Pseudomonas Aeruginosa

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

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) Good Enough Characteristics as an Anti-Acinetobacter Lead ?

Half-life (t1/2) in the rat liver microsomes 100 90 80 70 60 50 40 30

Concentration (ng/mL) Concentration 20 UT-490 10 0 0 10 20 30 40 50 60 Time (min)

Strains MIC or MBC (mg/mL)

UT-490 Valnemulin Tobramycin Colistin

MIC MBC MIC MBC MIC MBC MIC MBC

A. baumannii ATCC 19606 1.75 6.25 3.13 12.5 6.25 12.5 3.13 6.25

MDR- A. baumanniiESCMID eLibrary ATCC BAA-1800 6.25 12.5 6.25 25.0 >25.0 >25.0 >12.5 25.0 © by author Pharmacokinetic Data for UT-490

10000 Mean (± SD) Concentration of Mouse IV and PO

1000 IV 3 mg/kg

PO 30 mg/kg 100

UT-490 10 Therapeutic concentrations 1.5-6.25 mg/mL against A. baumannii ATCC 19606 1

Plasma Concentration (ng/mL) Concentration Plasma 0.1 0 5 10 15 20 25 Time (hr) IV administration (3 mg/kg)

No. of pts AUC AUC AUC Extr Animal used for t (hr) C (ng/mL) last Inf V (L/kg) V (L/kg) CL (mL/min/kg) MRT (hr) 1/2 0 (hr*ng/mL) (hr*ng/mL) (%) z ss Inf t1/2 N 3 3 3 3 3 3 3 3 3 3 Mean 9.98 1908 993 1046 4.95 39.6 10.8 48.4 3.78 SD 5.68 579 114 135 1.79 19.7 1.6 6.7 0.79 CV% 56.9 30.3 11.5 12.9 36.1 49.7 14.3 13.9 20.8

PO administration (30 mg/kg) No. of pts AUC AUC AUC /D Animal used for t (hr) t (hr) C (ng/mL) last Inf AUC Extr (%) MRT (hr) Inf F (%) 1/2 max max (hr*ng/mL) (hr*ng/mL) Inf (hr*kg*ng/mL/mg) t1/2 N ESCMID3 3 3 3 3 eLibrary3 3 3 3 3 Mean 4.02 0.83 1344 2869 2891 0.758 2.60 96.4 27.6 SD 0.76 0.29 473 583 587 0.031 0.60 19.6 5.6 CV% 18.9 34.6 35.2 20.3 20.3 4.13 22.9 20.3 20.3 © by authorAUC: the area under the curve Survival Rate in C57BL/6 Mice Infected with A. baumannii

UT-490

The mice were infected intraperitoneally with A. baumannii (ATCC19606) at a dose that lead to >75% of death in a day. The test molecules were intraperitoneally administered once after 1 h of the infection. Mortality was monitored for 5 days for all groups. P < 0.05.

ESCMID eLibrary © by author Checkerboard Synergy Analyses of UT-490 Gentamycin Amikacin

