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

Abstract

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

A Novel Proline-Rich Cathelicidin from the Alpaca Vicugna pacos with Potency to Combat Antibiotic-Resistant Bacteria: Mechanism of Action and the Functional Role of the C-Terminal Region

“…Last decade, it was becoming apparent that bacteria can evolve resistance to AMPs, although specific mechanisms of bacteria-killing by cationic AMPs are much more favorable than those of conventional antibiotics to prevent resistance evolution [ 41 ]. In particular, PrAMPs can interact with several conservative targets within bacterial cells like ribosomes [ 4 ] or DnaK [ 42 ], and therefore the probability of the spontaneous resistance emergence concerned with such targets might be rather low. On the other hand, resistance to PrAMPs may arise as a result of the inactivation of transporter proteins like SbmA or MdtM, which was shown for a number of relatively short peptides.…”

Evaluation of proline-rich antimicrobial peptides as potential lead structures for novel antimycotics against Cryptococcus neoformans

“…All samples were corrected for the background absorbance of the medium. Relative cell viability was calculated as the ratio of absorbances recorded for treated and untreated cells ( Kolano et al, 2021 ; Brakel et al, 2022a , b ).…”

“…Short PrAMPs were originally isolated from insects, such as the milkweed bug ( Oncopeltus fasciatus ) or the honeybee ( Apis mellifera ), and further optimized for therapeutic applications by improving their activity against various bacteria and their protease (serum) stability ( Knappe et al, 2010 ; Berthold et al, 2013 ; Knappe et al, 2016 ). Synthetic derivatives such as Api137, Onc112, or Chex1Arg20 bind to the chaperone DnaK and the 70S ribosome after active transport into the cell, thereby inhibiting protein folding, and translation during protein biosynthesis ( Krizsan et al, 2014 , 2015a , b ; Brakel et al, 2022a , b ). PrAMPs have proven to be valid lead compounds due to their high efficacy in murine infection models and their low adverse effects in animals, all of which can be attributed to their bacteria-specific uptake and targeting of the bacterial ribosome ( Holfeld et al, 2015 ; Schmidt et al, 2017 ; Brakel et al, 2019 ).…”

Antimicrobial Mechanisms and Clinical Application Prospects of Antimicrobial Peptides

“…Class I PrAMPs, such as Bac7, Onc112, pyrrhocoricin, and metalnikowin, interfere with the initial step of translation to block the peptide transferase center and the peptide exit channel of the ribosome [128], whereas class II PrAMPs, such as apidaecin 1b and Api137, act during translation termination and inhibit protein synthesis by trapping release factors on the 70S ribosome following hydrolysis of the nascent polypeptide chain [129]. There are quite a few recent studies on PrAMPs, which attempt to enhance the antibacterial activity of PrAMPs against bacteria, even drug-resistant bacteria, through de novo synthesis, chemical modification and other methods [130][131][132]. A recent study showed that the PrAMP dimers produced by the bifunctional linker significantly enhanced their antibacterial and anti-biofilm activities against a variety of Gram-negative bacilli, including drug-resistant Acinetobacter baumannii strains [132].…”