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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,

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Presentation on theme: "November 2007 The development of novel broad- spectrum anti-bacterials for intracellular BW threats Terry L. Bowlin, Ph.D. CEO, Microbiotix, Inc. Worcester,"— Presentation transcript:

1 November 2007 The development of novel broad- spectrum anti-bacterials for intracellular BW threats Terry L. Bowlin, Ph.D. CEO, Microbiotix, Inc. Worcester, MA

2 November 2007 The development of novel broad- spectrum anti-bacterials for intracellular BW threats Microbiology Mechanism Animal studies

3 November 2007 The development of novel broad-spectrum anti-bacterials for intracellular BW threats Broad-spectrum anti-bacterials discovered by Sina Bavari, Ph.D.

4 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 (CC 50 ) of compounds

5 November 2007 Average MIC (µg/mL) Bacterial StrainMBX 1066MBX 1090MBX 1113MBX 1128 Bacillus subtilis BD540.1170.156 0.068 B. cereus ATCC 43420.0780.156 0.521 B. thuringiensis ATCC 107920.0780.3130.2350.182 B. anthracis Sterne0.2350.3130.1561.25 B. anthracis Ames  ANR (pXO1-, pXO2-) 0.0980.313 36.3 B. megaterium ATCC 128720.0780.1560.0780.176 B. licheniformis ATCC 145800.0590.3130.1560.117 Staphylococcus aureus ATCC 259230.1170.6250.3130.283 S. aureus (Smith) ATCC 137090.0780.3130.1560.078 Meth-res S. aureus (MRSA) 1094, clinical0.1370.6250.3130.508 S. aureus MT23142 NorA++0.0390.3130.2350.088 Enterococcus faecalis ATCC 292120.1370.313 0.107 Vanc-res E. faecalis (VRE) ATCC 515750.1170.6250.4690.107 E. faecium ATCC 194340.0590.1560.2740.088 VRE faecium B42762, clinical0.0390.3130.1560.068 MBX Gram Positive MIC Data (BSL 2)

6 November 2007 Average MIC (µg/mL) Bacterial StrainMBX 1066MBX 1090MBX 1113MBX 1128 Escherichia coli J53, lab strain0.3910.6250.31353.3 E. coli XL1Blue, lab strain0.0780.156 1.8 E. coli 700 TolC+1.250.6250.31380 E. coli 701 TolC-0.156 21.3 Klebsiella pneumoniae 5657, clinical0.2350.5800.35216.3 Pseudomonas aeruginosa PAO17.525 >80 P. aeruginosa PAO1 ΔmexAB-oprM1.15>20ND P. aeruginosa 278532.512.51.09>80 Burkholderia thailandensis E2646.25>8035>80 Stenotrophomonas maltophilia ATCC 136370.1760.6250.31311.3 MBX Gram Negative MIC Data (BSL 2)

7 November 2007 Average MIC (  g/mL) Bacterial StrainTest SiteMBX 1066MBX 1090MBX 1113MBX 1128 Burkholderia pseudomallei 1026bCalgary0.653.2>8>8* Burkholderia mallei GB3Calgary120.67>8* Bacillus anthracis AmesUSAMRIID0.0670.0990.110.145 Burkholderia mallei ATCC 23344USAMRIID0.421.61.8>9.7 Burkholderia pseudomallei DD503USAMRIID1.73.11.8>9.7 Francisella tularensis Schu4USAMRIIDND1.560.924.9 Yersinia pestis CO92USAMRIID3.4>12.5>7.4>9.7 USAMRIID and U. Calgary MIC Data (BSL 3) *Value determined only once.

8 November 2007 Average MIC (µg/mL) Bacterial StrainMBX 1066MBX 1142MBX 1143MBX 1162 Bacillus subtilis BD540.1170.0680.0340.063 B. cereus ATCC 43420.0780.1070.0390.054 B. thuringiensis ATCC 107920.0780.1560.0780.117 B. anthracis Sterne0.2350.2150.0780.088 B. anthracis Ames  ANR (pXO1-, pXO2-) 0.0980.1560.0390.063 B. megaterium ATCC 128720.0780.0980.0340.037 B. licheniformis ATCC 145800.0590.1560.0490.088 Staphylococcus aureus ATCC 259230.1170.2740.1170.156 S. aureus (Smith) ATCC 137090.0780.2150.0780.102 Methicillin-res. S. aureus (MRSA) 1094, clinical0.1370.2930.1560.127 S. aureus MT23142 NorA ++0.0390.1780.0780.166 Enterococcus faecalis ATCC 292120.1370.1760.0780.156 Vanc-resistant E. faecalis (VRE) ATCC 515750.1170.1820.1560.137 E. faecium ATCC 194340.0590.2350.1370.186 VRE faecium B42762, clinical0.0390.2350.1560.137 MBX Gram Positive MIC Data for selected MBX 1066 Analogs