50 4.0312 4.0624 4.1248 4.2504 4.5 5 6 8 50 4.0312 4.0624 4.1248 4.2504 4.5 5 6 8 25 2.0312 2.0624 2.1248 2.2504 2.5 3 4 6 25 2.0312 2.0624 2.1248 2.2504 2.5 3 4 6 12.5 1.0312 1.0624 1.1248 1.2504 1.5 2 3 5 12.5 1.0312 1.0624 1.1248 1.2504 1.5 2 3 5 6.25 0.5312 0.5624 0.6248 0.7504 1 1.5 2.5 4.5 6.25 0.5312 0.5624 0.6248 0.7504 1 1.5 2.5 4.5 3.13 0.2816 0.3128 0.3752 0.5008 0.7504 1.2504 2.2504 4.2504 3.13 0.2816 0.3128 0.3752 0.5008 0.7504 1.2504 2.2504 4.2504 1.56 0.156 0.1872 0.2496 0.3752 0.6248 1.1248 2.1248 4.1248 1.56 0.156 0.1872 0.2496 0.3752 0.6248 1.1248 2.1248 4.1248 0.78 0.0936 0.1248 0.1872 0.3128 0.5624 1.0624 2.0624 4.0624 0.78 0.0936 0.1248 0.1872 0.3128 0.5624 1.0624 2.0624 4.0624 0.39 0.0624 0.0936 0.156 0.2816 0.5312 1.0312 2.0312 4.0312 0.39 0.0624 0.0936 0.156 0.2816 0.5312 1.0312 2.0312 4.0312 0.39 0.78 1.56 3.13 6.25 12.5 25 50 0.39 0.78 1.56 3.13 6.25 12.5 25 50 UT-490 UT-490 Colistin Doxycycline 25 4.0312 4.0624 4.1248 4.2504 4.5 5 6 8 0.8 4.0312 4.0624 4.1248 4.2504 4.5 5 6 8 12.5 2.0312 2.0624 2.1248 2.2504 2.5 3 4 6 0.4 2.0312 2.0624 2.1248 2.2504 2.5 3 4 6 6.25 1.0312 1.0624 1.1248 1.2504 1.5 2 3 5 0.2 1.0312 1.0624 1.1248 1.2504 1.5 2 3 5 3.13 0.532 0.5632 0.6256 0.7512 1.0008 1.5008 2.5008 4.5008 0.1 0.4994 0.5624 0.6248 0.7504 1 1.5 2.5 4.5 1.56 0.2808 0.312 0.3744 0.5 0.7496 1.2496 2.2496 4.2496 0.05 0.2812 0.3124 0.3748 0.5004 0.45 1.25 2.25 4.25 0.78 0.156 0.1872 0.2496 0.3752 0.6248 1.1248 2.1248 4.1248 0.025 0.1562 0.1874 0.2498 0.3754 0.42 1.125 2.125 4.125 0.39 0.0936 0.1248 0.1872 0.3128 0.5624 1.0624 2.0624 4.0624 0.0125 0.0937 0.1249 0.1873 0.3129 0.35 1.0625 2.0625 4.0625 0.2 0.0632 0.0944 0.1568 0.2824 0.532 1.032 2.032 4.032 0.00625 0.06245 0.09365 0.15605 0.28165 0.53125 1.03125 2.03125 4.03125 0.39 0.78 1.56 3.13 6.25 12.5 25 50 0.39 0.78 1.56 3.13 6.25 12.5 25 50 UT-490 UT-490 Tygecyclin 200 4.0312 4.0624 4.1248 4.2504 4.5 5 6 8 SFIC is the sum of fractional inhibitory concentration calculated 100 2.0312 2.0624 2.1248 2.2504 2.5 3 4 6 by the equation SFIC = FICA + FICB = CA/MICA + CB/MICB. 50 1.0312 1.0624 1.1248 1.2504 1.5 2 3 5 25 0.5312 0.5624 0.6248 0.7504 1 1.5 2.5 4.5 12.5 0.2812 0.3124 0.3748 0.5004 0.75 1.25 2.25 4.25 6.25 0.1562 0.1874 0.2498 0.3754 0.625 1.125 2.125 4.125 Synergy analyses with 25 drugs. Only doxycycline 3.13 0.0938 ESCMID0.125 0.1874 0.313 0.5626 1.0626 2.0626 4.0626eLibrary 1.56 0.0624 0.0936 0.156 0.2816 0.5312 1.0312 2.0312 4.0312 displayed synergy with UT-490 at a wide range of 0.39 0.78 1.56 3.13 6.25 12.5 25 50 concentrations. UT- 490 © by author Role of Doxycycline in Combination with UT-490?

(UT-490/Dox)R-A. baumannii ATCC 19606

UT-490

MIC100 6.25 mg/mL against A. baumannii ATCC 19606 UT-490R-A. baumannii ATCC 19606 (MIC 100 mg/mL or higher)

UT-490 (50.0 mg/mL) Dox (3.13 mg/mL)

Concentrations of Doxycycline 6.25 mg/mL 12.5 mg/mL 25.0 mg/mL

(UT-490/Dox)R-A. baumannii ATCC 19606 0 0 0 Number of colonies

Concentrations of UT-490 12.5 mg/mL 25.0 mg/mL 50.0 mg/mL

(UT-490/Dox)R-A. baumannii ATCC 19606 3.8 x 106 3.2 x 106 1.5 x 106 Number of colonies

