Antibiotic-Based Conjugates Containing Antimicrobial HLopt2 Peptide: Design, Synthesis, Antimicrobial and Cytotoxic Activities

Abstract

Recent studies have shown that modified human lactoferrin 20–31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain–heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity

“…The antifungal activity and mammalian cytotoxicity of redox-sensitive [10] 3 and 5 were very similar to that of their redox-resistant analogues 2 and 4. On the other hand, the activity of cleavable 3 against S. epidermidis, P. aeruginosa, and especially against E. coli was better than that of non-cleavable 2.…”

“…They corresponded to intact conjugate (m/z 2685.56), HS-(CH 2 ) 2 CO-TP10-NH 2 (m/z 2268.41), its dimer (m/z 4534.86), and CIP-CO(CH 2 ) 2 -SH (m/z 420.14; [M+H] + ) ( Figure S1). Unlike antimicrobial peptide HLopt2, which was quickly further degraded [10], TP10-NH 2 was resistant inside the bacterial cells. The identified dimer of HS-(CH 2 ) 2 CO-TP10-NH 2 is a product of peptide oxidation inside the MS spectrometer.…”

“…In our recent paper [10], we showed that ciprofloxacin conjugated through a disulfide bridge with the antimicrobial peptide named HLopt2 entered microbial cells. After 2 h of incubation, in 2/3 of the conjugate molecules that entered microbial cells, the linker was reduced but remained intact when incubated for 2 h with supernatant obtained from S. aureus culture.…”

“…The only example of similar derivatisation of fluoroquinolones was enrofloxacin and ciprofloxacin conjugates with β-octaarginine, showing a similar or lower in vitro antibacterial potential than the mother chemotherapeutics [ 8 ]. On the other hand, in our previous studies, we demonstrated some beneficial results of the conjugation of CIP and LVX with antimicrobial peptides (AMPs), namely the lactoferricin truncated analogues [ 9 ] and lactoferrin HLopt2 fragment [ 10 ].…”

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy

“…was >250 mg/L, but when tested in BHI diluted 1:100 the minimum microbicidal concentrations (MMC) were 2–31 mg/L. Similarly, the MIC in CA-MH vs. P. aeruginosa and S. aureus was much higher (125 and 63 mg/L, respectively) than the MMC when tested in 1:100-diluted BHI (4 and 2 mg/L, respectively) (Ptaszynska et al, 2019 ). In some cases, the choice of media type can have detrimental effects on AMP activity.…”