Neue Antibiotika gegen resistente Bakterien

Transcription

1 Neue Antibiotika gegen resistente Bakterien Bad Honnef-ymposium 2009 Dr. Imke Wiedemann University of Bonn Institute of Medical Microbiology Immunology and arasitology - harmaceutical Mirobiology ection - Meckenheimer Allee 168 D Bonn, Germany

2 New antibiotics against resistant bacteria Introduction art I: Are there new compounds with new targets/modes of action? ADEs (acyldepsipeptides) Friulimicin (Lipopeptide) art II: Can lantibiotics provide new strategies for the design of future antibiotics? tructure, biosynthesis, mode of action of lantibiotics Multiple activities of the lantibiotic nisin ummary I.Wiedemann, University Bonn 2

3 Antibiotics- The past The Golden Age of antibiotic discovery was very brief: 1930s-1950s New antibacterial agents approved in the United tates, , per 5-year period enicillin Cephalosporin treptomycin Erythromycin Tetracycline Vancomycin Boucher HW et al., Clin Infect Dis Jan 1;48(1):1-12. Review I.Wiedemann, University Bonn 3

4 An innovation gap for antibiotic classes C.Walsh, Harvard Medical chool, 2008 I.Wiedemann, University Bonn 4

5 New antibiotics against resistant bacteria Introduction art I: Are there new compounds with new targets/modes of action? ADEs (acyldepsipeptides) Friulimicin (Lipopeptide) art II: Can lantibiotics provide new strategies for the design of future antibiotics? tructure, biosynthesis, mode of action of lantibiotics Multiple activities of the lantibiotic nisin ummary I.Wiedemann, University Bonn 5

6 ADEs - New antibiotics from old patent literature ADEs (acyldepsipeptides) are a new class of antibiotics U patent in 1985: - a mixture of acyldepsipeptides was originally isolated from the culture broth from treptococcus hawaiiensis - report suggested notable in vitro activity against staphylococci and streptococci -tructure determination de novo synthesis synthetic analogs natural product IC µg/ml against gram-positive pathogens Brötz-esterhelt et al., (2005) Nature Medicine 11, 382 (Bayer) I.Wiedemann, University Bonn 6

7 ADEs: mechanism of action The target of ADEs is the bacterial protease roteases degrade improperly folded, damaged or unnecessary proteins (consist of seven Clp subunits and six monomers of AT-dependent ClpX; central pore size is 10 angström) roteins interact with ClpX, are unfolded in an AT-dependent manner and pass through the protease. Inside, the protein is degraded to di- and tripeptides. ADEs bind to Clp inducing degradation of cellular proteins in an unregulated manner Brötz-esterhelt et al., (2005) Nature Medicine 11, 382 (Bayer) I.Wiedemann, University Bonn 7

8 Lipopeptides: Friulimicin and Daptomycin Actinoplanes friluiensis MW 1453 treptomyces roseosporus MW 1620 exocyclic aa = Asn - branched-chain fatty acid (C13-C15) - cis3 double bond 3 exocyclic aa = Trp Asn - Asp -decanoyl fatty acid - aa-sequence of the peptide core differs markely active against gram-positive bacteria antimicrobial activity depends on the presence of Ca 2+ I.Wiedemann, University Bonn 8

9 Friulimicin- Mode of action Friulimicin- but not Daptomycin C 55 - inhibits cell wall biosynthesis LII FRI C 55 - : peptide ratio B 1: 0 1: 0.5 1: 0.7 1: 1 1: 0.5 1: 0.7 1: 1 UD MraY UM UD MurG UD FemXAB lipid II synthesized (%) FRI DA C 55 - : peptide ratio In vitro formation of lipid II using membrane preparations of M.luteus DM1970 1: 0 1: 0.5 1: 1 1: 0.5 1: 1 chneider et al., Antimicrob. Agents Chemother. I.Wiedemann, University Bonn 9

10 Friulimicin forms a complex with the lipid carrier C 55 - Friulimicin blocks WTA- and cell wall biosynthesis reactions - Molecular target that is not shared by any other antibiotic chneider et al., Antimicrob. Agents Chemother. I.Wiedemann, University Bonn 10

11 New antibiotics against resistant bacteria Introduction art I: Are there new compounds with new targets/modes of action? ADEs (acyldepsipeptides) Friulimicin (Lipopeptide) art II: Can lantibiotics provide new strategies for the design of future antibiotics? tructure, biosynthesis, mode of action of lantibiotics Multiple activities of the lantibiotic nisin ummary I.Wiedemann, University Bonn 11

