REBELREpotentiating BEta Lactam antibiotics

Mariël Pikkemaat Paris, 13-01-2017

REBEL

�Mariël Pikkemaat (coordinator)

RIKILT – Wageningen UR, Netherlands

�Natalie Strynadka,

University of British Columbia, Canada

� Fredrik Almqvist,

Umeå University, Sweden

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REBEL project

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�REpotentiating BEta-Lactam antibiotics

�Restoration of the effectivity of a proven

successful class of antibiotic drugs

Beta-lactam antibiotics

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Penicillins

Cephalosporins

Carbapenems

Monobactams(Aztreonam)

Antibacterial mechanism

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Enzyme establishing cross-links PG cell wall

β-lactam antibiotic

Deactivated enzyme

Cross-linked proteinProtein (not cross-linked)

Polysaccharide backbone

Beta-lactam resistance

�Bacteria start producing beta-lactam hydrolysing

enzymes

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Beta-lactamases

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Wright 2005, Adv. Drug Deliv Rev 57(10) 1451

A. Serine beta-lactamase

B. Metallo beta-lactamase

Beta lactamase classification

� Structural and biochemical classification

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Based upon Bush Ann NY Acad Sci. 1277 (2013) 84-90

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10

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Carbapenemases

12NDM producers, Dortet et al. 2014 BioMed Res Int

KPC producing Klebsiella pneumoniaeLee et al. 2016 Front Microbiol

Circumventing resistance

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Supplement the beta-lactam antibiotic

with a beta-lactamase inhibitor

Beta-lactamase inhibition

�β-lactamase inhibitors currently used in

combination with β-lactam antibiotics:

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Clavulanic acid Sulbactam Tazobactam

Avibactam

Beta-lactamase inhibition

�Similarity to the antibiotic > risk of inhibitor

resistant β-lactamases

�Not effective against metallo β-lactamases (and

Oxa’s)

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Urgent need for novelbeta-lactamase inhibitors

REBEL project

�Isolation of new beta-lactamase inhibitors from

botanical origin

�Plant Extract Collection Kiel in Schleswig-

Holstein (PECKISH)

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PECKISH library

>4600 aqueous, ethanolic and other extracts

>860 different plant species, representing ~190 plant

families, 11 different plant tissues

�Origin: health shops, outdoor cultivated plants,

marine algae, herbs from Traditional Chinese

Medicine, African plants and known medical plants

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Screening strategy

Development of bacterial tester strains

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Development tester strains

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Screening strategy

� Measure inhibition of bacterial growth with resazurin

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t = 0 t ~20 hBacterial growth

Growth inhibited

Screening strategy

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Duplicate plates

Non-specific antimicrobial activity

Beta-lactamase inhibition

No Ampicillin Ampicillin

Screening strategy

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- β lactam + β lactam

Bacterial tester strain

Library Botanical extracts

Replica plates

Results

� 15-30 % of extracts inhibit bacterial growth

� unexpectedly large number of hits

� significant number of extracts showed inhibition across the entire set of available beta lactamases

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KPC-2 OXA-48 OXA-23 SHV-2 VIM-2 IMP-1 TEM-24 CMY-2 CTX-M-15

HIT 197 125 486 70 218 387 76 196 316

E-HIT 69 50 79 361 159 334 106 257 133

Selection

� Check expression:

In Situ Screening of Bacterial Colonies

� T

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- ampicillin

Transfer of 10 µl to PVDF membrane

Microwave induced lysis on top of Whatman paper soaked in 5% SDS

Electro transfer to remove SDS at (50 volts for 50 min)

BSA Blocking, mouse anti-His AB, anti-mouse Alkaline phosphatase AB

> colour development

Selection

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Selection

� Expression results confirmed our suspicion: many of the “broad spectrum” HITs caused by inhibition of expression

� Starting from each individual beta lactamase, scored:

(a) similar beta lactamase hit patterns

(b) similar pattern with other extracts of the same botanical

� Check expression > final selection

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75 Extracts

Potency testing

Bioassay guided fractionation

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Gradient Liquid chromatography

Flow split

Bioassay plateDuplicate – identificationUPLC/QTOFMS(/MS) GC/TOFMS, NMR

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New lead compounds

Structure-guided lead optimization

NewBeta lactamase

inhibitors

Kinetic characterization

Structural characterization

Cytotoxicity testing

Efficacy on clinical isolates

Isolation or synthesis of standards

Synthesis of analogues

Acknowledgements

� Funding organizations:

● Swedish Research Council

● Canadian Institutes of Health Research

● ZonMw - The Netherlands Organisation for Health

Research and Development

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