A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon FLAVIA MARINELLI. DBSM, University of Insubria, Varese Italy

Size: px

Start display at page:

Download "A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon FLAVIA MARINELLI. DBSM, University of Insubria, Varese Italy"

Julius Horton
6 years ago
Views:

FLAVIA MARINELLI DBSM, University of Insubria, Varese Italy

1 A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon Actinomycete Planomonospora sp. FLAVIA MARINELLI DBSM, University of Insubria, Varese Italy Vicuron Pharmaceuticals, Gerenzano Research Center, Varese, Italy

The emergence of multidrug-resistant microbial pathogens is driving the search for novel and more effective antibiotics High throughput screening using assays based on validated and

2 The emergence of multidrug-resistant microbial pathogens is driving the search for novel and more effective antibiotics High throughput screening using assays based on validated and established specific vital targets Chemical diversity produced by novel species of actinomycetes and fungi Streptomyces 15% ther actinomycetes 48% ther bacteria 1% Fungi 35% Myxobacteria 0.4%

3 A validated established specific antibiotic target is the bacterial peptidoglycan biosynthesis Cell wall biosynthesis pathway Antibiotics that act on PG biosynthesis Walsh C. 2003, Antibiotics. Actions, origins, resistance, ASM Press, Washington, D.C.

4 Mode of action of clinically relevant glycopeptides and β-lactams: Inhibition of peptidoglycan synthesis D-Ala Glycopeptide: vancomycin or teicoplanin D- Ala L-Lys Transglycosilation NAM NAG D-Glu L-Ala Transpeptidation Bactoprenol β-lactams Cell membrane

5 Glycopeptide antibiotics are active against multiresistant Gram positive pathogens Amycolatopsis orientalis Actinoplanes teichomyceticus Drugs of last resort against nosocomial infection diseases: staphylococci streptococci pneumococci enterococci

6 Multiresistant emerging Gram-positive pathogens: a nosocomial USA survey 25% of Streptococcus pneumoniae are resistant to penicillin 35% of Staphylococcus aureus are resistant to methicillin and all availableβ-lactams. Many of them are resistant to aminoglycosides,fluoroquinolones, macrolides, tetracyclines 12% of Enterococcus faecalis are resistant to vancomycin (first clinical isolates in 1988) Vancomycin-resistant Staphylococcus aureus (VRSA) (first clinical isolates in 2002) Appelbaum, P. C., and Jacobs, M. R. (2005) Recently approved and investigational antibiotics for treatment of severe infections caused by Gram-positive bacteria, Curr. pin. Microbiol. 8,

7 PG biosynthesis still offers remarkable opportunities for identifying unique inhibitors capable of bypassing existing resistance mechanisms Design of a HTS procedure to identify inhibitors targetting PG biosynthesis steps other than those blocked by β-lactams and glycopeptides

8 ur HTS procedure : Selection of PG inhibitors Primary screening of 120,000 extracts from 40,000 uncommon actinomycetes by a differential activity assay versus S.aureus and its L-form Elimination of β-lactams and glycopeptides Reversion of antimicrobial activity versus S.aureus by adding a β-lactamase cocktail or a D-Ala-D-Ala affinity resin Elimination of known compounds (LC-MS) and of false positives (hit refermentation and retesting)

10 Lantibiotics identified in 35 broth extracts by LC-MS: characteristics Lan and Melan amino acids in the acid hydrolysates Five showed strucutral characterisitc that did not match with any known microbial metabolite

11 Lantibiotics are ribosomally synthetized and posttranslationally modified peptides produced by Gram positives Ca.50 known structures Type A: flexible amphiphilic long peptides forming pores in bacterial membranes, following interaction with Lipid II 3353 Da The prototype is nisin produced by Lactococcus lactis subsp.lactis: strong bactericidal agent used as food preservant

Pure PG inhibitors less bactericidal than nisin under

12 Type B: rigid globular peptides blocking PG synthesis by binding to Lipid II 1825 Da, produced by Bacillus spp. Pure PG inhibitors less bactericidal than nisin under evaluation in systemic chemotherapy 1890 Da, produced by Actinoplanes spp.

