Pegylated LyeTx I-b peptide is effective against

carbapenem-resistant Acinetobacter baumannii in an

in vivo model of pneumonia and shows reduced toxicity.
Júlio César Moreira Brito, William Gustavo Lima, Jarbas Magalhães Resende, Débora Cristina
Sampaio de Assis, Daiane Boff, Valbert Nascimento Cardoso, Flávio Almeida Amaral, Elaine
Maria Souza-Fagundes, Simone Odília Antunes Fernandes, Maria Elena de Lima,
International Journal of Pharmaceutics, Volume 609, 2021, 121156, ISSN 0378-5173,

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Carbapenem-resistant Acinetobacter baumannii (CRAB)

Biofilms

Resistance to several antimicrobials (especially carbapenem)

E.g., quinolones, aminoglycosides, β-lactams and polymyxin
(Lima et al., 2020)

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LyeTx I-b
• Synthetic and modified peptide
• Derived from LyeTx I (isolated from the venom of Lycosa erythrognatha spider
• Broad-spectrum and high activity
(Santos et al., 2010; Reis et al., 2018)

However, it shows high cytotoxicity and hemolysis

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PEGylation
• Mask the protein surface by covalent coupling of soluble
poly(ethylene glycol) (PEG)

Prolong the residence time of drugs

Reduces toxicity LyeTxl-b  LyeTxI-bPEG

Reduces immunogenicity

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 AIM
Evaluate the toxicity and effectiveness of LyeTxI-bPEG against CRAB,
both in vitro and in vivo

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• LyeTx I-bPEG Modification

• In Vitro Antimicrobial Activity

• In Vivo Effect (CRAB pneumonia model)

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LyeTx I-bPEG Modification
LyeTx I-bcys
PEG -- Maleimide

Succinimide ring
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CD spectra of
LyeTx I-bPEG
α-helix

α-helix
α-helix

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In vitro antimicrobial activity
• MIC/MBC
• Efficacy (Time-kill curve)
• Efficacy against mature biofilm
• Synergism assay (with conventional antibiotics)
• Stability
• Multi-step resistance
• Cytotoxicity (HEK-293) & Hemolytic (HBC)

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 0.5 2
| |
8 16

PEG-form was slightly less active than Lye but it still performed some antibacterial effects.
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Time-kill curve of peptides and colistin with CRAB

Microbial load: 1x106 CFU/mL

5X MIC 3X MIC

LyeTx I-b <1h 3h

3 LyeTx I- 3h 6h
bPEG
3
Colistin 6h

PEG-form has maintained the fast and efficient

microbicide effect of this analogue.

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Efficacy against biofilm of peptides and colistin with CRAB

improved the anti-biofilm activity of LyeTx I-b AMP anti-biofilm effects are more stable
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Synergism assay
FICI ≤ 0.5 (synergic)
0.5 < FICI ≤ 1 (additive)
1 < FICI ≤ 4 (indifferent)

PEGylation did not influence the synergistic effect of the peptide when combined
with conventional antibiotics.

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 Reduced
antibacterial
activity

Stable
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Potential for resistance induction in the multi-step resistance test

LyeTx I-b: 1 8

Colistin: 1 4

PEGylation increases antibiotic’s stability against microbial resistance 19

In vitro toxicity In vivo toxicity
toxicity

toxicity
mortality

Intravenous (tail vein), n=3 (3 mice per conc.),

each circle represents a tested mice.
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In vivo antibacterial activity timeline
8 groups (5 mice each)

108 CFU of CRAB (20 μl –intranasal)

2h

AMP treatments (intravenous –tail vein)

24h
Lung macerated and plated on nutrient agar

24h, 37 〫 C

CFU/Lung
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In vivo antibacterial activity of LyeTx I-b and LyeTx I-bPEG on pulmonary
infection caused by CRAB in mouse.
Loss of activity in vivo
Colistin

Colistin

Reduced bacterial load

“*” indicate not statistically different from untreated control group, “**” not statistically different from CMC,
“***” not statistically different from LyeTx I-bPEG, at concentrations of 0.5, 1 and 2 mg.kg − 1 22
8 groups, n=5 (6 week BALB/c)
Conclusion
In Vitro Antimicrobial Activity (LyeTx I-bPEG)
MIC/MBC
Efficacy (Time-kill curve)
In vivo antimicrobial activity
≈
Efficacy against mature biofilm
Cytotoxicity
Synergism assay (with conventional antibiotics) Lung bacterial load
≈
Stability Stable in plasma
Multi-step resistance PEGylation was efficient in maintaining the
Cytotoxicity (HEK-293) peptide activity through the intravenous
route.

The above parameters suggest that LyeTx I-bPEG may constitute a good model of a new
antibiotic against CRAB, to be used alone or in combination with classic drugs.
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