MICROBIOTIX

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

2. DTRA01-06-BAA-01 Project Title The development of novel broad-spectrum anti-bacterials for intracellular BW threats

3. Introduction – Goals of Program • DTRA Mission: Protect the warfighter from conventional or genetically engineered biological threats • Program Mission: Discover and develop broad spectrum anti-bacterials for military use against category A&B biowarfare pathogens • Microbiotix Contract Objective : “Develop a new class of therapeutic agents, the bis-(imidazolinylindole) series discovered in preliminary studies, for use against intracellular bacterial warfare threats”

4. Introduction – Project Strategy • Microbiotix originally structured this extremely rapid anti-bacterial development program to provide the greatest chance of success within the two year time-frame. • The program was initially designed using the best case scenario with no complicating issues anticipated, based upon the data available at contract initiation.

5. 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.

6. Synthesis of bis-(imidazolinylindole) compounds

7. Synthesis of Lead Compounds for Anti-Infective Studies

8. Representative Synthesis: MBX 1066

9. SAR Example: MBX 1066/1162 Tether Modifications

10. Lead and Backup Compounds and Salt Forms

11. IND-Enabling Studies: Scale-up of Lead Compound Precursors

12. IND-Enabling Studies: Scale-up of Lead Compounds

13. IND-Enabling Studies: Radiolabeled Drug

14. Chemistry Summary • Established synthetic route for original 4 compounds • Small scale synthesis of ~100 analogs in SAR program to improve potency, solubility, and minimize cytotoxicity • MBX 1066 analog MBX 1162 identified as a potential backup • Scaled-up synthesis of three compounds • Performed salt selection studies • Synthesized radiolabeled MBX 1162 free base (MBX 1143)

15. In vitro activity (MICs) against category A and B pathogens

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

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

18. MIC90 values Against Multiple Isolates of Gram-positive and Gram-negative Species

19. Gram-Positive (Staphylococci) Laboratory/Clinical Strains

20. Gram-Positive (Enterococci) Laboratory/Clinical Strains

21. Gram-Positive (Streptococci) Laboratory/Clinical Strains

22. Gram-Negative (Nonfermentors) Laboratory/Clinical Strains

23. Gram-Negative (Enterobacteriaceae) Laboratory/Clinical Strains

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

25. MBX compounds are rapidly bactericidal

26. Mammalian cytotoxicity values consistent with favorable selectivity indices Method: Human HeLa cells were exposed for 72 hours to serial dilutions of compounds, then assessed for cell viability using an MTT assay

27. Microbiology Summary • Potent in vitro activity against category A or B bioterrorism pathogens • Potent in vitro activity against a broad-spectrum of Gram-pos. and Gram-neg. strains, including clinical isolates and multiple-drug resistant strains • Potency vs. >10 isolates/species (MIC90 values) • Rapidly bactericidal mechanism of action • Low 3-day cytotoxicity (CC50) of compounds

28. In vivo Potency in Murine Infection Models

29. MBX compounds are efficacious in a murine S. aureus infection model (i.p./i.v.) 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

30. MBX compounds are efficacious in a Yersinia pestis infection model when administered single-dose i.p. Infection:Y. pestis (100 cfu, CO92 strain) given i.p Treatment: Compound (1.5 mg/kg /injection in 1.5 % DMSO in water) given i.p. qid starting at 6 hours post-infection and ending 5 days post-infection

31. MBX compounds are efficacious in a Yersinia pestis infection model when administered i.p. or i.m. Infection:Y. pestis (100 cfu, CO92 strain) given i.p Treatment: 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

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

33. MBX 1090 and 1162 are somewhat efficacious in a Burkholderia mallei murine infectionmodel (intranasal/i.v.) 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 *MBX 1066 was not tested due to the deaths observed in the B. pseudomallei model.