13 Interaction with Lipid II: no cross-resistance with glycopeptides no cross-resistance between mersacidin and nisin P.D. Cotter et al. 2005, Curr.Protein Pept Sci 6, 61-75

14 Planosporicin is a novel lantibiotic produced by the uncommon actinomycete Planomonospora H 1 Ile Dhb Ala 4 S Val Abu Ala Dha Ala Trp S 8 S Thr Ala Glu 12 Gly Gly Gly Ala Gly 16 Ala Ala S 21 Ala His Ala Pro Gly H 1890 Da 19 S -Type B globular and rigid structure -Different from mersacidin and actagardine -NH 2 sequence identical to nisin: Lipid II pyrophoshate cage

15 Planosporicin as type B lantibiotics and glycopeptides inhibits PG synthesis as confirmed by inhibiton test of macromolecular syntheses in whole cells of Staphylococcus aureus. A Planosporicin B Teicoplanin PG RNA % inhibition 50 % inhibition 50 Protein DNA conc (µg/ml) conc (µg/ml) C A ctagardine D Nisin % inhibition 50 % inhibition 50 Nisin inhibits all the syntheses conc (µg/ml) conc (µg/ml)

16 Planosporicin as type B lantibiotics and glycopeptides causes the accumulation of the cytoplasmatic UDP-pentapeptide precursor in Bacillus megaterium growing cells H H N H 2 N N H H N H N H H N H H NH P H P H H H N N H ua 3, H UDP-MurNAc-L-Ala-D-Glu-mDap-D-Ala-D-Ala A ,4 4,5 Planosporicin-treated cells , ,15 15,5 12,2 9,6 15, Time (min) ua 3, ,4 B ,8 4,0 4,5 6,1 Teicoplanin-treated cells ua ,0 3,7 Time (min) Untreated cells 4,2 4,47 24,4 5,5 6, Time (min) C

17 The in vitro spectrum of activity of planosporicin covers Gram-positive pathogens, including MRSA and enterococci Van, which are of increasing medical importance because of the spread of resistance mechanisms. Planosporicin antimicrobial activity against Gram-positive pathogens is better than that of actagardine and comparable to that of mersacidin. Lower MICs of nisin may be explained by its dual mechanism of action, combining sequestering of lipid II and pore formation

Planosporocin is produced by a novel species of Planomonospora deposited as DSM 14920 300 Time course of Planomonospora sp DSM 14920 fermentation at 300 l scale: the antibiotic is produced

18 Planosporocin is produced by a novel species of Planomonospora deposited as DSM Time course of Planomonospora sp DSM fermentation at 300 l scale: the antibiotic is produced extracellularly with a maximum productivity of 280 mg/l biomass [pmv%] and potency [mg/l] 250 antibiotic ph 150 biomass fermentation time (hours) ph D. Losi et al 2004 European Patent A1 F. Castiglione et al Biochemistry 46:

19 Conclusions Planosporicin is a novel patentable chemical structure belonging to type B lantibiotics The class of type B lantibiotics is currently under evaluation for chemotherapeutic expolitation: -novelty and complexity of the binding target -no cross resistance with β-lactams and glycopeptides -activity against MRSA and VRSA pathogens -efficacy in vivo in protecting mice from septicaemia

20 Conclusions Planosporicin vs other Type B lantibiotics: -soluble and stable at physiological phs -improved antimicrobial activity and spectrum of action -improved in vivo efficacy -additional insight into the molecular determinants of the biological activity of Lipid II-targeting antibiotics Planosporicin is a potential new agent for the treatment of emerging resistant Gram-positives

21 Franca Castiglione, Linda Cavaletti, Daniele Losi, Ameriga Lazzarini, Lucia Carrano, Marina Feroggio, Ismaela Ciciliato, Emiliana Corti, Gianpaolo Candiani, Enrico Selva

Novel Tetrapeptide Inhibitors of Bacterial Protein Synthesis Produced by a. Streptomyces sp. FLAVIA MARINELLI

Novel Tetrapeptide Inhibitors of Bacterial Protein Synthesis Produced by a. Streptomyces sp. FLAVIA MARINELLI ovel Tetrapeptide Inhibitors of Bacterial Protein Synthesis Produced by a Streptomyces sp. FLAVIA MARIELLI DBSM, University of Insubria, Varese Italy Vicuron Pharmaceuticals, Gerenzano Research Center,