Levofloxacin (LVX) and ciprofloxacin (CIP) are broad-spectrum fluoroquinolone synthetic chemotherapeutics, active against both Gram-positive and Gram-negative bacteria. LVX and CIP are used worldwide to treat a number of bacterial infections, including sinusitis, pneumonia, urinary tract infections, tuberculosis, and meningitis. The antimicrobial spectrum of fluoroquinolones is limited to bacteria and does not include human pathogenic eukaryotic microorganisms [1
]. Their mammalian toxicity is generally low. Both are on the WHO List of Essential Medicines containing the most effective and safe medicines needed in the health system [2
]. Like all fluoroquinolones, LVX, and CIP function by inhibiting bacterial gyrase and topoisomerase IV [3
]. Resistance to fluoroquinolones, which is common in staphylococci, enterococci, and Pseudomonas
spp., occurs in multiple ways, including alterations in DNA gyrase subunit A, topoisomerase IV, as well as overexpression of multidrug-resistance (MDR) efflux pumps [4
One of the possibilities to alter the biological properties of chemotherapeutic agents is their chemical modification, including conjugation with oligopeptides, especially those known as the cell-penetrating peptides (CPPs). Such conjugates may possess additional advantages, including an increased selectivity of drug delivery, enhanced efficacy, reduced systemic toxicity, as well as improved pharmacokinetics and pharmacodynamics. Several examples of CPP:drug conjugates have been reported, although most of them concern those of CPPs with anticancer agents [5
]. Among very few CPP:antimicrobial agent congeners, those worth mentioning are conjugates of arginine oligomers with triclosan exhibiting promising chemotherapeutic effect in the murine model of toxoplasmosis [6
] and peptide-vancomycin conjugates demonstrating enhanced effectiveness against vancomycin-resistant Acinetobacter baumannii
and Enterococcus faecalis
]. 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
Cell-penetrating peptides (CPPs), serving as nanocarriers, are considered the fundamental part of a new concept of drug delivery systems [11
], which has attacted great interest from many research groups. The unique ability of CPPs to penetrate the plasma membrane makes them a useful tool for the delivery of a vast range of different biologically active compounds to eukaryotic and prokaryotic cells, including proteins, nucleic acids, oligonucleotides, liposomes, nanoparticles, peptides, and low molecular weight chemotherapeutic agents (e.g., doxorubicin, methotrexate, cyclosporine A, paclitaxel [12
], and antibiotics [13
]). Since the discovery in 1988 of the first cell-penetrating peptide, trans-activator protein (TAT) [14
], more than 1700 CPPs have been described [16
]. One of the well-known CPPs is transportan (TP), a chimeric oligopeptide composed of the first 12 amino acid residues of the neuropeptide galanin and the 14-amino-acid-residue-long wasp venom peptide, mastoparan, connected via a lysine residue [17
]. A shorter variant of TP, with deletion of the N
-terminal hexapeptide, named TP10-NH2
, having an amide moiety at the C
-terminus, retains the efficient cell penetration property of the parent compound, with significantly less potential side effects [18
]. This peptide amide enters all cell types, including the mammalian ones, and inhibits the growth of some microorganisms, such as Candida albicans, Staphylococcus aureus
], Streptococcus pneumoniae
, and Mycobacterium tuberculosis
can transport cargo across cell membranes, applying different mechanisms, dependent on the size and character of the vectors delivered [21
]. Recently, Rusiecka and co-workers showed that TP10-NH2
improves the cytotoxic activity of cisplatin against cancer cell lines. Unconjugated TP10-NH2
induces a cytostatic effect on HeLa (cervical cancer) and OS143B (osteosarcoma) cancer cell lines. At the same time, TP10-NH2
, as well as TP10-NH2
in complex with cisplatin, had no impact on the cell viability of two non-cancer cell lines HEK 293 (embryonic kidney) and HEL 299 (lung fibroblasts) [22
may thus not only facilitate the transport of an active agent across the microbial cell membranes, but, having an intrinsic antimicrobial activity, it may also potentiate an antimicrobial efficacy of any conjugated antibiotic.
Taking into consideration the literature data mentioned above, we decided to design, synthesise, and determine the antimicrobial activity of conjugates composed of TP10-NH2 and levofloxacin or ciprofloxacin. The series of synthesised compounds consisted of five CIP- and two LVX-based conjugates. The conjugation did not improve the antibacterial activity of the mother fluoroquinolones on average. Surprisingly though, they gained activity against human pathogenic yeasts.
