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MICROBIOTIX

MICROBIOTIX. A product-focused, small molecule, anti-infective drug discovery company. CONFIDENTIAL. The development of novel broad-spectrum anti-bacterials for intracellular BW threats. AGENDA. Terry Bowlin, Ph.D. – Introduction/Welcome John Williams, Ph.D. – Chemistry

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MICROBIOTIX

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  1. MICROBIOTIX A product-focused, small molecule, anti-infective drug discovery company CONFIDENTIAL

  2. The development of novel broad-spectrum anti-bacterials for intracellular BW threats

  3. AGENDA • Terry Bowlin, Ph.D. – Introduction/Welcome • John Williams, Ph.D. – Chemistry • Michelle Butler, Ph.D. – Microbiology/Cytotoxicity • Donald Moir, Ph.D. – Mechanism • Terry Bowlin, Ph.D. – Animal Studies • Jennifer Brooks – Development Plan • Terry Bowlin, Ph.D. – Goals/Milestones

  4. 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 safetyevaluation.

  5. CHEMISTRY John Williams, Ph.D.

  6. Synthesis of MBX 1066 5 Steps overall

  7. Synthesis of MBX 1090 7 Steps overall

  8. Synthesis of MBX 1113

  9. Synthesis of MBX 1113 8 Steps overall

  10. Synthesis of MBX 1128

  11. Synthesis of MBX 1128

  12. Synthesis of MBX 1128 13 Steps overall

  13. Analogs of MBX 1066

  14. MICROBIOLOGY/CYTOTOXICITY Michelle Butler, Ph.D.

  15. Aim 1/2 Microbiology Studies • Microbiology -- Original 4 compounds plus MBX 1066 analogs • MICs against standard Gram-pos. and Gram-neg. lab strains • MICs against category A or B bioterrorism pathogens • Cytotoxicity (CC50) of compound

  16. MBX BLS1/2 MIC Data

  17. MBX BLS1/2 MIC Data (continued)

  18. U. Calgary and USAMRIID BSL3 Data for MBX Compounds *Value determined only once.

  19. BLS1/2 MIC Data for MBX 1066 Analogs

  20. BLS1/2 MIC Data for MBX 1066 Analogs (continued)

  21. U. Calgary and USAMRIID BSL3 Data for MBX 1066 Analogs

  22. CC50 of MBX 1066 and its analogs on HeLa cells

  23. MICROBIOLOGY SUMMARY • Accomplishments: • All four of the original lead compounds have been remade and retested in an independent laboratory with similar antibacterial potencies, especially with relevant BSL3 strains • MBX 1066 displays the most favorable in vitro selectivity index with low mammalian cell cytotoxicity • 14 analogs of MBX 1066 have been tested to date and several maintain activity against the Gram-positive strains while displaying greater potency against Gram-negative strains • Future work: • We will continue to acquire and test other relevant bacterial strains against the current compounds and new series as they are synthesized

  24. MECHANISMDonald Moir, Ph.D.

  25. 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

  26. Antibacterial Mechanism of bis-(imidazolinylindole) compounds • Favorable in vitro therapeutic index (CC50/MIC) indicates selectivity for bacteria • Rapid bactericidality implicates DNA, RNA, cell wall or membrane targets • DNA synthesis is the most sensitive of the macromolecular pathways to MBX 1066 (effects observed at >10x MIC) • The bis-(imidazolinylindole) compoundsinteract with DNA • Fluorescence enhancement in the presence of DNA (Max1/2~0.4 μM) • Inhibition of ReplixTM (IC50 ~2 μM) & replicativehelicase (IC50~1 μM) • ~2x preference for AT-rich B. anthracisDNA vs. calf thymus DNA • Target appears to be cytoplasmic • Fluorescence enhancement of compound observed within bacterial cells • MIC is significantly lower in efflux mutant of P. aeruginosa • Very low frequency of mutation to resistance • Minimal effects on cell membranes • No lysis of membranes • No perturbation of the membrane potential near the MIC for some compounds • Working Hypothesis: The bis-(imidazolinylindole) compounds enter bacterial cells, bind preferentially to AT-rich DNA, and inhibit one or more DNA replication functions

