Vectorisation of Gram-positive antibiotics into Gram-negative bacteria ECCMID Amsterdam, Netherlands 2019 S0331 Marvin J. Miller Department of Chemistry and Biochemistry University of Notre Dame Notre Dame, IN 46556, USA [email protected] eLibrary & Hsiri Therapeutics, LLC © by author The world is running out of antibiotics, WHO report confirms - 20 September 2017 IF YOU WEREN'T taking antibiotic resistance seriously before, now would be a good time to start. A project commissioned by the British government has released estimates of the near-future global toll of antibiotic resistance that are jaw-dropping in their seriousness and scale: 10 millions deaths per year, more than cancer, and at least $100 trillion in sacrificed gross national product. WHO Priority 1: CRITICAL AcinetobacterESCMIDbaumannii, carbapenem eLibrary-resistant Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae© by, carbapenem author-resistant • Changing membrane permeability via porin modification • Enzymatically deactivating the antibiotic (e. g. beta-lactamase) • Decreasing intracellular drug concentration by effluxESCMID pump systems eLibrary • Inadequate diagnostics Clatworthy, A. E. et. al.© Nature Chemical by biology 2007 author, 3, 541-548; Michael A. F., Walsh C. T. Science 2009, 325, 1089. • However, microbes still need to assimilate nutrients, especially IRON (Fe) • Bacteria need MICROMOLAR amounts of Fe to grow, multiply and sustain an infection • However, Fe is very insoluble & only 10-18 to ESCMID-24 eLibrary 10 molar free Fe is available in serum • How can bacteria assimilate enough Fe? Clatworthy, A. E. et. al.© Nature Chemical by biology 2007 author, 3, 541-548; Michael A. F., Walsh C. T. Science 2009, 325, 1089. Siderophores & Bacterial Fe(III) Acquisition Siderophore: multidentate Fe3+ chelator used by bacteria for extracellular solubilization of otherwise insoluble Fe3+ followed by protein-mediated active transport into cells. Siderophore-Mediated Iron(III) Transport Outer Membrane Receptors ESCMID eLibraryGram-negative Model 5 Sandy & Butler; Chem.© Rev. 2009by, 109, 4580. author Examples of Natural Siderophores Siderophores are multidentate Fe(III) chelators used by bacteria for extracelluar solubilization of otherwise insoluble Fe(III) followed by molecular recognition based protein-mediated active transport into cells. ESCMID eLibrary Hider, R. C.; Kong,© X. Chemistry by and Biology author of Siderophores. Nat. Prod. Rep. 2010, 27, 637-657. Siderophores for “Trojan Horse” Drug Delivery Sideromycins use the microbe’s iron acquisition system to smuggle a drug into the cell. (‘Trojan Horse’ approach) -Siderophore: Help to overcome the permeability problem by active transport. -Linker:ESCMID With or without drug release function. eLibrary -Drug: Activity may or may not be retained after attachment. Wencewicz, T. A.*; Miller, M. J. "Sideromycins as Pathogen-Targeted Antibiotics." in Topics in Medicinal Chemistry. 2017, Springer, Berlin, Heidelberg.© DOI: 10.1007/7355_2017_19by author “Controversy” – Can Sideromycins be Useful Antibiotics? Read the Old Literature – 1955 Effective Natural Sideromycins – in people! Albomycin, a new antibiotic, has been manufactured during O recent years by the pharmaceutical industry of the Soviet O O siderophore -linker- drug Union. It has been studied both in the laboratory and in N Fe Fe O N clinical practice, and it is the purpose of this paper to N O O HO summarize the results of the most important studies. OH O O H H N N N H N H HO Albomycin was obtained by Gause and Brazhnikova (1951) H O S H2N O from cultures of a new species of streptomycetes, O HN Y N CO2H Actinomycessuibtropicus. This antibiotic strongly inhibits H albomycins, OH Peptidase required the growth of Gram-positive cocci, chiefly pneumococcus natural siderophore for drug release or antibiotics no antibiotic activity and staphylococcus. More important, it inhibits the growth (V. Braun - Biometals 2008 of staphylococci resistant to other antibiotics, including G. Benz - series of papers 1980s) penicillin,ESCMID streptomycin, the tetracyclines, and erythromycin. eLibrary It is also effective against a number of Gram-negative bacteria. 1 mg of albomycin inhibits© the growthby of staphylococci author about ten times more strongly than an equal amount of penicillin. “Controversy” – Can Sideromycins be Useful Antibiotics? Read all the Relevant Literature Salmycins – natural amino glycoside sideromycins O O O siderophore -linker- drug N Fe Fe O N N O O Discovery & early report: Gause, G. F. British Medical HO OH O O H H Journal 1955, 1177- N N N In vivo efficacy: PEOPLE 1950s- H N H HO DiscoveryH : All of these compoundsO S are highly active H2N O Subsequent in vitro and in vivo studies: Braun, V. et al against StaphylococciO HN and Streptococci Y N CO2H Biometals. 