3. Materials and Methods
3.1. Solid-Phase Peptide Synthesis (SPPS)
Peptides were synthesised by a solid-phase approach using the Fmoc/Boct chemistry at 50 μmole scale, applying a Prelude peptide synthesiser (Gyros Protein Technology, Inc., USA). A TentaGel S RAM resin (substitution 0.24 meq/g, Rapp Polymere, Germany) was used as a solid support, yielding, after cleavage, peptides with amide on their C-termini. Peptide chains were elongated in the consecutive cycles of deprotection and coupling. Deprotection was performed with 20% piperidine in N,N-dimethylformamide (DMF) and the peptide chain elongation was affected using a reaction mixture composed of N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uroniumtetrafluoroborate (TBTU), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM), and a three-fold molar excess of each N-α-Fmoc protected amino acid derivative (GL Biochem, Shanghai, China). After completing the synthesis, peptides were cleaved from the resin and the protecting groups were removed in a one-step procedure using a mixture of TFA:phenol:triisopropylsilane:H2O (88:5:2:5, v/v/v/v). The crude peptides were purified on Beckman Gold System (Beckman, Miami, FL, USA) equipped with an RP Supelco Discovery BIO, Wide Pore C8, 10 mm column (10 × 250 mm, Sigma Aldrich, St. Louis, MO, USA) or by PLC 2050 Gilson HPLC with Gilson Glider Prep. Software (Gilson, France), equipped with Grace Vydac C18 (218TP) HPLC column (22 × 250 mm, 10 µm, 300 Å, Resolution Systems). The solvent systems were 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in 80% acetonitrile in water (B). Different linear gradients were applied (flow rate 5.6 or 20 mLmin−1, monitored at 226 nm). The purities of the synthesised peptides were checked with an HPLC Pro Star system (Varian, Australia) and use of a Kinetex 5 μm XB-C18 100 Å column (4.6 × 150 mm, Phenomenex®, Torrance, CA, USA). The solvent system was as described above. A linear gradient from 10% to 90% B for 40 min, flow rate 1 mLmin−1, monitored at 226 nm was used. All described peptides had purities of at least 95%. In order to confirm the correctness of the molecular masses of the synthesised peptides, mass spectrometry analysis was carried out by a MALDI-TOF/TOF, Autoflex MAX spectrometer, (Bruker, Billerica, MA, USA) with an α-cyano-4-hydroxycinnamic acid (CCA) and/or 2,5-dihydroxybenzoic acid (DHB) matrix.
3.2. Synthesis of Levofloxacin and Ciprofloxacin-Based Conjugates
In the case of CIP-TP10-NH2
) and LVX-TP10-NH2
), Boc-CIP (obtained by the reaction of CIP with di-tert-butyldicarbonate in the presence of NaOH) and LVX were manually attached to the peptidyl-resin. An equimolar mixture of N
′-diisopropylcarbodiimide (DIPCI), N
′-diisopropylethylamine, and LVX or Boc-CIP (3 equiv. each) were dissolved in DMF/DCM (v
; 1/1) and added to the SPPS vessel with peptidyl-resin and allowed to stir for 90 min. The procedure was repeated until the chloranil test gave a negative result. In order to obtain CIP-CH2
), ciprofloxacin was coupled to the peptide via the submonomeric approach as described previously [36
]. After assembling the peptide chain and the removal of the Fmoc group, bromoacetic acid (5 equiv.) was attached to the peptidyl-resin using DIPCI (5 equiv.) in DMF. The procedure was repeated twice. Next, 1.5 equiv. of both ciprofloxacin and triethylamine in DCM/DMF was used to attach the constituent antimicrobial. The reaction mixture was stirring for 24 h at room temperature. In the case of CIP-S-S-TP10-NH2
), a solution of Lomant’s reagent (3,3′-dithiodipropionic acid di(N
-hydroxysuccinimide ester))–DSP (1.2 equiv.) was dissolved in DMF, added to the SPPS vessel with peptidyl resin, and shaken overnight. The procedure was repeated twice. Ciprofloxacin and triethylamine (1.5 equiv. each) were dissolved in DMF/DCM and added to peptidyl-resin with coupled DSP. The reaction mixture was stirred for another 24 h at room temperature. All conjugates were detached from the solid support together with the removal of side-chain protection groups and purified as described above. To obtain LVXC-S-S-CTP10-NH2
), the disulfide bridge between LVX-Cys (described below in Section 3.2.1
) and CTP10-NH2
was formed. Briefly, CTP10-NH2
(46 mg, 0.02 mmol) was dissolved in 5 mL of DMF and LVX-Cys(Npys) (12 mg, 0.02 mmol) was added. The mixture was stirred for 4 h at room temperature. The reaction was monitored by analytical HPLC. After 4 h, the solvent was removed in vacuo, and the conjugate was purified by semi-preparative HPLC; yield 20%; the product was characterised by HPLC and MALDI-TOF MS.
In the case of CIPC-S-S-CTP10-NH2 (6)
, the disulfide bridge was formed during the reaction of CTP10-NH2
and Cys(Npys)-CIP (see below Section 3.2.2
.) as described above for 5
. The total yield was 19 %. The product was characterised by HPLC and MALDI-TOF MS.
