D-Amino Acid-Containing Lipopeptides Derived from the Lead Peptide BP100 with Activity against Plant Pathogens

Abstract

From a previous collection of lipopeptides derived from BP100, we selected 18 sequences in order to improve their biological profile. In particular, analogues containing a D-amino acid at position 4 were designed, prepared, and tested against plant pathogenic bacteria and fungi. The biological activity of these sequences was compared with that of the corresponding parent lipopeptides with all L-amino acids. In addition, the influence of the length of the hydrophobic chain on the biological activity was evaluated. Interestingly, the incorporation of a D-amino acid into lipopeptides bearing a butanoyl or a hexanoyl chain led to less hemolytic sequences and, in general, that were as active or more active than the corresponding all L-lipopeptides. The best lipopeptides were BP475 and BP485, both incorporating a D-Phe at position 4 and a butanoyl group, with MIC values between 0.8 and 6.2 µM, low hemolysis (0 and 24% at 250 µM, respectively), and low phytotoxicity. Characterization by NMR of the secondary structure of BP475 revealed that the D-Phe at position 4 disrupts the α-helix and that residues 6 to 10 are able to fold in an α-helix. This secondary structure would be responsible for the high antimicrobial activity and low hemolysis of this lipopeptide.

Peptide Conjugates Derived from flg15, Pep13, and PIP1 That Are Active against Plant-Pathogenic Bacteria and Trigger Plant Defense Responses

“…PIP1-BP475 and BP475-PIP1 followed the opposite trend, with the conjugate containing the elicitor peptide PIP1 at the N-terminus being the least hemolytic. In contrast, despite it having been reported that the presence of a d -amino acid decreases the hemolysis ( 46 , 48 ), in the present work this trend has only been observed for conjugates Pep13-BP143 and BP143-Pep13 that are less hemolytic than their l -counterparts.…”

“…The acylation of a peptide sequence is regarded as a means of increasing membrane affinity and, consequently, antimicrobial activity, since the acyl chain may act as a membrane anchor ( 47 , 48 , 73 ). However, in the present study, the incorporation of a fatty acid chain at the side chain of a Lys residue in BP100 did not lead to an improvement of the antibacterial activity.…”

“…In particular, from a 125-member library (CECMEL11), we identified the peptides KKLFKKILKKL-NH 2 (BP16) and KKLFKKILKYL-NH 2 (BP100) ( 45 ). The biological profile of BP100 was optimized by incorporating a d -amino acid and/or a fatty acid chain into its sequence, providing derivatives KKLfKKILKYL-NH 2 (BP143) ( 46 ), Ac-KKLFKKIK(COC 3 H 7 )KYL-NH 2 (BP387) ( 47 ), and Ac-KKLfKKILKK(COC 3 H 7 )L-NH 2 (BP475) ( 48 ). These derivatives displayed improved biological activities in terms of antibacterial activity and hemolysis.…”

Table_1.DOCX

“…The PAC-MBHA resin was employed to prepare C-terminal carboxylic acid peptides whereas the Fmoc-Rink-ChemMatrix and the Fmoc-Rink-MBHA resins served for C-terminal peptide amides. Peptide elongation was carried out through sequential steps of Fmoc removal and coupling of the corresponding amino acid as previously described (Caravaca-Fuentes et al, 2021;Oliveras et al, 2021). Once the peptide sequence was completed, each resulting peptidyl resin was treated with trifluoroacetic acid (TFA)/H 2 O/triisopropylsilane (TIS) (95:2.5:2.5).…”

“…The new sequences were designed by reducing the peptide length [magainin 2(1-10)], replacing the Trp residues by D-Phe (BP525), incorporating an acyl group (BP526 to BP529), preparing the amidated C-terminal analogs (RR2-NH 2 , RR3-NH 2 , and RR4-NH 2 ), replacing D-amino acids by their L-enantiomers (RJK2), or replacing a Lys by an Arg (AamAP-R). These modifications have been reported to increase the antimicrobial activity of peptides (Guell et al, 2011;Cutrona et al, 2015;Vasilchenko et al, 2017;Oliveras et al, 2021).…”