Vectorisation of Gram-Positive Antibiotics Into Gram-Negative Bacteria

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

O O O siderophore -linker- drug N Fe Fe O N N O O HO OH O O H H N N N H N H HO H O S H2N O O HN Y N CO H H 2 albomycins, OH Peptidase required Salmycins – natural natural siderophore for drug release or amino glycoside sideromycins antibiotics no antibiotic activity (V. Braun - Biometals 2008 G. Benz - series of papers 1980s)

Discovery & early report: Gause, G. F. British Medical Journal 1955, 1177- In vivo efficacy: PEOPLE 1950s- Discovery: All of these compounds are highly active Subsequent in vitro and in vivo studies: Braun, V. et al against Staphylococci and Streptococci Biometals. 2009, 22, 3-13. “The in vivo efficacy of at 0.01 µg/mL concentrations, including resistant albomycin … has been examined in a mouse (infection) strains. model. Albomycin is effective in clearing infections... Vertesy, L.; Aretz, W.; Fehlhaber, H.-W., Kogler, The recovery rate of albomycin resistant mutants is Helv. Chim. Acta 1995, 78, 46-60. 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

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. Drug-Resistant A. baumannii MICs in µM vs beta-lactamase producers

ATCC BAA BAA BAA BAA ISR ISR ARC ARC ARC ARC 17961 1710 1793 1797 1800 14- 14-006 3484 3486 5079 5081 005

HT-06 <0.02 >25 >25 >25 >50 >50 >50 >50 >50 >50 >50 5 HT-06 + <0.01 .025 0.01 0.2 0.1 >50 1.5 <0.1 12.5 6.25 Sulbactam 2 2 (1.2 µg/ml) Loracarbef + >100 >100 >100 >100 Sulbactam (1.2 µg/ml) SulbactamESCMID 0.6 5 5 10 5eLibrary20 >20 6 20 10 10 (µg/ml)Can we circumvent beta-lactamase problems without adding a beta-lactamase inhibitor? © by author Synthetic Sideromcyins – circumventing Beta-lactamases A new monobactam-siderophore conjugate against resistant Ab Organism Aztreonam 42 (+ tazo) (+ tazo) OH

O S. aureus SG 511 >50 >50 OH P. aeruginosa 01 3 0.4 OH P. aeruginosa KW799/wt 1.6 0.4 O O P. aeruginosa ARC 3502 >50 >50 N N a H (50) (1.6 ) N S NH E. coli DC0 0.1 <0.025 N O O OH Proteus mirabilis X235 0.05 <0.025 S O N OH Salmonella typhimurium 0.1 0.025 H2N O SO3H N ATCC13311 H Enterobacter aerogenes 0.2 0.075b 42 X816 Citrobacter freuendii 0.1 0.05 ATCC 29063 Potent, broad spectrum Gram-negative antibiotic A. baumannii ATCC 17961 25 0.2 A. baumannii ATCC BAA 25 0.4 Active against carbapenemase and 1793 (12.5) (0.1) cephalosporinase producing A. baumannii A. baumannii ATCC BAA 25 0.4 1797 WHO highest concern A. baumannii ATCC BAA 25 0.4 1800 (12.5) (0.1) A. baumannii ATCC 17978- 12.5 0.4 PNT-165c Good, but we also need narrow spectrum A. baumannii ATCC 17978- 50 0.4 PNT-320d antibiotics a plus some growth at 50, clear between 1.6-50 b plus some slight growth in a well between c ESCMID eLibrary carbapenemase producing organism d cephalosporinase producing organism Carosso, S.; Liu,© R.; Miller, by P. A.; Hecker author, S. J.; Glinka, T.; Miller, M. J. J. Med. Chem. 2017 Synthetic Sideromcyins - Imparting Microbe Selectivity by utilizing Outer Membrane Receptor Recognition: mixed ligand gives select activity against Acinetobacter!

