Functional Effects of ARV-1502 Analogs Against Bacterial Hsp70 and Implications for Antimicrobial Activity

Abstract

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 Gram-negative 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.

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity

“…A recent study focusing on DnaK binding and inhibition of the ATPase activity of DnaK by ARV-1502 and its analogs could not explain the observed in vitro antibacterial activities. Thus, we broadened the scope of our research and studied the binding to the 70S ribosome isolated from E. coli , which was reported as one of the main intracellular targets of several PrAMPs [ 11 ]. Cf-ARV-1502 bound to the 70S ribosome preparation with a K d of 201 ± 16 nmol/L ( Figure 1 ), which was slightly higher than the K d determined for E. coli DnaK (K d = 140 ± 10 nmol/L).…”

“…Previous studies identified residues D 3 K 4 and Y 8 LPRP 12 as important for DnaK binding. However, substitution of these seven residues with basic (Lys), acidic (Asp), hydrophilic (Ser), aliphatic (Leu), and aromatic canonical amino acids (Phe) showed only minor effects on DnaK binding [ 11 ]. The ATPase activity of DnaK and the chaperone activity of DnaK in a refolding assay using DnaK and co-chaperones were affected, but these data do not correlate to the MIC values obtained for E. coli and S. aureus .…”

“…ARV-1502 binds with residues YLPRP to the nucleotide binding domain of DnaK and thereby restricts the functional activity of DnaK [ 10 ]. Some ARV-1502-derived peptides showed an enhanced binding to DnaK and a better antibacterial activity against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphlyococcus aureus [ 11 ]. As ARV-1502 and other PrAMPs are also active against the DnaK knockout mutant E. coli JW0013 ∆ dnaK , it was suggested that further lethal bacterial targets exist [ 12 , 13 ].…”

“…In general, increasingly active PrAMPs can be obtained by improving the (i) target binding, (ii) bacterial uptake rates, and (iii) protease stability [ 6 , 20 , 21 ]. In vitro screening of substituted ARV-1502 peptides showed that the antimicrobial activity against E. coli can be improved only by replacing the Asp 3 Lys 4 -motif mainly by hydrophobic motifs, such as Phe 3 Phe 4 , which also significantly improves the activity against S. aureus [ 11 ]. It needs to be mentioned that the low antibacterial activity against S. aureus in vitro cannot be correlated with the high efficacy of ARV-1502 against this strain in mouse models, suggesting yet additional modes of action such as immunostimulation of the mammalian host [ 22 ].…”

Evaluation of Potential DnaK Modulating Proline-Rich Antimicrobial Peptides Identified by Computational Screening

“…This aspect was highlighted by a recent publication in which 47 analogues of oncosin were evaluated across several different modes of activity including 70S ribosomal binding, inner and outer membrane transport and antimicrobial activity. The lack of clear correlation between these variables led to the conclusion that several parameters, including additional targets such as DnaK, are in continuous play with respect to ultimate antimicrobial action ( Brakel et al, 2022 ; Kolano et al, 2022 ). Our data confirms this inability to reply solely on one or two in vitro assays for the design and prediction of novel antimicrobial peptide analogues.…”