses of DNA binding activity of selected bis-(imidazolinylindole) compounds

Slope = Kapp

November 7, 2008

Correlation between DNA binding and biological activity

y = 0.0484x-2.144

R² = 0.74290.01

0.1

1

10

0.1 1 10

Kap

px

106

(M-1

)

MIC (µg/ml) in 20 µg reserpine/ml

10901066

1196

1162

1195 (estimated Ka)y = 134.55x-2.206

R² = 0.3401

0.01

0.1

1

10

1 10 100K

app

x 10

6(M

-1)

CC50 (µg/ml)

10901066

1196

1162

1195 (estimated Ka)

antibacterial cytotoxicity

Compound 0 μg reserpine/ml 20 μg reserpine/ml CC50 (μg/ml) Kapp x 106 (M-1)MBX-1066 0.25 0.125 32.5 0.5MBX-1090 1 0.5 15 0.25MBX-1195 16 2 4 ND (too weak)MBX-1196 1 0.25 10 1MBX-1162 0.5 0.125 15 31

MIC (μg/ml)

Weak correlation between MIC and DNA binding

No correlation between cytotoxicity and DNA binding

November 7, 2008

Profiling the changes in gene expression in response to MBX-1066 and -1090 for MOA (in progress)

Grow S. aureus NCTC 8325 in presence of MBX-1066, MBX-1090 and a compendium of antibiotics that affect RNA/DNA syntehsis at 1-2X MIC for 1 doubling time (3 h in MHB) in triplicate

Harvest cells and prepare RNA. Microarray analyses at NimbleGen Identify genes up- and down-regulated

by MBX-1066 and -1090. Compare profile to other antibiotics using statistical methods.

antibiotic concentration1 control NA2 1090Rd20A1 NA3 MBX 1066 1X MIC4 MBX 1090 1X MIC5 Distamycin 2X MIC6 Novobiocin 2X MIC7 Trimethoprim 1X MIC8 Nalidixic Acid 1X MIC9 Ciprofloxacin 2X MIC10 actinomycin D 1X MIC11 Rifampicin 1X MIC12 Phleomycin 2X MIC

Samples prepared for profiling

November 7, 2008

Transcription profiling results-analyses in progress

Identify genes affected by treatment (induced and repressed)

Analyze metabolic pathways affected by MBX-1066 and MBX-1090 treatment

Hierarchical clustering

November 7, 2008

Genes affected by MBX-1066 and -1090

MBX-1066691 MBX-1090

13174

Numbers of genes Up- and Down-regulated (4X, 90% confidence)

Up=333Down=358conserved hypothetical protein = 325

Up=44Down=87conserved hypothetical protein = 65

peptide ABC transporter, ATP-binding protein, putative 5.511 upsuperoxide dismutase, putative 5.103 upchaperonin, 10 kDa, GroES, putative 9.694 upprophage genes 5-23 upphosphoribosylformylglycinamidine synthase, PurS protein 4.687 upphosphoribosylformylglycinamidine synthase II 4.924 up

capsular polysaccharide synthesis enzyme Cap5B 9.603 downacetyl-CoA acetyltransferase, putative 5.995 downiron (chelated) ABC transporter, permease protein, putative 9.980 downclumping factor 4.173 down

MBX-1090 selected genes

November 7, 2008

+4.8 +15.9

DIS

T-R

EP

1066

-REP

NA

-REP

TMP-

REP

RIF

-REP

CIP

-REP

PHLE

O-R

EP

NO

VO-R

EP

1090

-REP

AC

TD-R

EP

Hierarchical clustering: MBX-1066 genes 4X UP and DOWN (691)

MBX-1066 clusters with Distamycin (DNA minor croove binder)MBX-1090 weakly associated with DNA/RNA inhibitors

November 7, 2008

+4.8 +15.8

DIS

T-R

EP

1090

-REP

1066

-REP

NA

-REP

TMP-

REP

CIP

-REP

PHLE

O-R

EP

RIF

-REP

NO

VO-R

EP

AC

TD-R

EP

Hierarchical clustering: MBX-1090 genes 4X UP and DOWN (131)