12 Lantibiotics - to date approx. 60 different lantibiotics were described - gene-encoded peptides, posttranslationally modified - thioether amino acids lanthionine/methyllanthionine - size ranging from 19 to 38 amino acids (1825 to 4635 Da) Actagardine Trp er Val 5 H 1 2 N- Glu Val 15 -CH 10 - produced by and act mainly on Gram positive bacteria Gallidermin 5 he Mersacidin H 2 N- Dhb NH ro I ro he Tyr 20 CH 5 10 II Val Asn CH H 2 N- he 1 Glu I NH ep5 15 Dha I CH II 30 CH CH 3 10 I CH 2 5 Val Gln Asn I C - ro Arg Gln Dhb Dhb Arg he II Val -CH 25 lantaricinc Asp Glu His Asp Dha er Thr Asn Trp Val Epidermin 5 he H 2 N- Dhb ro he Tyr 20 Asn NH I CH II CH I.Wiedemann, University Bonn 12

13 Lantibiotics with potential applications Lantibiotic Nisin A Lacticin 3147 Inhibitory activity of commercial interest Gram-positive bacteria, Gram negative bacteria including Helicobacter pylori Gram positive bacteria otential Biomedical application Bacterial mastitis, Treatment of methicillin-resistant MRA and enterococcal infections, ral hygiene, peptic ulcer treatment Bacterial mastitis, Treatment of MRA and enterococcal infections, oral hygiene, Acne Gallidermin Epidermin ropionibacterium acnes, staphylococci, and streptococci Acne, eczema, folliculitis Mersacidin Actagardine taphylococci including methicillin resistant strains, streptococci Treatment of MRA and streptococcal infections MRA: Methicillin resistant taphylococcus aureus Cotter et al., Current rotein and eptide cience 6, I.Wiedemann, University Bonn 13

14 Lantibiotics- Mode of action Lantibiotics interfere with cell wall biosynthesis 30 min 60 min Electronmicrograph of taphylococcus simulans 22 taphylococcus simulans 22 after addition of 10 x MIC Mersacidin I.Wiedemann, University Bonn 14

15 The Mersacidin-Lipid II-binding motif mersacidin (type-b) H 2 N- he 1 ro 10 5 Val hinge Glu 15 Dha I NH I CH II CH plantaricinc (type-aii) Asp Glu His Asp Dha er Thr Asn Trp Val Lacticin3147 (two component lantibiotic) Trp 15 Asn Asn Tyr LtnA Asp H 2 N- he Dha Asn Dhb Trp Met Glu His Trp 30 LtnA2 Asn Thr Thr Arg 2-ob Dhb ro Dhb ro Tyr ro Thr I.Wiedemann, University Bonn 15

16 mersacidin: Inhibition of cell wall biosynthesis mersacidin peptidoglycan network XB2 outside pyrophosphorylase cytoplasm MraY MurG FemXAB N-acetyl muramic acid (MurNAc) N-acetyl glucosamine (GlcNAc) lipid I lipid II L-,D-Glu,L-, D-, D- 5 undecaprenyl (C 55 ) Brötz,H., (1998) AAC. 42: I.Wiedemann, University Bonn 16

17 Classification of Lantibiotics about 80% (54 of 66) of the lantibiotics interact with lipid II Type subgroups examples No. of members Interaction with Lipid II Ref. Type-AI nisin group nisina, nisinz, subtilin ericina 9 Wiedemann, I. et al., J. Biol. Chem. 276: epidermin group epidermin, streptin, mutacin Bonelli, R.R. et al., Agents Chemother. 50: ep5 group ep5, epilancink7, epicidin280 4 Type-AII lacticin481 group lacticin481, nukacin IK-1, plantaricinc 17 Wiedemann, I. et al., Appl. Environ. Microbiol. 72: Type-B mersacidin group mersacidin, actagardine, michiganina 4 Brötz, H. et al., Eur. J. Biochem. 246: cinnamycin group cinnamycin, duramycin, ancovenin 5 others class III morphogenetic peptides apb, apt, Amf 3 two peptide lantibiotics lacticin3147, haloduracin, mb 7 Wiedemann,I., et al., (2006) Mol.Microbiol. 61: peptides which have not been assigned to a group planosporicin, lactocin, carnocinu /2009 I.Wiedemann, University Bonn 17

18 nisin: Lipid II-dependent mode of action Lipid II targeted pore formation Inhibition of cell wall biosynthesis 100 % 80 % 60 % 40 % 20 % 0 control Nisin ΔN20/ΔM21 M17K 3T 15 µm 30 µm 60 µm Nisin mutant peptides 3T and N20/ M21: reduced antimicrobial and pore forming ability The activity of nisin (squares) and magainin (circles) was measured by monitoring the leakage of carboxyßuorescein from model membranes composed of 1,2- dioleoyl-sn-glycero- 3-phosphocholine in the presence (filled symbols) or absence (open symbols) of mol % purifed Lipid II. M17K 5 15 Met 25 1 Dha E er Dhb A B C Asn Met Asn D ro His T2 ΔN20/ΔM21 T13C T2A 3T N20/M21 T2V M21G Val Dha 30 V32E V32K er the N- terminal part binds to lipid I/lipid II the hinge- region is necessary for pore formation Breukink, Wiedemann et al., cience 1999 Dec 17;286:2361 Wiedemann et al., J Biol Chem Jan 19;276(3): I.Wiedemann, University Bonn 18

