Extending the Spectrum of Pleuromutilins to MDR Gram-negatives 28th ECCMID April 24, 2018 Michio Kurosu, Professor College of Pharmacy University of Tennessee Health Science Center ESCMIDMemphis, eLibrary TN https://mkurosu2.wixsite.com/kurosulab© by author Antibiotic Resistance Threats Antibiotic Resistance Antibiotic Identified Introduced ESKAPE pathogens Penicillin-R Staohylococcus 1940 1943 Penicillin CDC assessment (7 factors) Urgent threats Clostridium difficile 1950 Tetracycline Carbapenem-resitstant Enterobacteriaciacease 1953 Erythromycin Drug-resistant Neisseria gonorrhoeae Serious Threats Tetracycline-R Shigella 1959 1960 Methicillin MDR-Acinetobacter baumannii Methicillin-R Staphylococcus 1962 MDR-Pseudomonas aeruginosa Penicillin-R Pneumococcus 1965 1967 Gentamicin MDR-Mycobacterium tuberculosis plus 10 others (1 fungus) 1972 Vancomycin Concerning Threats (3 bacteria) Gentamicin-R Pneumococcus 1979 Number of Antibacterial Approval 19 1985 Imipenem and 20 Ceftazidime-R Enterobacteriaceae 1987 Ceftazidime Vancomycin-R Enterococcus 1988 18 16 Levofloxacin-R Pneumococcus 1996 1996 Levofloxacin 14 11 XDR Tuberculosis 2000 2000 Linezolid 12 Linezolid-R Staphylococcus 2001 10 Vancomycin-R Staphylococcus 2002 2003 Daptomycin 8 6 MDR Acinetobacter and Pseudomonas 2004 6 4 3 Ceftriaxone-R Neisseria 2009 2010 Ceftaroline 4 MDR Enterobacteriaceae 2009 2012 Bedaquline (TB) 2 Cetaroline-R Staphylococcus 2011 ESCMID2014 eLibraryDalbancin (iv) 0 Tedizolid 1980- 1995- 2000- 2005- 2010- Cefdtobiprole (USA) 1984 1999 2004 2009 2014 Ceflozane-tazobactam (iv) Fewer options to treat © by2015 Ceftazidime author-avibactam resistant bacteria Development of New Antimicrobial Agents Objective • A significant public health problem: the increasing multidrug resistance (MDR) among Gram-negative bacteria (Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacteriaceae) Gram-positive bacteria (Spore-forming bacteria), Mycobacterium tuberculosis Challenges • New antibiotic profiles: effective against sensitive strains, safe • New antibiotics must not only be active against resistant strains but must outperform the products already marketed against sensitive strains • The results of over 10 years’ screening of strains and molecular targets from traditional sources (randomly generated library molecules, and secondary metabolites) have been disappointing (no drug leads have been identified against new targets) Strategy for the development of new drug leads for MDR bacteria • Essential and unexploited bacterial drug target(s) • Identification of new molecules effective • CreativeESCMID synthesis and assay methods eLibrary • In vitro and in vivo efficacy © by author Collections of Bacteria (BSL1-BSL-3) • Mycobacterium tuberculosis (H37Rv from BEI) • Mycobacterium avium Subsp. paratuberculosis • Drug resistant Mycobacterium tuberculosis (ATCC 27294) • Rifampicin resistant Mycobacterium tuberculosis STRAIN TMC 331(ATCC 35838) • Isoniazid resistant Mycobacterium tuberculosis STRAIN TMC 303(ATCC 35822) • Mycobacterium smegmatis (ATCC 607™) • Mycobacterium smegmatis (ATCC14468™) • Mycobacterium bovis BCG • Staphylococcus aureus. Subsp. Aureus (ATCC 25923) • Staphylococcus