OD600 0.1-0.2

UT-490-Dox (35/1) MIC100 2.0 mg/mL against A. baumannii ATCC 19606 Effect of Dox (0.06 mg/mL): no inhibition UT-490-Dox (20/1) MIC100 2.0 mg/mL against A. baumannii ATCC 19606 Effect of Dox (0.09 mg/mL): no inhibition Dox MIC 0.4ESCMID mg/mL against A. baumannii ATCC 19606 eLibrary Conclusion: (UT-490/Dox)R-A. baumannii ATCC 19606 (MIC 50 mg/mL Max) (UT-490/Dox)R-A. baumannii ATCC 19606 is not a Dox resistant strain, but shows resistance to UT-490 (100 © by mg/mL).author Role of Doxycycline in Combination with UT-490?

Amino acid alignment of the 50S ribosomal protein L3 (RplC) from the resistant A. baumannii strains

UT-490R-A. baumannii ATCC 19606

UT-490 UT-490R-A. baumannii ATCC 19606 (MIC 100 mg/mL or higher) (Query) and wild-type control (Sbjct) C456A single mutation : N152K mutation

(UT-490/Dox)R-A. baumannii ATCC 19606

No mutation in the rplC gene

ESCMID eLibraryUT-490/Dox (35/1) 50 mg/mL Conclusion: We need perform whole-genome analysis to identify R (UT-490/Dox) -A. baumannii ATCC 19606 potential resistant mechanisms of UT-490/Dox (MIC 50 mg/mL Max ) © by author MICs of Old Drugs against A. baumannii

Rifampicin Colistin Tygecyclin Erythromycin Clarithromycin

A. baumannii 3.12 3.12-6.25 25.0-50.0 (50%) 6.25 (50%) 12.5 (50%) ATCC 19606 A. baumannii MDR 3.12 >6.25 64.0 - - (BAA-1800)

Azithromycin Gentamycin Amikacin Tobramycin Clindamycin Tunicamycin

A. baumannii 25 12.5 12.5 6.25 (50%) >50 >50 ATCC 19606 A. baumannii 12.5 (50%) >64.0 >64.0 >64.0 - - MDR (BAA-1800)

Meropenem Vancomycin Linezolid Trimethoprim Capreomycin Retapamulin

A. baumannii 2.0 >50 50 (50%) 25 (50%) >50 50 ATCC 19606 A. baumannii 8.0 - - - - - MDR (BAA-1800)

Minocycline Doxycycline Demeclocycline Ethinamide Ethambutol Isoniazid

A. baumannii 1.58 0.4-3.13 3.12 >50 >50 >50 ATCCESCMID 19606 eLibrary A. baumannii 25 >25 - - - - MDR (BAA-1800) © by authorAcinetobacter baumannii (ATCC19606) Synergy of Doxycycline with Four Dugs