ESCMID eLibrary © by author Synthetic Sideromcyins - Demonstrating Microbe Selectivity and that Active Transport Works: E. coli DC0, K. pneumoniae ATCC8303 X68 & E. aerogenes X816

E. coli DC0 K. pneumoniae

E. aerogenes Molecular recognition is key:

bis-catechol conjugates are broadly recognized ESCMID eLibraryby Gram-negative bacteria but mixed ligand conjugate is selective for Acinetobacter © by authorbaumannii Designing a TB-selective Sideromycin: IRON Assimilation by Mycobacteria uses Extracellular and Membrane Bound Siderophores

Extracellular Cell Envelope Intracellular Medium Medium

Carboxymycobactin Salicylate Reductase Fe(III) • Mycobacteria use 3 types of Carboxymycobactin Fe(II) Salicylate 3+ Fe(III) siderophores to acquire Fe from Source Porin-mediated diffusion host or environment Reductase (Fe(II))n Mycobactin Protein Receptor Bacterioferritin IREP • Iron supplies are closely regulated Fe(III) Fe(II) Salicylate Exochelin Reductase Exochelin • Iron transport is a possible target ESCMIDSalicylate eLibrary Fe(III) for anti-TB drugs Source Vergne, A. F.; Walz, A. J.;© Miller, M. J. Nat.by Prod. Rep. 2000 ,author 17, 99-116. Ratledge, C. Tuberculosis 2004, 84, 110-130. IC50 (mg/mL) vs P. faciparum strains compd HB3 7G8 3D7 Dd2

Conj 0.0047 0.0051 0.0040 0.0041 Art 0.0013 0.0004 0.0036 0.0022 MIC(mg/mL) vs. M. smegmatis >5 No activity vs. other Gram (+) or Gram (-) strains of bacteria! SELECTIVE

MIC(mg/mL) vs. MDR Mtb MIC(mg/mL) vs. XDR Mtb HRESP HREZSP HCPTTh HREKP HRERb HREZSKPTh HREZRbTh HRESPO HREPKO HRESPO HRESPO CTh Th CTh 1.25 0.625 0.625 0.625 0.3125 0.625 0.3125 ESCMID eLibrary0.31 0.078 0.625 0.31 H, isoniazid: R, rifampicin; E, ethamubtol; Z, pyrazinamide; S, streptomycin; C, cycloserine; T, ethionamide; K, kanamycin; P, p-aminosalicylic acid; Rb rifabutin; Th, thioacetazone; O, oflaxacin Miller, M. J.; Walz, A. J.: Zhu, H.; Wu, C.: Moraski,G.; Möllmann, U.; Tristani, E. M.; Crumbliss, A.; Ferdig, M.; Checkley, L.:© Edwards, R. L.;by Boshoff, H. I. J. Am.author Chem. Soc. 2011, 133, 2076-2079. Examples of Natural Sideromycins

Sideromycins are siderophores covalently linked to an antibiotic that enter bacterial cells via siderophore uptake pathways and deliver the toxic antibiotic in a ‘Trojan Horse’ fashion.

ESCMID eLibrary Wencewicz, T. A.;© Long, T. E.;by Möllmann, U.;author Miller, M. J. Biometals 2009, 22, 633-648. Synthetic Sideromcyins – Non- beta-lactam conjugates: HT-10 a Mixed Ligand – Daptomycin Conjugate Designed to Target Gram Negative A. baumannii HO

Daptomycin is Gram-positive active. HO O HO First demonstration of activity OH O NH N OH

against Gram-negative bacteria O O O NH HN N by Daptomycin N H O O O H H N N N N H H O CONH O NH O O 2 O HN H HO2C 0.5mM 0.4mM N O N N HO2C H H NH O O O HO NH CO H O 2 H O N N N O H H HT-10 O MW = 2334 O NH HO O 0.3 0..2 0.1 0.05mM 2

