The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring
short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone
DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-
DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the
binding motif and characterize the influence of the chaperone on protein refolding activity,
especially in stress situations. In continuation of this research, 182 analogs of ARV-1502
were designed by substituting residues involved in antimicrobial activity against Gramnegative
pathogens. The peptides synthesized on solid-phase were examined for their
binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding
characteristics for at least one DnaK. These 15 analogs were distinguished from the
original sequence by their increased hydrophobicity parameters. Additionally, the influence
of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on
refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides
showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing
protein refolding by up to 64%. However, these more hydrophobic peptides had only a
minor effect on the ATPase activity. The most dramatic changes on the ATPase activity
involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a
59% reduction of the ATPase activity in the E. coli chaperone system whereas they
stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the
improvement of the antimicrobial activity against S. aureus from originally >128 μg/mL to
as low as 16 μg/mL. Only a single analog exhibited improved activity over the original value
of 8 μg/mL against E. coli. Overall, the various moderate-throughput screenings
established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2)
the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with
co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:84529 |
| Date | 03 April 2023 |
| Creators | Brakel, Alexandra, Kolano, Lisa, Kraus, Carl N., Otvos Jr, Laszlo, Hoffmann, Ralf |
| Publisher | Frontiers Research Foundation |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:article, info:eu-repo/semantics/article, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 2296-2646, 798006 |
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