Replacement of l-Amino Acids by d-Amino Acids in the Antimicrobial Peptide Ranalexin and Its Consequences for Antimicrobial Activity and Biodistribution

Abstract

Infections caused by multidrug-resistant bacteria are a global emerging problem. New antibiotics that rely on innovative modes of action are urgently needed. Ranalexin is a potent antimicrobial peptide (AMP) produced in the skin of the American bullfrog Rana catesbeiana. Despite strong antimicrobial activity against Gram-positive bacteria, ranalexin shows disadvantages such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting exclusively of d-amino acids (named danalexin) was synthesized and compared to the original ranalexin for its antimicrobial potential and its biodistribution properties in a rat model. Danalexin showed improved biodistribution with an extended retention in the organisms of Wistar rats when compared to ranalexin. While ranalexin is rapidly cleared from the body, danalexin is retained primarily in the kidneys. Remarkably, both peptides showed strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria of the genus Acinetobacter with minimum inhibitory concentrations (MICs) between 4 and 16 mg/L (1.9-7.6 µM). Moreover, both peptides showed lower antimicrobial activities with MICs ≥32 mg/L (≥15.2 µM) against further Gram-negative bacteria. The preservation of antimicrobial activity proves that the configuration of the amino acids does not affect the anticipated mechanism of action, namely pore formation.

Keywords: Ranalexin; antibiotics; antimicrobial activity; configuration; peptide therapeutics.

Figure 1

Time-kill curves of ranalexin and danalexin (n = 1). Time-kill curves were determined at 1×, 2× and 4× MIC against E. coli ATCC 25922 and S. aureus ATCC 25923. Ranalexin (red) and danalexin (blue) showed a fast, concentration-dependent mode of action. Both substances were more bactericidal compared to cefuroxime (green).

Figure 1

Time-kill curves of ranalexin and danalexin (n = 1). Time-kill curves were determined at 1×, 2× and 4× MIC against E. coli ATCC 25922 and S. aureus ATCC 25923. Ranalexin (red) and danalexin (blue) showed a fast, concentration-dependent mode of action. Both substances were more bactericidal compared to cefuroxime (green).

Figure 2

Scintigraphic imaging of ranalexin-d-Tyr in rats. Images were recorded 10–20 min, 1 h, 2 h and 3 h post injection into the tail vein of a Wistar rat. The peptide is excreted by the kidneys. At 1 h post injection, the majority of the substance is found in the bladder.

Figure 3

Scintigraphic images of d-Tyr-danalexin. Images were recorded 10–20 min, 1 h, 2 h, 3 h, 5 h and 24 h post injection into the tail vein of a Wistar rat. An accumulation of d-Tyr-danalexin in the kidneys is clearly visible. Even 24 h post injection, radioactivity is still visible in the kidneys.

Figure 4

Positron emission tomography (PET) images of DOTA-d-Tyr-danalexin in a Wistar rat. After injection into the tail vein, distribution in the circulation (as reflected by the perfusion of the heart, kidneys, liver and the blood vessels) is visible. At 20 min after injection, an accumulation in the kidneys is clearly visible. This accumulation in the kidneys remains for at least 3 h. Smaller amounts of DOTA-d-Tyr-danalexin are taken up by the liver.

Figure 5

Standard uptake values (SUVs) of DOTA-d-Tyr-danalexin after injection into the tail vein of a Wistar rat. The SUV is used for the quantification of radioactivity in the individual organs. The accumulation of the substance in the kidneys is clearly visible. Smaller amounts are found in the liver. DOTA-d-Tyr-danalexin is excreted via the bladder.