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

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

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

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

4. 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

5. MBX Gram Positive MIC Data (BSL 2)

6. MBX Gram Negative MIC Data (BSL 2)

7. USAMRIID and U. Calgary MIC Data (BSL 3) *Value determined only once.

8. MBX Gram Positive MIC Data for selected MBX 1066 Analogs

9. MBX Gram Negative MIC Data for selected MBX 1066 Analogs

10. USAMRIID and U. Calgary MIC Data for Selected MBX 1066 Analogs (BSL 3)

11. Rapid Bactericidal Activity of MBX 1066, 1090, 1142 and 1162 A B 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). Summary of time kill results

12. Comparison of MBC and MIC values for infectious B. anthracis strains

13. Compound Cytotoxicity on HeLa cells

14. 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. Mechanism Studies • MMS • DNA Binding • Replix • Helicase • In Situ • Efflux • Resistance • Membrane Effects

16. 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. 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)

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

19. 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)

20. 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

21. 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

22. 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

23. 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. 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

25. 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

26. 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. 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

28. 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. ANIMAL STUDIES • B. Anthracis • Y.Pestis • B.Pseudomallei • S. Aureus

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

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

32. 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

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

34. In vivo testing in a S. aureus murine infection model with i.p. or IV treatment

35. Toxicity Determination in Mice

36. 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

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