epidermidis Evans (ATCC® 35984™) • Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 25923™) • Enterococcus faecalis (ATCC 19433™) • Enterococcus faecalis carrying pam373 (ATCC43062) • Streptococcus salivarius (ATCC 7073) • Streptococcus pneumoniae (ATCC 6301) • MRSA BAA-2094 • MRSA BAA-41 Regional Biocontainment • Staphylococcus aureus subsp. aureus (BAA-44) • Staphylococcus aureus subsp. aureus (ATCC-33592) Laboratory (RBL) at UTHSC • Staphylococcus aureus, STRAIN M10/0148 (BAA-2313) • Staphylococcus aureus (BAA-1683) • Clostridium difficile (ATCC 43596) • Staphylococcus aureus (BAA-42) • Clostridium perfringens (ATCC13124) • Staphylococcus aureus (ATCC6538) • Lactobacillus casei (Orla-Jensen) Hansen and Lessel (ATCC 393™) • Enterococcus faecium (ATCC 349) • Lactobacillus acidophilus (Moro) Hansen and Mocquot (ATCC 4356™) • Enterococcus durans (ATCC 6056) • Bifidobacterium longum subsp. longum Reuter (ATCC 15707™) • Enterococcus hirae (ATCC 49135) • Eggerthella lenta (Eggerth) (ATCC 43055™) • Bacillus cereus (No 2045) • Bacillus subtilis subsp. subtilis (Ehrenberg) Cohn (ATCC 6051) • Bacillus cereus (FERM BP-01719) • Streptomyces aurantiacus (ATCC19822™) • Bacteroides fragilis (ATCC 25285) • E. coli (ATCC 10798) • Fusobacterium peridonticum (ATCC 33693) ESCMID• E. coli (ATCC 35218) eLibrary • E. coli (Migula) Castellani and Chalmers (ATCC 10798™) • Pseudomonas aeruginosa (ATCC 27853™) • Klebsiella pneumoniae subsp. Pneumonia (ATCC 8047™) • Acinetobacter baumannii (ATCC 19606) Antimicrobial Susceptibility Tests • MDR Acinetobacter © baumannii by (ATCC BAA -1800)author Antimycobacterial Pleuromutilin Analogues UT-800 Efficacy against dormant Mtb Efficacy against intracellular Mtb (Wayne mode) Mycobacterium tuberculosis UT-800 ESCMID eLibraryFluorescence confocal microscopy of J774A.1 murine macrophages infected with tdTomato (red) expressing Mtb after the treatment of the mlecules (at 4xMIC). Fixed cells were stained with DAPI (4',6-diamidino-2- © by authorphenylindole) to visualize nuclei. Spectrum of Activity against Gram-negative Bacteria K. pneumoniae K. pneumoniae K. pneumoniae E. coli E. coli PCI 602 GN69 GN4081 BE1121 JR66/W677 (TC, CM, SM, KM, APC) (tolC mdtK) Meropenem 0.25 8.0 0.031 0.031 0.031 UT-490 0.25 32.0 64.0 0.25 8.00 P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa A3 GN17433 K-Ps102 GN17433 Meropenem 1.0 0.25 4.0 0.25 UT-490 64.0 32.0 >64.0 32.0 A. baumannii A. baumannii A. baumannii A. baumannii ATCC19606 JCM6841 ATCC BAA-1710 ATCC BAA-1800 (MDR) (MDR) Meropenem 2.0 2.0 1.0 8.0 UT-490 6.25 2.0 6.25 6.25-12.5 ESCMID eLibrary Acinetobacter baumannii UT-490 (ATCC19606) © by author Pleuromutilin • Pleuromutilin was first isolated in 1952 from two Basidiomycete spp. (Pleurotus mutilus and Pleurotus passeckerianus) • Tiamulin and Valunemulin, were successfully developed as therapeutic agents for veterinary use • Retapamulin (GSK) is the first antibacterial agent of the pleuromutilin class, approved in 2007 by FDA for the topical treatment, mainly caused by the Gram-positive Pleuromutilin bacteria (S. aureus and S. pyogenes) • BC-3781 (Nabriva Therapeutics) entered Phase II clinical studies, demonstrating