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MICROBIOTIX. A product-focused, small molecule, anti-infective drug discovery company. CONFIDENTIAL. The development of novel broad-spectrum anti-bacterials for intracellular BW threats. AIMS.
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MICROBIOTIX A product-focused, small molecule, anti-infective drug discovery company CONFIDENTIAL
The development of novel broad-spectrum anti-bacterials for intracellular BW threats
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.
Aim 1 Demonstrate potent, selective inhibitory activity of one or more bis-(imidazolinylindole) compounds in animal models of infection (year 1).
MBX Compounds Have Potent in vitro Activities Against Category A & B Biowarfare Agents
MIC90 Values for 20 Strains each of B. pseudomallei and B. mallei
Demonstrate in vivo Potency in Various Murine Efficacy Models
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
Aim 3 Demonstrate structure-activity relationships for the potency and selectivity of the bis-(imidazolinylindole) class of compounds (year 2).
Task 4.3.2 Synthesis of bis-(imidazolinylindole) analogs 4.3.2.1 Synthesis of NSC 317,880 (MBX 1090) and analogs
Evaluation of bis-(imidazolinylindole) analogs Potency measurement of MBC/MIC values on intact cells
MBX compounds act with a bactericidal mechanism of action with rapid killing kinetics observed
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
MBX 1066 and 1162 Maintain Excellent Potencies Against Multiple Isolates of Gram-positive and Gram-negative Species
Gram-Negative (Enterobacteriaceae) Laboratory/Clinical Strains
Gram-Negative (Atypical) and Gram-positive (Anaerobe) Laboratory/Clinical Strains
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
Aim 2 Establish the mechanism of action of the bis-(imidazolinylindole) class of compounds (year 1).
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
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)
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
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)
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)
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….
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
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
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