MBX-1066 and MBX-1090 clusters with Distamycin (DNA minor groove binder)

November 7, 2008

+4.8 +15.8

DIS

T-R

EP

1066

-REP

1090

-REP

NA

-REP

TMP-

REP

CIP

-REP

PHLE

O-R

EP

RIF

-REP

NO

VO-R

EP

AC

TD-R

EPHierarchical clustering: Intersection MBX-1090 and 1066 genes 4X UP and DOWN (74)

MBX-1066 and MBX-1090 clusters with Distamycin (DNA minor groove binder)

November 7, 2008

Biolog-MOA using Phenotypic Microarrays

1. Measure magnitude of synergy/antagonism (SAVs) of experimental compound against a panel of 60 antibiotics with known MOA using PM technology

2. Generate matrix of SAVs that describes the chemical interaction between the chemicals in the PM plate and the added inhibitors being tested.

3. Use statistical clustering program to group antibiotics based on SAVs. Antibiotics with like MOAs cluster together.

November 7, 2008

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94

130

109

125

110

145

57

65

202

67

55

212

206

196

8

231

282

280

244

245

217

152

224

2

101

384

457

179

179

179

226

163

163

348

132

240

74

458

270

52

139

515

516

10

81

313

351

361

4

211

Spiramycin

Tylosin

Erythromycin

Erythromycin

Erythromycin

Josamycin

Dirithromycin

Dirithromycin

Roxithromycin

Clindamycin

Lincomycin

Cadmium Chloride

Tyrothricin

Monensin

Antimony (III) Chloride

Coumarin

MBX-1066

MBX-1090

2,4-Dintrophenol

CCCP

Pentachlorophenol

Salicylanilide

Sodium Azide

1,10-Phenanthroline

Hexachlorophene

Macrolide, 16 ring

Ketolide

Macrolide, 14 ring

Macrolide, 14 ring

Macrolide, 14 ring

Macrolide, 16 ring

Macrolide, 14 ring .

Macrolide, 14 ring .

Macrolide

Lincosamine

Lincosamine

toxic cation

Polyether

toxic cation

AminoCoumerins

phenol

Phenanthroline

Ribosome 50S, 23S RNA,.


Ribosome, 50S, 23S RNA,.







Ribosome, 50S, Peptidyltr.

Ribosome, 50S, Peptidyltr.

Cation toxicity

membrane, cyclic peptide

Ionophore, K?

Cation toxicity

DNA Topoisimerase II (D.

Uncoupler, Respiration

Uncoupler, Respiration

membrane permeability, .


respiration, uncoupler

Chelator, Fe (Zinc?)


Phenotypic Microarray-Clustering results

MBX-1066 and MBX-1090 cluster with toxic cations—suggests non-specific MOA

November 7, 2008

Summary: Antibacterial Mechanism of bis-(imidazolinylindole) compounds

• Bottom Line: MOA(s) unknown (but, we know what it isn’t)• Activity against DNA-dependent macromolecular synthesis

• Inhibition of DNA and RNA synthesis at >10x MIC in cell-based MMS assay • Inhibition of replicative helicase (IC50~1 μM; 4X MIC)• Inhibition of ReplixTM (IC50 ~2 μM; 8X MIC) • Above activities NOT correlated with antibacterial activity

• Minimal effects on bacterial and mammalian cell membranes• Extremely low frequency of resistant mutants• The bis-(imidazolinylindole) compounds interact with DNA• Fluorescence enhancement in the presence of DNA (Max1/2~0.4 μM)• Intracellular fluorescence observed at 1X MIC• AATT is optimal binding site• DNA binding affinity is NOT correlated with antibacterial activity• Microarray studies suggests non-specific MOA

november 7, 2008 microbiotix a product-focused, small molecule, anti-infective drug discovery...

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