34. MBX compounds were not effective in a multiple-dose F. tularensis infectionmodel (i.p./i.p.) Infection:F. tularensis (Schu4 strain) given i.p Treatment: 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

35. Demonstrated efficacy with MBX-1066 in a single-dose Bacillus anthracis infectionmodel (i.p./i.p.) Infection: B. anthracis Ames spores (500 cfu) given IP Treatment: MBX 1066 (10 mg/kg/inj. dissolved in aq. 4% DMSO) given IP qid starting at the indicated post-infection time and ending after 5 days

36. Efficacy in a single-dose Bacillus anthracis infectionmodel (i.p./i.v.) 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

37. Efficacy in a multiple-dose Bacillus anthracis murine infectionmodel (i.p./i.v.) 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

38. Summary of MBX-1066 & 1162 Efficacy in Animal Models of Infection • Both are active against S. aureus in 1-10 mg/kg range, single-dose i.p. or i.v., with no toxicity observed • Both prolonged survival inY. pestis infection model at 6-8 mg/kg/day x5d, multiple-dose i.p. and i.m. • MBX-1162 is active against B. pseudomallei at 10 mg/kg, single-dose i.p. • Slight prolongation of survival in B. mallei intranasal infection model at 10 mg/kg, single-dose i.v. • Not active against F. tularensis under conditions examined • Both are active against B. anthracis in 10-40 mg/kg range i.v. or i.p.

39. Mechanism of action studies of the bis-(imidazolinylindole) class of compounds

40. Macromolecular Synthesis Assays S. aureus Controls MBX-1066 • None of the MMS pathways affected at killing dose (5x MIC) • Target not identified by MMS studies • DNA synthesis is inhibited at >10X MIC (secondary effect)

41. Membrane perturbation assays Bacterial membrane perturbation DiOC(2)/FACS Mammalian membrane lysis LDH release assay MBX-1066 does not perturb bacterial or mammalian cellular membranes at bactericidal concentrations

42. Map loci responsible for bis-(imidazolinylindole) resistance Serial passage of S. aureus NCTC-8325 in subinhibitory compound concentrations to select resistance mutants A B C D E F G H MBX-1066 MBX-1090 MBX-1162 Passage Number Passage Number Passage Number MBX-1066 resistance is not detectable Resistant mutants-16X MIC MBX-1162 resistance is not detectable

43. MBX-1090 resistance maps to mepR, regulator of a drug-sodium antiporter No cross resistance vs. other bis-(imidazolinylindole) compounds • MBX-1090 is a MepA substrate • Other bis-(imidazolinylindole) compounds are NOT MepA substrates Model confirmed by extensive genetic and transcription profiling analyses

44. Compound MBX-1162 is not susceptible to bacterial efflux

45. Analysis of DNA binding activity of bis-(imidazolinylindole) compounds DNA Interaction with MBX-1066 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracis Genomic DNA Fluorescence Enhancement of MBX-1066 in the Presence of DNA – Concentration Dependence Half-maximal DNA interaction by MBX-1066 occurs at about 0.4 μM (~0.3 μg/ml) Affinity of MBX 1066 for AT-rich B. anthracis DNA is ~2-fold stronger than for calf thymus DNA

46. 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 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

47. DNA sequence preference for MBX-1162 binding and affinity constant Fluorescent displacement assay MBX-1162 Relative affinity for AATT Scatchard plot (Kapp) 136 possible sequences A A 5’-CGXXXXC 3’-GCXXXXG A A A Slope = Kapp Dr. Eric Long (IUPUI) Preference for A/T rich sequences Highest affinity for AATT

48. Absence of correlation between DNA binding and biological activity cytotoxicity antibacterial MIC and cytotoxicity correlate poorly with DNA binding

49. Profiling the changes in gene expression in response to MBX-1066 and -1090 for MOA (in progress) Samples prepared for profiling • Grow S. aureus NCTC 8325 in presence of MBX-1066, MBX-1090 and a compendium of antibiotics that affect RNA/DNA synthesis 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.

50. Genes affected by MBX-1066 and -1090 MBX-1090 selected genes Numbers of genes Up- and Down-regulated (4X, 90% confidence) MBX-1066 691 MBX-1090 131 74 Up=44 Down=87 conserved hypothetical protein = 65 Up=333 Down=358 conserved hypothetical protein = 325