Rifampicin Colistin

A. baumannii 3.12 3.12-6.25 ATCC 19606 A. baumannii MDR 3.12 >6.25 (BAA-1800)

Azithromycin Tobramycin

A. baumannii 25 6.25 (50%) ATCC 19606 A. baumannii 12.5 (50%) >64.0 MDR (BAA-1800)

DS 6.25 3.125 1.5625 0.78125 0.39063 0.19531 0.09766 0.04883 0.02441 0.01221 0.0061 0.00305 RIF:Dox 1.5:1 0.125 0.184 0.199 0.225 0.238 0.24 0.247 0.352 0.507 0.86 1.517 1.617 RIF:Dox 12.5:1 0.151 0.28 0.308 0.348 0.415 0.454 0.741 1.368 1.612 1.657 1.69 1.69 RIF:Dox100:1 0.15 0.283 0.351 0.404 0.437 0.544 1.39 1.597 1.651 1.68 1.706 1.701 Tobra:Dox100:1 0.169 0.31 0.519 0.708 1.339 1.615 1.662 1.689 1.674 1.688 1.692 1.692 Tobra:Dox25:1 0.163 0.386 0.388 0.426 0.469 0.811 0.696 1.061 1.5 1.659 1.685 1.722 Tobra:Dox6.25:1 0.198 0.287 0.38 0.424 0.492 0.851 0.793 1.27 1.581 1.678 1.698 1.715 Azithro:Dox50:1 0.191 0.282 0.339 0.511 1.022 1.355 1.516 1.595 1.646 1.67 1.669 1.658 Azithro:Dox12.5:1 0.192 0.203 0.201 0.244 0.338 0.453 0.677 1.301 1.392 1.409 1.548 1.612 Col:dox (1.56:1) 0.161 0.202 0.225 0.308 0.343 0.47 0.686 0.643 1.025 1.035 1.124 1.051 Col:dox (6.25:1) 0.167 0.187 0.202 0.193 0.358 0.679 1.068 1.124 1.075 1.097 1.104 1.098 Col:dox ESCMID(100:1) 0.151 0.748 1.107 1 eLibrary1.163 1.139 1.143 1.136 1.129 1.107 1.108 1.087 Acinetobacter baumannii (ATCC19606) : remained blue in colorimetric assays using risazurin : turned red in colorimetric © assays by using risazurin author Design of New Pleuromutilins Analogues-Docking Study ?

ESCMID eLibraryTiamulin Overlay of PDB 3g4s (tiamulin co-crystallized structure with the large ribosomal subunit; carbons are red) © with a novelby analog author using Maestro from Schrodinger Design of New Pleuromutilins Analogues-Docking Study ?

A. baumannii S. aureus Vero cell Compound ATCC19606 ATCC 6538

UT-490 6.25 0.024 45.0

UT-490

A. baumannii S. aureus Vero cell Compound ATCC19606 ATCC 6538

UT-1100 >50.0 50.0 >100

UT-1100

A. baumannii S. aureus Vero cell Compound ATCC19606 ATCC 6538

UT-1095 50.0 25.0 >100

UT-1095 A. baumannii S. aureus Vero cell ESCMIDCompound eLibraryATCC19606 ATCC 6538

S. aureus ATCC 6538

Concentration 25.0 12.50 6.25 3.13 1.56 0.78 0.39 0.19 0.098 0.049 0.024 0.012

UT-910 0.125 0.184 0.199 0.185 0.238 0.24 0.247 0.189 0.160 0.135 0.145 0.165 Ref: Retapamulin MIC 0.19-0.098 Linezolid MIC 0.19 Colistin MIC >25

A. baumannii (ATCC19606)

Concentration 25.0 12.50 6.25 3.13 1.56 0.78 0.39 0.19 0.098 0.049 0.024 0.012 UTESCMID-910 0.125 0.184 0.199 0.185 0.238eLibrary0.220 0.569 1.10 1.10 1.50 1.20 1.40 Ref: : remained blue in colorimetric assays using risazurin Tobramycin MIC 12.5 Colistin MIC 3.12 : turned red in colorimetric assays using risazurin Linezolid MIC 50 © by50 author Design of New Pleuromutilins

On-going Studies

Whole genome analysis of a UT-490-Dox resistant strain

UT Health Science Center University of Minnesota

Dr. Katsuhiko Mitachi Dr. Yinduo Ji Ms. Shakiba Eslamimehr Mr. Junshu Yang

Mr. Clayton Stewart BIKAKEN Ms. Maddie Lemieux Dr. Katsuhisa Yamazaki Dr. Shajila Siricilla Dr. Yoshimasa Ishizaki

Financial Supports ($$$) University of Tennessee (CORNET) ESCMIDNational InstituteeLibrary of Health NIAID © byNIGMS author ESCMID eLibrary © by author A Mechanism of Resistance

Inhibition of Transcription/Translation Genetic Analysis of a UT-490 to Study the Resistance Mechanism

100 90 80 70 60 50 40 30

Luminescence (%) Luminescence C456A single nucleotide mutation, which corresponded to 20 10 N152K mutation 0 0 0.2 0.4 0.6 0.8 1 Concentration (ug/mL)

The amino acid alignment of the 50S ribosomal protein L3 (RplC) from a UT-490- ESCMIDresistant A. baumannii strain (UT-490 ReLibrary) UT-490R (Query) and wild-type control (Sbjct): the red color represents the site mutation in RplC . © by author