0.025 0.0125 0.00625mM Daptomycin control

ESCMIDH2O ciprofloxacin eLibrary Ghosh, M.; Miller, P. A.; Möllmann, U.; Claypool, W. D.; Schroeder, V. A.; Wolter, W. R.; Suckhow, M.; Yu, H.; Li, S.; Huang, W.; Zajicek, J.; Miller, M. J. ©A. baumannii by 17961 authorJ. Med. Chem. 2017, 60, 4577-4583 Synthetic Sideromcyins – Non- beta-lactam conjugates: HT-10 a Mixed Ligand – Daptomycin Conjugate Designed to Target Gram Negative A. baumannii First demonstration of in vitro & HO HO O in vivo activity against HO OH O NH N Gram-negative bacteria OH O O O NH by Daptomycin HN N N H O O O H Molecular recognition based H N N N N H H O CONH O NH O selectivity for O 2 O HN H HO2C N O N N HO2C Acinetobacter baumannii H H NH O O O HO NH CO H O 2 H O N as designed N N O H H HT-10 O (no drug release necessary) MW = 2334 O

NH2 HO O Strain Selectivity (MICs in µM): Note: retains Gram + activity

A. baumannii A. baumannii A. baumanniiI ISR P. aeruginosa Burkholderia S. aureus ISR S. aureus E. coli ATCC 17961 ARC 3486 14-006 PA01 multivorans AU0100 14-001 ISR-002 DCO HT-10 ESCMID<0.0125 0.05 0.4 >100eLibrary>100 6 6 >100 Daptomycin >100 >100 >100 >100 >100 0.8 0.8 >100 Cipro 0.5 >13 >13 0.5 >13 0.5 >13 0.5 J. Med. Chem. 2017©, 60, 4577 -4583by author Synthetic Sideromcyins – Non- beta-lactam conjugates: Siderophore Teicoplanin Conjugates Designed to Target Gram Negative A. baumannii

Compound S. P. E. A. A. A. A. aureus aeruginosa coli baumannii baumannii baumannii baumannii SG 511 01 DC0 ATCC ATCC ATCC ATCC BAA 1793 BAA 1797 BAA 1800 17978- ESCMID eLibrary PNT-165 Bis Catechol 0.8 µM >50 >50 0.8-1.6 3 0.8-3 0.8 Mixed 1.6 >50 >50 0.8-1.6 0.8 0.8-1.6 0.4 Ghosh, M.; Miller, P. A.; Miller, M. J. Ligand unpublished © byTeicoplanin <0.025author>50 >50 >50 >50 >50 25 Synthetic Sideromycin LINKER Considerations

-Siderophore: Helps to overcome the permeability problem by active transport. Molecular recognition imparts first level of microbe selectivity -Linker: With or without drug release function. Many antibiotics that act on cell walls tolerate peripheral substitution and are compatible with non-releasable linkers. Most antibiotics with cytoplasmic targets do not tolerate large substituents like directly attached siderophores. Thus, conjugates with cytoplasmic drug targets may require RELEASABLE linkers. CHOICE OF LINKER?? Exploit bacterial weakness. -Drug: Activity may or may not be retained after attachment. Must be able toESCMID reach and be recognized by its target .eLibrary

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 Sideromcyins – Chemically Designed Conjugates with Enzyme-Triggered Drug Release Inducing Bacterial Suicide: A Siderophore-Oxazolidinone Conjugate with a b-Lactamase Triggered Releasable Linker Allows Delivery of the Usually Gram-positive only Oxazolidinone to and Activity Against Gram-Negative Bacteria

ESCMID eLibrary

Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. J. Med Chem. 2018. Highlighted in C&E News©, April 9, 2018.by author Sideromcyins – Synthesis of a Conjugate with a b–Lactamase Triggered Drug Release: Siderophore-Cephalosporin-Oxazolidinone

Siderophore-Linker-Drug (1):

