Combination of Amphiphilic Cyclic Peptide [R4W4] and Levofloxacin against Multidrug-Resistant Bacteria

Abstract

Bacterial resistance is a growing global concern necessitating the discovery and development of antibiotics effective against the drug-resistant bacterial strain. Previously, we reported a cyclic antimicrobial peptide [R₄W₄] containing arginine (R) and tryptophan (W) with a MIC of 2.67 µg/mL (1.95 µM) against methicillin-resistant Staphylococcus aureus (MRSA). Herein, we investigated the cyclic peptides [R₄W₄] or linear (R₄W₄) and their conjugates (covalent or noncovalent) with levofloxacin (Levo) with the intent to improve their potency to target drug-resistant bacteria. The physical mixture of the Levo with the cyclic [R₄W₄] proved to be significantly effective against all strains of bacteria used in the study as compared to covalent conjugation. Furthermore, the checkerboard assay revealed the significant synergistic effect of the peptides against all studied strains except for the wild type S. aureus, in which the partial synergy was observed. The hemolysis assay revealed less cytotoxicity of the physical mixture of the Levo with [R₄W₄] (22%) as compared to [R₄W₄] alone (80%). The linear peptide (R₄W₄) and the cyclic [R₄W₄] demonstrated ~90% and 85% cell viability at 300 µg/mL in the triple-negative breast cancer cells (MDA-MB-231) and the normal kidney cells (HEK-293), respectively. Similar trends were also observed in the cell viability of Levo-conjugates on these cell lines. Furthermore, the time-kill kinetic study of the combination of [R₄W₄] and Levo demonstrate rapid killing action at 4 h for MRSA (ATCC BAA-1556) and 12 h for E. coli (ATCC BAA-2452), P. aeruginosa (ATCC BAA-1744), and K. pneumoniae (ATCC BAA-1705). These results provide the effectiveness of a combination of Levo with cyclic [R₄W₄] peptide, which may provide an opportunity to solve the intriguing puzzle of treating bacterial resistance.

Keywords: antibacterial resistance; checkerboard assay; combination therapy; cyclic antimicrobial peptides; drug-resistant bacteria.

Scheme 1

Synthesis of cyclic peptide-Levo conjugate [Levo-C₁₀-KR₃W₄].

Figure 1

Chemical structures of synthesized peptides and conjugates.

Figure 2

The data represent the percentage hemolysis of the Levo and the peptides (linear and cyclic) alone and their chemical conjugates and physical mixture at 500 µg/mL. The experiment was conducted in triplicate.

Figure 3

Cytotoxicity of peptides and their conjugates in (A) MDA-MB-231 cells and (B) in HEK-293 cells after 48 h incubation.

Figure 4

Time-dependent kill study of the test compounds against drug-resistant bacterial strains of (A) S. aureus (ATCC BAA-1556), (B) E. coli (ATCC BAA-2452), (C) P. aeruginosa (ATCC BAA-1744), and (D) K. pneumoniae (ATCC BAA-1705). The data represent the results of the experiment performed in triplicate.