Structure-activity study of oncocin: On-resin guanidinylation and incorporation of homoarginine, 4-hydroxyproline or 4,4-difluoroproline residues.

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.

Macrocyclic Peptidomimetic Plasmepsin X Inhibitors with Potent In Vitro and In Vivo Antimalarial Activity.

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

Peptide/ß-Peptoid Hybrids with Ultrashort PEG-Like Moieties: Effects on Hydrophobicity, Antibacterial Activity and Hemolytic Properties.

PEGylation of antimicrobial peptides as a shielding tool that increases stability toward proteolytic degradation typically leads to concomitant loss of activity, whereas incorporation of ultrashort PEG-like amino acids (sPEGs) remains essentially unexplored. Here, modification of a peptide/ß-peptoid hybrid with sPEGs was examined with respect to influence on hydrophobicity, antibacterial activity and effect on viability of mammalian cells for a set of 18 oligomers. Intriguingly, the degree of sPEG modification did not significantly affect hydrophobicity as measured by retention in reverse-phase HPLC. Antibacterial activity against both wild-type and drug-resistant strains of Escherichia coli and Acinetobacter baumannii (both Gram-negative pathogens) was retained or slightly improved (MICs in the range 2-16 µg/mL equal to 0.7-5.2 µM). All compounds in the series exhibited less than 10% hemolysis at 400 µg/mL. While the number of sPEG moieties appeared not to be clearly correlated with hemolytic activity, a trend toward slightly increased hemolytic activity was observed for analogues displaying the longest sPEGs. In contrast, within a subseries the viability of HepG2 liver cells was least affected by analogues displaying the longer sPEGs (with IC50 values of ~1280 µg/mL) as compared to most other analogues and the parent peptidomimetic (IC50 values in the range 330-800 µg/mL).