  27. Rapid Bactericidal Activity of MBX 1066, 1090, 1142 and 1162

  28. 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

  29. Fluorescence Enhancement of MBX 1066 in the Presence of DNA – Concentration Dependence Conclusion: Half-maximal DNA interaction by MBX 1066 occurs at about 0.4 μM (~0.3 μg/ml)

  30. MBX 1066, 1090 and 1113 are Potent Inhibitors of Replix™, a Permeable Cell DNA Replication Assay

  31. Helicase Inhibition by MBX 1066 & 1090 as Measured by 32P-Based Unwinding Assay – Comparison to Other Helicase Inhibitors Conclusion: MBX 1066 & 1090 are very potent B. anthracis helicase inhibitors with IC50’s of <1 μM (<0.6 μg/ml)

  32. DNA Interaction with MBX 1066 & Hoechst 33258 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracisGenomic DNA Average A+T content: 64% for B. anthracis DNA vs. 58% for calf thymus DNAc Conclusion: Affinity of both MBX 1066 and Hoechst 33258 for AT-rich B. anthracisDNA is ~2-fold stronger than for calf thymus DNA

  33. 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 DIC DAPI 4 X MBX 1113 Intracellular fluorescence readily detected at 1X MIC Consistent with DNA-dependent fluorescence enhancement DIC 1 X MBX 1066 cytoplasmic localization DAPI Contrast enhanced 10X zoom

  34. MBX MIC Data for MBX 1066 & Analogs IsogenicP. aeruginosaStrains +/- 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

  35. Mutation to Resistance to MBX 1066 is Rare in S. aureus NCTC-8325 Serial Passage S. aureus NCTC 8325 E D B F G H A C MBX 1066 MBX 1113 MBX 1090 0.125 0.125 0.125 0.25 0.25 0.25 0.5 0.5 0.5 1 1 1 2 2 2 4 4 4 8 8 8 Highest Sublethal Concentration (Fold MIC) 16 16 16 32 32 32 64 64 64 128 128 128 1 5 10 15 20 1 5 10 15 20 1 5 10 15 20 Time (days) Time (days) Time (days) Resistant mutants-16X MIC

  36. 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

  37. Bacterial membrane perturbation assay using DiSC3(5) Ex-622 Em-670 2H+ DiSC3(5) No membrane potential perturbation by compound e-transport QUENCH Membrane potential perturbation by compound 2H+ Ex-622 Em-670 Membrane potential perturbation Membrane disrupter

  38. Summary of Membrane effects of bis-(imidazolinylindole) Compounds in DiSC3(5) assay Conclusion: MBX 1066 & 1128 do not perturb membrane potential at concentrations near the MIC

  39. 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

  40. MBX 1066 & 1090 do 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

  41. Favorable Features of MBX 1066 Antibacterial Mechanism • In vitro therapeutic index (CC50/MIC >170) is favorable for MBX 1066 • MBX 1066 is rapidly bactericidal • DNA synthesis is the most sensitive macromolecular pathway to MBX 1066 (effects observed at >10x MIC) • Interacts with DNA • MBX 1066 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 • Very low frequency of mutation to resistance • Minimal effects on cell membranes • MBX 1066 does not lyse membranes or perturb the membrane potential at <4x MIC • Conclusion: MBX 1066 is less cytotoxic, exhibits fewer membrane effects, and is less susceptible to mutation to resistance than are MBX 1090, 1113, or 1128

  42. 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

  43. ANIMAL STUDIESTerry Bowlin, Ph.D.

  44. In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis Note: MBX 1066 protected 5/5 mice for 14 days in a previous Ames challenge experiment

  45. In Vivo Testing Against F. Tularensis

  46. In Vivo Testing in Y. Pestis Murine Model

  47. Efficacy of MBX 1162 in a murine IP/IP B. pseudomalleiinfection model Three groups of 5 Balb/C mice (female, 20-22g) were inoculated intraperitoneally with 106 cells of Burkholderiapseudomalleistrain 1026b. Mice were treated intraperitoneally ten minutes post infection with tetracycline (10 mg/kg), MBX 1162, or vehicle alone

  48. Efficacies of MBX compounds in a murine IP/IP S. aureus infection model

  49. Efficacies of MBX compounds in a murine IP/IV S. aureus infection model

  50. Toxicity Determination in Mice

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