ESCMID eLibrary Cephalosporinase (ADC-1)-induced hydrolytic release of oxazolidinone 3 from conjugate 1. LC/MS Base Peak Chromatograms showing pure conjugate (A), product© formed followingby reaction author with ADC-1 (B) and ADC-1 only (C). Sideromcyins – Synthesis of a Conjugate with a b–Lactamase Triggered Drug Release: Siderophore-Cephalosporin-Oxazolidinone

Siderophore-Linker-Drug:

strain 3 22 23 27 26 1 siderophore cephalosporin siderophore siderophore + oxazolidinone + cephalosporin + + cephalosporin + oxazolidinone oxazolidinone cephalosporin oxazolidinone

A. baumannii >50 >50 >50 >50 0.8 0.8 µM ATCC 17961

A. baumannii >50 >50 >50 >50 12.5 0.8-1.6 ATCC BAA 1793

A. baumannii >50 >50 >50 >50 >50 6.25 ATCC BAA 1797

A. baumannii >50 >50 >50 >50 25 0.8 ATCC BAAESCMID 1800 eLibrary E. coli DC0 >50 >50 50 >50 <0.025 <0.025

P. aeruginosa >50 >50 >50 >50 0.4 0.2-0.4 KW799/wt © by author Sideromcyins – Synthesis of a Conjugate with a b–Lactamase Triggered Drug Release: Siderophore-Cephalosporin-Oxazolidinone

Siderophore-Linker-Drug:

3 17 27 22 23 26 1 siderophore siderophore cephalosporin siderophore + oxazolidinone eperezolid + + cephalosporin + cephalosporin oxazolidinone oxazolidinone cephalosporin + oxazolidinone A. baumannii >500 >50 >50 >50 >50 0.4 0.4 ATCC 17978

A. baumannii ATCC 17978 >500 >50 >50 >50 >50 0.4 0.4 pNT255

A. baumannii ATCC 17978ESCMID eLibrary >500 >50 >50 >50 >50 >50 6 pNT225: ADC-1 Activity (in µM) of compounds© againstby A. baumannii authorATCC 17978 without and with plasmid-encoded β-lactamase (ADC-1). Sideromcyins – Synthesis of a Conjugate with a b–Lactamase Triggered Drug Release: Siderophore-Cephalosporin-Oxazolidinone

Siderophore-Linker-Drug

Fe3+

Active transport Outer Membrane

Inner Membrane ESCMID eLibrary

Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. J. Med Chem. 2018. Highlighted in C&E News©, April 9,by 2018. author Therapeutic Implications of Iron (Fe), Its Assimilation and Metabolism - the Creation of Designer Sideromycins •Exploit microbial iron assimilation processes for development of new antimicrobial agents:

Antibiotics of the past and future??

Phillip Gregory Mattingly Charles K. Zercher Mark G. Stocksdale Katherine Waring Melinda Jean King-Morris Matthew A. Williams Mark J. Mulvihill Brian Bodnar Janet Marie Pachta Francine Farouz Yong Lu Joshua Bolger Peter J. Maurer Julia Ann McKee Yun-Ming Lin Ossama Darwish Robert Thomas Ronau Catherine M. Gasparski Raynauld L. Bishop Leslie Holtzclaw-Patterson Marjorie Anne Morrison Bruce Timothy Lotz John M. Roosenberg, II Larry Tardibono Byung Hyun Lee Chun Ma Anne F. Vergne Baiyuan Yang Therese Debiak Allen R. Ritter Brock Shireman Cara Cesario Bruce Allan Steinbaugh Sylvie Lebrat Hui Li Jerod Patzner Atanu Biswas Margaret Stauber Matthew D. Surman Brian Tobben Mark Alan Krook Peter R. Guzzo Timothy B. Durham Timothy Wencewicz Steven Raymond Woulfe Gordon C. Savela Li Dong Shanshan Yan Michael Roger Dick John Francis Okonya Vinay Girijavallabhan Raul Juarez Hernadez G. Rajendra Jingdan Hu Clara Schous Cheng Ji Debra A. Ulanowicz Alain A. Priour Aaron Murray Andrew Ward Charles Andrew Giglio John R. Bellettini Gaiying Zhao Elizabeth Springer Sushil L. Sharma Anjanette J. Koritnik Fangzheng Li Maria Drietz-Jobbins Li Liu Paul F. Vogt George Nora Nicole Windmon Chia-En Lin Deyi Zhang Kelley Snelgrove-Fennell Mark Majewski Deirdre Powell Tracey A. Gibson Weiqiang Huang Kyle Watson