19 The Nisin-Lipid II-binding motif nisin (type-ai) H 2 N- Dhb 5 Dha ro 10 Met hinge His Asn Met er His Val Dha -CH Hsu,.T., et al., (2004) novel antibiotics Nat.truct.Mol.Biol. 11: I.Wiedemann, University Bonn 19

20 Nisin- a dual mode of action a) Inhibition of cell wall biosynthesis b) ore formation eptidoglycan network metabolites, salts, ions, potassium outside C X nisi n N G M lipid II G M N N G M cytoplasm C C I.Wiedemann, University Bonn 20

21 Lipid II targeted pore-formation by nisin Black Lipid Membrane Bilayer ystem (schematic) Black lipid membrane: Artificially formed bilayer separates two chambers microelectrodes for applying potentials and measuring current flows between chambers chambers filled with identical conductive aqueous solution (KCl) glass window seal steel-block Teflon-chamber ingle channel recording: Diph C membrane 0.1 µm nisin applied voltage: 100 mv 6 s 4 n 6 s Diph C membrane with 1 mol % lipid II 0.1 µm nisin applied voltage: 5 mv oresize: nm ore-lifetime: 6 s Estimated distance of Lipid II 18 Å. During or after assembly of four 1:1 (nisin:lipid II) complexes four additional nisin molecules are recruited to form the pore complex. Wiedemann et al., J Bacteriol May;186(10): Breukink and de Kruiff, 2006, Nat Rev Drug Discov Apr;5(4): I.Wiedemann, University Bonn 21

22 Nisin withdraws lipid II from the septum in bacteria fluorescein-labeled vancomycin Lipid II target-sites GUV s with lipid II Bacillus megaterium Bacillus subtilis fluorescein-labeled nisin glucosamine CH 3 muramic acid C H H H NH CH 2 CH 2 NH H 3 C CH H C C CH 3 L- D-Glu pentapeptide- L- sidechain D- nisin D- 8 2 vancomycin bactoprenol Bacillus megaterium (0.5 µg/ml fl-nisin) Bacillus megaterium (0.1 µg/ml fl-nisin) Bacillus subtilis Hasper,H.E., et al., (2006) cience 313: I.Wiedemann, University Bonn 22

23 Nisin-induced changes in Bacillus morphology Electron micrographs showing morphogenesis (cale bars: 200 nm, except B Top and Bottom and F, 500 nm.) A: control untreated B. subtilis B: multiple septation after 30 min 5µg/ml nisin C,D: 50 µg/ml nisin multiple septation; corkskrew growth; septal malformation G: Minicells Deregulation of Mbl (cell shape) and Min-ystem (FtsZ formation) Hyde,A.J.,et al., (2006) roc.natl.acad.ci.u..a 103: I.Wiedemann, University Bonn 23

24 Multiple effects of Nisin cell wall inhibition lipid II-mediated pore formation (nm concentration) segregation of lipid II deregulation of proteins involved in cell shape determination and cell division carpet mechanism (ren & hai,1998 and Tossi et al., 2000) pore formation (µm concentration) activation of autolysins (50 nm) (Bierbaum et al., 1987) The combination of these mechanisms depend on the lantibiotic and the specific strain. I.Wiedemann, University Bonn 24

25 ummary art I: Are there new compounds with new targets/modes of action? ADEs Friulimicin bacterial protease lipid carrier C55- art II: Can lantibiotics provide new strategies for the design of future antibiotics? one antibiotic X one mode of action The combination of different mechanisms of action in one molecule leads to potent antimicrobial activity making lantibiotics interesting model systems for the design of new antibiotics I.Wiedemann, University Bonn 25

26 Acknowledgements Dr. R.R. Bonelli Tim Böttiger hd student rof. Dr. Hans-Georg ahl harmaceutical Microbiology University of Bonn Germany Dr. Tanja chneider ost Doctoral Fellow) Collaborators: B.De Kuiff and E. Breukink, University Utrecht, Netherlands C. Hill and.ross, University Cork, Ireland U.eydel and T.Gutsmann, Forschungsinstitut Borstel, Germany R. Benz, University Würzburg, Germany B.B. Berger-Bächi, University Zürich, witzerland