TY - JOUR

T1 - Structure-activity study of oncocin

T2 - On-resin guanidinylation and incorporation of homoarginine, 4-hydroxyproline or 4,4-difluoroproline residues

AU - Shaikh, Ashif Y.

AU - Björkling, Fredrik

AU - Zabicka, Dorota

AU - Tomczak, Magdalena

AU - Urbas, Malgorzata

AU - Domraceva, Ilona

AU - Kreicberga, Agrita

AU - Franzyk, Henrik

N1 - Publisher Copyright: © 2023 The Authors

PY - 2023

Y1 - 2023

N2 - Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = 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 Arg → 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-Arg → 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 Pro → Dfp modifications proved to possess the best activity profile: MICs of 1–2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC50 above 1280 µg/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.

AB - Antimicrobial peptides (AMPs) often display guanidinium functionalities, and hence robust synthetic procedures are needed to facilitate access to analogues with unnatural homologues of arginine (Arg = 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 Arg → 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-Arg → 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 Pro → Dfp modifications proved to possess the best activity profile: MICs of 1–2 µg/mL against E. coli and Klebsiella pneumoniae, less than 1% hemolysis at 800 µg/mL, and an IC50 above 1280 µg/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.

KW - 4,4-Difluoroproline

KW - Antibacterial activity

KW - On-resin guanidinylation

KW - Oncocin

KW - Stability to enzymes

KW - Structure-activity relationships

KW - Trans-4-Hydroxy-L-proline

U2 - 10.1016/j.bioorg.2023.106876

DO - 10.1016/j.bioorg.2023.106876

M3 - Journal article

C2 - 37797458

AN - SCOPUS:85173093853

VL - 141

JO - Bioorganic Chemistry

JF - Bioorganic Chemistry

SN - 0045-2068

M1 - 106876

ER -