9 November 2007 Average MIC (µg/mL) Bacterial StrainMBX 1066MBX 1142MBX 1143MBX 1162 Escherichia coli J53, lab strain0.3910.430.1950.274 E. coli XL1Blue, lab strain0.0780.2150.0980.254 E. coli 701 TolC-0.1560.2540.1560.137 Klebsiella pneumoniae 5657, clinical0.2350.2540.1370.146 Pseudomonas aeruginosa PAO17.50.9380.2350.293 P. aeruginosa PAO1 ΔmexAB-oprM1.150.3130.1560.254 P. aeruginosa 278532.50.7810.2150.254 Burkholderia thailandensis E2646.2522.50.352 Stenotrophomonas maltophilia ATCC 13637 0.1760.1560.0780.156 MBX Gram Negative MIC Data for selected MBX 1066 Analogs

10 November 2007 Average MIC (  g/mL) Bacterial StrainTest SiteMBX 1066MBX 1142MBX 1143MBX 1162 Burkholderia pseudomallei 1026bCalgary0.65110.375 Burkholderia mallei GB3Calgary1ND Burkholderia mallei ATCC 23344USAMRIID0.421.8 0.6 Burkholderia pseudomallei DD503USAMRIID1.71.80.6ND Francisella tularensis Schu4USAMRIIDND1.8ND1.8 Yersinia pestis CO92USAMRIID3.43.5ND3.5 USAMRIID and U. Calgary MIC Data for Selected MBX 1066 Analogs (BSL 3)

11 November 2007 CompoundMBX 1090MBX 1066MBX 1142MBX 1162 Time to reach cidal effect (hours) for S. aureus 421.51 Time to reach cidal effect (hours) for Y. pestis ≤1 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 ~10 3 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 AB

12 November 2007 Strain MBC (µg/mL) MBX 1090 MIC (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 cipro R 1.1680.58421.292 1 B. anthracis 10242.3361.16820.6460.1624 B. anthracis vollum 1.1680.58420.324 1 Comparison of MBC and MIC values for infectious B. anthracis strains

13 November 2007 Compound Cytotoxicity on HeLa cells Compounds CC 50 (µg/mL) MIC S. aureus 25923 (µg/mL) Selectivity Index (in vitro) MBX 1066 >200.117>170 MBX 1090 100.62516 MBX 1113 30.3139.6 MBX 1128 170.28360 MBX 1142 140.27451 MBX 1143 130.117111 MBX 1162 40.15626 HB-EMAU 3557

14 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

15 November 2007 Mechanism Studies MMS DNA Binding Replix Helicase In Situ Efflux Resistance Membrane Effects

16 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

17 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

18 November 2007 MBX 1066, 1090 and 1113 are Potent Inhibitors of Replix™, a Permeable Cell DNA Replication Assay IC 50 µM (µg/mL) Against Permeable Bacteria CompoundB. subtilisB. 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)

19 November 2007 Conclusion: MBX 1066 & 1090 are very potent B. anthracis helicase inhibitors with IC 50 ’s of <1 μM (<0.6 μg/ml) Helicase Inhibition by MBX 1066 & 1090 as Measured by 32 P-Based Unwinding Assay – Comparison to Other Helicase Inhibitors

20 November 2007 In situ Fluorescence of MBX 1066 in S. aureus cells is Consistent with Cell Penetration & DNA Binding None1 X MBX 10664 X MBX 10661 X MBX 10904 X MBX 1090 4 X MBX 1113 DIC DAPI DIC DAPI Intracellular fluorescence readily detected at 1X MIC Consistent with DNA-dependent fluorescence enhancement 1 X MBX 1066 Contrast enhanced 10X zoom cytoplasmic localization

21 November 2007 Average MIC (µg/mL) Bacterial StrainMBX 1066MBX 1142MBX 1143MBX 1162 Pseudomonas aeruginosa PAO17.50.9380.2350.293 P. aeruginosa PAO1 ΔmexAB-oprM1.150.3130.1560.254 MBX MIC Data for MBX 1066 & Analogs Isogenic 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