2009, 22, 3-13. “The in vivo efficacy of at 0.01 µg/mL concentrations,H including resistant albomycins, OH Peptidase required albomycin … has been examined in a mouse (infection) nastrains.tural siderophore for drug release or antibiotics no antibiotic activity model. Albomycin is effective in clearing infections... Vertesy, L.; Aretz, W.; Fehlhaber(V., BraH.u-nW., - Bi oKoglermetals 2,0 08 The recovery rate of albomycin resistant mutants is Helv. Chim. Acta 1995, 78, 46-G60.. Benz - series of papers 1980s) lower thanESCMID that of the wild-type which suggests a eLibrary Synthetic studies: Roosenberg, J. M.; Dong, L.; reduced fitness of the mutants. Albomycin could be a Miller, M. J. J. Am. Chem. Soc. 2002, 124, 15001-05 useful antibiotic provided sufficient quantites can be In vivo efficacy: Braun, V. et al Biometals. 2009, 22, isolated ... or synthesized© chemically by” author3-13. Therapeutic Implications of Iron (Fe), Its Assimilation and Metabolism - the Creation of Designer Sideromycins Exploitation of microbial iron assimilation processes for development of new antimicrobial agents Key points: 1. Siderophore based molecular recognition 2. Linker considerations – releasable or not 3. Intracellular Drug target ESCMID eLibrary © by author Synthetic Sideromcyins - Imparting Microbe Selectivity by correct choice of conjugate components: Siderophore recognition Enterobactin as a xenosiderophore used by many Gram negative bacteria ESCMID eLibrary Zheng, T.; Bullock, J.© L.; Nolan, E.by M. J. Am. Chem. authorSoc., 2012, 134, 18388. Synthetic Sideromcyins - Imparting Microbe Selectivity by correct choice of conjugate components: Tripodal catechol ampicillin conjugate is selectively active against Pseudomonas ! COOH O O O N H Both conjugates 7 and 8 exhibited significantly N HN N S enhanced (>256-4000 fold) in vitro antibacterial H O activity against Gram-negative species compared to the parent drugs, especially against P. aeruginosa. R MIC in mM O NH O NH HN O 7 8 Ampicillin Amoxicillin OAc OAc AcO +Fe -Fe +Fe -Fe +Fe -Fe +Fe -Fe P. aeruginosa KW799/wt 33 0.05 25 0.05 >200 >200 >200 >200 OAc OAc AcO P. aeruginosa KW799/61 12.5 0.067 12.5 0.083 0.52 0.78 0.46 0.39 7 R=H ampicillin 8 R=OH amoxicillin P. aeruginosa PAO1 50 0.39 50 0.39 >200 >100 >200 >100 P. aeruginosa Pa4 25 0.39 25 0.21 >200 >100 >200 >100 P. aeruginosa Pa6 >200 >200 >200 >200 >200 >200 >200 >200 ESCMIDE. coli ATCC 25922 eLibrary150 1.56 100 6.25 16.7 12.5 4.17 4.17 K.pneumoniae ATCC >200 >100 >200 >100 >200 >100 100 >100 Ji, C. Miller, P. A.; Miller, M. J 8303 J. Am. Chem. Soc. 2012, ©134, 9898-9901 .by author Synthetic Sideromcyins - Results from Other Labs Verify the Iron Transport-Mediated Trojan Horse Concept ESCMID eLibrary Not all iron chelators are siderophores and one must consider proper iron binding© bystoichiometry author and molecular recognition Design, Syntheses & Studies of Sideromycins, Novel Antibiotics that Target Specific Infections – HT06 OH OH OH OH OH OH siderophore -linker- drug NHS/EDC Fe O O OH DMF/2-MeTHF OH HN HN O N O rt, 16 h N O Microbe Target Target OH O selectivity selectivity reactivity Bis-catechol core N (releasable 960 g O O or not) (PracticaChem) O NaHCO3,THF/H2O O O O H2N H2N NH NH H2N S NH S Ph Ph Ph N N Cl N O O O Cl CO2H CO2H Lorabid Ampicillin CO2H (carbacephem) Ceclor OH OH OH OH OH OH OH OH OH O O OH O OH HN OH HN N O HN N O O N O O H O H N H N NH N NH ESCMID eLibraryNH S O Ph S O Ph Sideromycin N Sideromycin O Ph Sideromycin N Cl YML-1-60 O Cl YML-1-62 N YML-1-109 O O CO H CO H HT-06 © byCO2H HTauthor-11 2 HT-07 2 ESCMID eLibrary © by author Design, Syntheses & Studies of Sideromycins, Novel Antibiotics that Target Specific Infections – HT06 HT-06 –IV Tolerability in ICR mice •Single IV dose tolerability from 25 mg/kg to 250 mg/kg •Two IV doses of 250 mg/kg – at zero time and 3.5 hours Results:ESCMID All doses up to 250 mg/kg eLibrary dosed once or twice were well tolerated with no observed adverse effect © by author Design, Syntheses & Studies of Sideromycins, Novel Antibiotics that Target Specific Infections – HT06 Effect of HT-06 on Mouse Survival (100% survival!) - IP Acinetobacter baumannii 17961 Sepsis Model 7 6 5 HT-06 250 mpk x 2 4 HT-06 50 mpk x 2 Ciprofloxacin 50 mpk x 2 3 Loracarbef 50 mpk x 2 Vehicle 2 Number of Mice of Number 1 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 ESCMID• 6 to 8 week old female ICR mice eLibrary • IP inoculation with 108 CFU Acinetobacter baumannii ATCC 17961 • IV Rx at 30 min and 4 hrs • Rx - HT-06 250 mg/kg, HT-06 50 mg/kg, Ciprofloxacin 50 mg/kg, Loracarbef© 50 mg/kg, by Vehicle author Design, Syntheses & Studies of Sideromycins, Novel Antibiotics that Target Specific Infections – HT06 HT-06 (with/without sulbactam) vs.