Postdocs, Research Associates, Visiting Scientists

ESCMID eLibrary © by author Acknowledgments Siderophore Collaborators Patty Miller (ND) Ute Möllmann (Jena, Germany) Francois Malouin (Sherbrooke, Canada) Al Crumbliss (Duke)

National Institutes of Health NIH Merit Award DOD George and Winifred Clark Chair ESCMIDUniversity of eLibraryNotre Dame © byHsiri authorTherapeutics Grandpa, remember that Grandma said to use a cephalosporin linker like this

ESCMID eLibrary © by authorMadeline born 07/18/2016 ESCMID eLibrary © by author Synthetic Sideromcyins - Observation of Iron Transport Deficient Mutants Wild Type E. coli is Pathogenic, but Mutants are Not Pathogenic (Iron Starved!)

ESCMID eLibrary

Pathogenic Non-pathogenic Non-pathogenic A. A. Minnick, E. K.© Dolence, J. A.by McKee-Dolence, author F. Malouin Synthetic Sideromcyins – Non- beta-lactam conjugates: HT-10 a Mixed Ligand – Daptomycin Conjugate Designed to Target Gram Negative A. baumannii HO

HO O HO First demonstration of activity OH O NH N against Gram-negative bacteria OH O O O NH by Daptomycin HN N N H O O O H Molecular recognition based H N N N N H H O CONH O NH O O 2 O HN selectivity for H HO2C N O N N HO2C H H Acinetobacter baumannii NH O O O HO NH CO H O 2 H O N as designed N N O H H HT-10 O Activity vs MDR Ab (µM) MW = 2334 O NH2 HO O ESCMID eLibrary © by author Synthetic Sideromcyins – Non- beta-lactam conjugates: HT-10 a Mixed Ligand – Daptomycin Conjugate Designed to Target Gram Negative A. baumannii HO Tolerability in mice: HO O HT-10 was dosed at 5, 10, 25, 50, HO OH O NH N and 250 mg/kg in single doses with OH no observed adverse effect. O O O NH HN N N H O O O H In vivo activity (preliminary study): H N N N N H H O 6 to 8 wk old female ICR mice CONH O NH O O 2 O HN 8 H HO2C IP inoculation with 10 Acinetobacter N O N N HO2C H H NH O O O HO NH baumannii ATCC 17961 CO H O 2 H O N Treatment administered IV at 30 min and 24 h N N O H H post inoculation HT-10 O MW = 2334 O

NH2 HO O Treatments – Ciprofloxacin 50 mg/kg, HT-10 @25 mg/kg, HT-10 @10 mg/kg, 4/5 HT-10 is the first mice healthy after 7 days HT-10 @5 mg/kg, 1/5 mice survived after 7 form of Daptomycin that is days ESCMID eLibraryintentionally targeted Daptomycin 25 or 50 mg/kg and Vehicle to and is very active against treated mice were all dead at Day 1 © by authorAcinetobacter baumannii “Controversy” – Can Sideromycins be Useful Antibiotics? Read the Old Literature – 1955 Effective Natural Sideromycins – in people!

O O O siderophore -linker- drug N Fe Fe O N N O O HO OH O O H H N N N H N H HO H O S H2N O O HN Y N CO H ESCMIDH 2 eLibrary albomycins, OH Peptidase required natural siderophore for drug release or antibiotics no antibiotic activity (V. Braun - Biometals 2008 © byG. Benz - series o f papauthorers 1980s)