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Development of Novel Bis-(Imidazolinylindole) Compounds for Broad-Spectrum Anti-Bacterial Efficacy

MICBIOTIX.A focuses on the discovery of small molecule anti-infectives targeting intracellular biological warfare threats. This project aims to establish potent, selective bis-(imidazolinylindole) inhibitors through animal models and elucidate their mechanisms of action. Key milestones include demonstrating in vivo efficacy against category A and B pathogens with safety studies leading to IND application submission for clinical trials. Successful candidates, MBX 1066 and MBX 1162, exhibit potent activity against severe bacterial threats, paving the way for innovative therapeutics.

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Development of Novel Bis-(Imidazolinylindole) Compounds for Broad-Spectrum Anti-Bacterial Efficacy

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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. 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.

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

  5. Lead and Back-up Compounds

  6. MBX 1066 Analogs

  7. Current Work

  8. Future Possibilities

  9. Exploration of Scale-up Conditions

  10. Large Scale and Radio Syntheses

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

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

  13. Demonstrate in vivo Potency in Various Murine Efficacy Models

  14. No efficacy was demonstrated in a F. tularensis infectionmodel 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

  15. MBX 1066 and 1142 were efficacious in a Yersinia pestis infection model Infection:Y. pestis (100 cfu, CO92 strain) given i.p Treatment: 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.

  16. 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.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 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.

  17. MBX 1162 demonstrated efficacy in a Burkholderia pseudomallei infection model 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.

  18. 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.

  19. MBX 1090 and 1162 demonstrated efficacy in a Burkholderia mallei infectionmodel 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.

  20. Demonstrated efficacy in a Bacillus anthracis infectionmodel 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.

  21. Demonstrated efficacy in a Bacillus anthracis infectionmodel 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.

  22. Demonstrated efficacies of MBX compounds in a murine S. aureusinfection 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.

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

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

  25. Task 4.3.1 Perform molecular modeling studies

  26. Task 4.3.2 Synthesis of bis-(imidazolinylindole) analogs 4.3.2.1 Synthesis of NSC 317,880 (MBX 1090) and analogs

  27. 4.3.2.2 Synthesis of NSC 317,881 (MBX 1066) and analogs

  28. 4.3.2.3 Synthesis of NSC 330,687 (MBX 1113) and analogs

  29. Evaluation of bis-(imidazolinylindole) analogs Potency measurement of MBC/MIC values on intact cells

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

  31. Mammalian cytotoxicity values provide good 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 Conclusion: MBX 1066 displayed the highest selectivity index, with 4 other compounds displaying indices >50

  32. MBX 1066 and 1162 Maintain Excellent Potencies Against Multiple Isolates of Gram-positive and Gram-negative Species

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

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

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

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

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

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

  39. The Effect of Efflux on Activity of Compounds

  40. Aims 1 and 3 Research Summary • Chemistry– • Original 4 compounds plus MBX 1066 analogs and salts • Small scale of > XX analogs in SAR program • Scale-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 action • Potent in vivo efficacy against several murine infection models • Low 3-day cytotoxicity (CC50) of compounds

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

  42. Aim 2 – Mechanism of Action NSC 317880, NSC 317881, NSC 330687, NSC 369718 & New analogs Completed 1. Macromolecular Synthesis Assays & cidal/static determination (DNA, RNA, protein, cell wall, & lipid biosynthesis) Inhibits >1 pathway DNA+RNA @ ≥10X MIC Inhibits 0 or 1 pathway Not Done Completed No 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 NO Completed Completed 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 6. Experiments not in original plan -- DNA binding studies (Eric Long, IUPUI) -- Phenotypic Macroarray (Biolog) Not Done (no discreet targets ID) 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 Membrane is target

  43. Macromolecular Synthesis Assays S. aureus Controls MBX-1066 • None of the MMS pathways affected at killing dose (5x MIC) • Unknown target • DNA synthesis is inhibited at >10X MIC (secondary effect)

  44. 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 therapeutically relevant concentrations

  45. Map loci responsible for bis-(imidazolinylindole) resistance Serial passage of S. aureus NCTC-8325 in subinhibitory compound concentrations to select resistance mutants E D B F G H A C MBX 1066 MBX 1113 MBX 1090 S. aureus NCTC 8325 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 16 16 16 Highest Sublethal Concentration (Fold MIC) 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 MBX-1113 resistance is rare MBX-1066 resistance is rare MBX-1162 resistance is rare (data not shown)

  46. Map loci responsible for bis-(imidazolinylindole) resistance 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 (see poster)

  47. 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….

  48. Analysis of DNA binding activity of bis-(imidazolinylindole) compounds Why?—structural similarity with DNA minor groove binders 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 both MBX 1066 for AT-rich B. anthracis DNA is ~2-fold stronger than for calf thymus DNA

  49. 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

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

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