RESUMO
Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Argâ¯=â¯R). Initially, a resin-bound Arg/Pro-rich fluoren-9-yl-methyloxycarbonyl-protected fragment (Fmoc-RPRPPR) of the AMP oncocin (i.e., VDKPPYLPRPRPPRRIYNR-NH2) was employed in a comparative on-resin assessment of commercial guanidinylation reagents head-to-head with the recently studied bis-Boc-protected triazole-based reagent, 1H-triazole-1-[N,N'-bis(tert-butoxycarbonyl)]-carboxamidine, which was synthesized by a chromatography-free procedure. This reagent was found to enable quantitative conversion in solid-phase peptide synthesis (SPPS) of peptides displaying homoarginine (Har) residues and/or an N-terminal guanidinium group. SPPS was used to obtain analogues of the 18-mer oncocin with single as well as multiple Argâ¯ââ¯Har modifications. In addition, the effect of replacement of proline (Pro) residues in oncocin was explored by incorporating single or multiple trans-4-hydroxy-l-proline (Hyp) or 4,4-difluoro-l-proline (Dfp) residues, which both affected hydrophobicity. The resulting peptide library was tested against both Gram-negative and Gram-positive bacteria. Analysis of the minimal inhibitory concentrations (MICs) showed that analogues, displaying modifications at positions 4, 5 and 12 (originally Pro residues), had retained or slightly improved antimicrobial activity. Next, an oncocin analogue with two stabilizing l-Argâ¯ââ¯d-Arg replacements in the C-terminal part was further modified by triple-replacement of Pro by either Dfp or Hyp in positions 4, 5, and 12. The resulting analogue displaying three Proâ¯ââ¯Dfp modifications proved to possess the best activity profile: MICs of 1-2⯵g/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800⯵g/mL, and an IC50 above 1280⯵g/mL in HepG2 cells. Thus, incorporation of bis-fluorinated Pro residues appears to constitute a novel tool in structure-activity studies aimed at optimization of Pro-rich AMPs.
Assuntos
Escherichia coli , Homoarginina , Hidroxiprolina/farmacologia , Homoarginina/farmacologia , Guanidina/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Triazóis/farmacologiaRESUMO
C4 -substituted fluoroprolines (4R)-fluoroproline ((4R)-Flp) and (4S)-fluoroproline ((4S)-Flp) have been used in protein engineering to enhance the thermodynamic stability of peptides and proteins. The electron-withdrawing effect of fluorine can bias the pucker of the pyrrolidine ring, influence the conformational preference of the preceding peptide bond, and can accelerate the cis/trans prolyl peptide bond isomerisation by diminishing its double bond character. The role of 4,4-difluoroproline (Dfp) in the acceleration of the refolding rate of globular proteins bearing a proline (Pro) residue in the cis conformation in the native state remains elusive. Moreover, the impact of Dfp on the thermodynamic stability and bioactivity of globular proteins has been seldom described. In this study, we show that the incorporation of Dfp caused a redox state dependent and position dependent destabilisation of the thioredoxin (Trx) fold, while the catalytic activities of the modified proteins remained unchanged. The Pro to Dfp substitution at the conserved cisPro76 in the thioredoxin variant Trx1P did not elicited acceleration of the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond. Our results show that pucker preferences in the context of a tertiary structure could play a major role in protein folding, thus overtaking the rules determined for cis/trans isomerisation barriers determined in model peptides.