22 November 2007 Mutation to Resistance to MBX 1066 is Rare in S. aureus NCTC-8325 Serial Passage A B C D E FGH Highest Sublethal Concentration (Fold MIC) S. aureus NCTC 8325 Resistant mutants-16X MIC

23 November 2007 MBX 1090 Resistant Mutants are not Cross- Resistant to MBX 1066 MICs vs MBX 1090, MBX 1066, and MBX 1113 No cross resistance to MBX 1066, suggesting different MOAs for MBX 1090 and MBX 1066

24 November 2007 Bacterial membrane perturbation assay using DiSC 3 (5) DiSC 3 (5) Ex-622 Em-670 e - transport QUENCH 2H+ Ex-622 Em-670 Membrane disrupter Membrane potential perturbation No membrane potential perturbation by compound Membrane potential perturbation by compound

25 November 2007 DiSC 3 (5) Membrane Perturbation Assay of MBX 1066 & Analog MBX 1162 Results of DiSC 3 (5) assay 10 min after compound addition Conclusion: MBX 1066 & 1162 do not perturb membrane potential at concentrations near the MIC

26 November 2007 MBX 1066 does not disrupt HeLa cell membranes 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

27 November 2007 Favorable Features of Lead Series Antibacterial Mechanism In vitro therapeutic index (CC 50 /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 (Max 1/2 ~0.4 μM) Inhibits Replix TM (IC 50 ~2 μM) & replicative helicase (IC 50 ~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

28 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

29 November 2007 ANIMAL STUDIES B. Anthracis Y.Pestis B.Pseudomallei S. Aureus

30 November 2007 In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis In vivo testing in a murine B. anthracis infection model MBX1066

31 November 2007 In Vivo Testing in Y. Pestis Murine Model (USAMRIID)

32 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 10 6 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, nTreatmentDose, mg/kg24 hours48 hours72 hours% survival 1, 10Vehicle control --106220 2, 10Tetracycline 10 990 3, 10MBX 1090 10 880 4, 10MBX 1066 10 6330 5, 15Vehicle control --153ND20 6, 15Tetracycline 1015 ND100 7, 15MBX 1162 1015 ND100

33 November 2007 survivors GroupntreatmentDose, mg/kg8 hr18 hr24 hr48 hr % survival 110 DMA/D5W, pH 3.52 -222220 210Dapto10 100 310MBX 106610988880 410MBX 109010 99990 52MBX 1113102 mice died immediately after injection 5’8MBX 11131211112.5 61MBX 1128101 mouse died immediately after injection 6'9MBX 1128152000 Efficacies of MBX compounds in a murine IP/IV S. aureus infection model

34 November 2007 Survivors TreatmentRouten Dose, mg/kg 8 hours18 hours24 hours48 hours% survival Vehicle control (D/PO) i.p.6--10000 Daptomycini.p.6106666100 MBX 1162i.p.101 100 MBX 1162i.p.10 100 Vehicle control (DMA/D5W) IV6--111117 DaptomycinIV6106666100 MBX 1162IV101966660 MBX 1162IV10 100 In vivo testing in a S. aureus murine infection model with i.p. or IV treatment

35 November 2007 NCI IDDose (mg/kg/ injection) Sched.Route#Mice#Surviving on Day 5 100Q04DX003i.p.66 MBX 1090200Q04DX003i.p.66 400Q04DX003i.p.66 100Q04DX003i.p.66 MBX 1066200Q04DX003i.p.66 400Q04DX003i.p.66 25Q04DX003i.p.66 MBX 111350Q04DX003i.p.66 100Q04DX003i.p.65 200Q04DX003i.p.65 400Q04DX003i.p.62 50Q01DX005i.p.66 MBX 1128100Q01DX005i.p.66 200Q01DX005i.p.66 Toxicity Determination in Mice

36 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 ED 50 <10mg/kg Well tolerated, with murine MTD >400mg/kg Easy and inexpensive to synthesize Next step: IND enabling GLP toxicology/parmacology

37 November 2007 ACKNOWLEDGMENTS USAMRIID: Sina Bavari, Ph.D., Rekha Panchal, Ph.D. University of Calgary: Donald Woods, Ph.D. Defense Threat Reduction Agency (DTRA)

38 November 2007

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