a therapeutic potential for the treatment of skin infections and lung including community-acquired bacterial pneumonia (systemic) Pleurotus mutilus MycoBank # 168967 Published in 1876 ESCMID eLibrary © by author Mode of Action of Pleuromutilin Pleuromutilins 1. Activation - Formation of 7. Termination – Release of amino acyl tRNA Linez_ olid polypeptide chain _ AA _ _ _ 5. Elongation – Peptidyl_ transfer Aminogly_ cosides _ _ _ _ 6. Elonga_ tion – 50S Translocation 5 0 PDB IDs 3g4s and 5v7q overlaid by using UCSF 3. Elongation – Entry of S aminoacyl tRNA Chimera software (The binding site: about 10 Å) A A e – S m n t so tio co o o a g S d p ib ng in 30 o AA R lo ad n E e 3 . f r 0 4 oo S Pr t S r 0 a n 5 t o S d o S c 0 3 A N R m RNAP S 0 3 5` 2. Initiation – 30S ribosomal subunit binds to mRNA ESCMID eLibrary Binding sites of pleuromutilin derivatives and linezolid on bacterial 50s ribosome. © byPleuromutilin author derivatives interfere with both the A- and P- sites Cross-resistance to LinedzolidR-strains? UT-490 MIC100 0.024 mg/mL against S. aureus ATCC 6538 MIC100 6.25 mg/mL against A. baumannii ATCC 19606 UT-490R-S. aureus ATCC 6538 (MIC 20 mg/mL) UT-490R-A. baumannii ATCC 19606 (MIC 100 mg/mL) Concentrations of UT-490 6.25 mg/mL 12.5 mg/mL 25.0 mg/mL LinezolidR-A. baumannii ATCC 19606 0 0 0 Number of colonies Concentrations of UT-490 1.0 mg/mL 5.0 mg/mL 10.0 mg/mL LinezolidR-S. aureus ATCC 6538 0 0 0 Number of colonies Linezolid MIC <1.0 g/mL against S. aureus ATCC 6538 ESCMID100 m eLibraryConclusion: MIC50 50 mg/mL against A. baumannii ATCC 19606 A mechanism of resistance of two strains against linezolid and UT-490 is different, although both share LinezolidR-S. aureus ATCC 6538 (MIC 20 mg/mL) LinezolidR-A. baumannii ATCC 19606 (MIC >500 mg/mL) the binding domain of the A-site partially, and cross © by authorresistance may not happen. Systemic Application of Pleuromutilin? Half-life (t1/2) in the rat liver microsomes 2500 Retapamulin-H2 2000 1500 Retapamulin 1000 Concentration (ng/mL) Concentration 500 0 0 20 40 60 80 100 ESCMID eLibraryTime (min) © by author Systemic Application of Pleuromutilin? Half-life (t1/2) in the rat liver microsomes 100 90 80 70 60 UT-490 50 40 30 Concentration (ng/mL) Concentration 20 UT-490 10 Valunemulin 0 0 10 20 30 40 50 60 Time (min) Cytotoxicity against Vero cells 1.2 1 1 Valnemulin 0.8 Valunemulin 0.6 0.4 UT-490 ESCMID (570nm) Absorbance eLibrary 0.2 Valunemulin 0 0 50 100 150 200 © by authorConcentration (mg/mL) Systemic Application of Pleuromutilin? Half-life (t1/2) in the rat liver microsomes UT-800 UT-810 ESCMIDUT-815 eLibrary © by author Systemic Application of Pleuromutilin? Half-life (t1/2) in the rat liver microsomes UT-800 UT-810 ESCMIDUT-815 eLibrary © by author In vitro Cytotoxicity of Pleuromutilin Analogues Cytotoxicity of selected analogues against Vero cells UT-490 Rifampicin Valunemulin UT-490 UT-800 Other UT-analogues Valnemulin IC50 45-50 mg/mL (UT-490), UT-810 IC50 15-35 mg/mL (UT-800, 810, 815, and several others), IC 15 g/mL (Valnemulin) ESCMID50 meLibrary Hemolysis 0% at 200 mg/mL concentration (UT-490) UT-815 © >50%by at 10 m g/mLauthor concentration (tunicamycin)