3.2.1. Synthesis of LVX-Cys(Npys)
LVX-Cys(Npys) was obtained in two steps. Firstly, levofloxacin-N-hydroxysuccinimidyl ester (LVX-NHS) was synthesised. The esterification reaction between the carboxylic group of LVX (119 mg, 0.33 mmol) and the hydroxyl group of N-hydroxysuccinimide (38 mg, 0.33 mmol) was carried out using N,N′-dicyclohexylcarbodiimide (DCC, 68 mg, 0.33 mmol) in DMF (6 mL). The solution was stirred overnight, and then DMF was evaporated under vacuum. The obtained residue was extracted with 10 mL of ethyl acetate and washed five times with water (5mL, portion each time) and twice with ice-cold saturated NaHCO3 (5 mL). Then, the washed extract was concentrated under vacuum and left overnight to crystallise at room temperature. The product isolated by recrystallisation in EtOH gave LVX-NHS in a 65% yield. In the next step, Cys(Npys) (34 mg, 0.12 mmol) and DIPEA (21 µL, 0.12 mmol) dissolved in 5 mL of DMF were added to the solution of LVX-NHS (55 mg, 0.12 mmol) (pH was adjusted to 8-9 by NaOH). The reaction mixture was stirred overnight. The solvent was removed in vacuo and the product was treated several times with ether to wash out the unreacted ester. The remaining material was dissolved in water and lyophilised (yield 83%, 89 mg).
3.2.2. Synthesis of Cys(Npys)-CIP
Boc-Cys(Npys)-OSu was obtained in the reaction of Boc-Cys(Npys)-OH with N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide (DCC) as the coupling reagent (see above). In the next step, CIP (66 mg, 0.2 mmol) in 5 mL of DMF/DCM (3/2; v/v) was added to the solution of Boc-Cys(Npys)-OSu (94 mg, 0.2 mmol) in 3 mL of DMF. The reaction mixture was stirred overnight. The solvent was removed in vacuo, and the product was treated several times with ether (containing 10% DCM) to wash out the unreacted ester. The Boc-protecting group was removed by TFA (5 mL, 15 min at room temperature). The solution was concentrated in vacuo, and then the remaining material was dissolved in water and lyophilised (yield 92%, 128 mg).
Synthesis of fluorescently labelled compounds
In order to obtain fluorescein-labelled ciprofloxacin, 5(6)-carboxyfluorescein N-succinimidyl ester was prepared. The solution of 5(6)-carboxyfluorescein and N-hydroxysuccinimide (0.2 mmol each) was stirred in the presence of N,N′-dicyclohexylcarbodiimide (0.2 mmol) in DMF/DCM, first for 15 min at 0 °C and then for 3 h at RT. In the next step, solvents were evaporated in vacuo, and the residue was crystallised from methanol. The yellowish precipitate was drained over reduced pressure and washed with methanol (yield 76%). Then, 5(6)-carboxyfluorescein N-succinimidyl ester and ciprofloxacin (0.05 mmol each) were suspended in DMF/DCM and allowed to stir overnight. The yellow precipitate was filtrated over reduced pressure, suspended in water, lyophilised, and analysed as described above (yield 87%).
3.3. Synthesis of Fluorescently Labelled Compounds
In order to attach fluoresceine (Cf) to TP10-NH2, an analogue of TP10-NH2 containing Lys(Mtt) (Mtt stands for 4-methyltrityl) in position 12 instead of Ala was synthesised on a solid phase. After completing the synthesis, the Mtt protection of an ε-amino group of Lys was removed by 3-min incubations in 1.8% TFA in DCM until reaction completion, indicated by the disappearance of the yellow color (Mtt cation) in the drained deprotection solution. Then, the fluorophore was attached manually using 3 equiv. of 5(6)-carboxyfluorescein (Novabiochem, Merck, Darmstadt, Germany), HATU, HOAt, and DIPEA (molar ratio 1:1:1:2). The procedure was repeated three times. Next, TP10(Cf)-NH2 was cleaved from half of the peptidyl-resin, and the crude peptide was purified as described above. The other half of the peptidyl-resin was used to obtain the labelled CIP-CH2CO-TP10(Cf)-NH2 (7). The synthesis was performed by manual attachment of bromoacetic acid to the peptidyl-resin, and then CIP was coupled via the sub-monomeric approach. Finally, the labelled conjugate was removed from the solid support. The crude product was purified by semi-preparative HPLC, and characterised by HPLC and MS analysis.
3.4. Microorganism Strains and Growth Conditions
The following strains were used for antimicrobial activity tests: C. albicans
ATCC 10231, C. albicans
SC5314, C. glabrata
DSM 11226, C. krusei
DSM 6128, C. albicans
ATCC 25922, P. aeruginosa
ATCC 27853, S. aureus
ATCC 29213, and S. epidermidis
ATCC 12228. C. albicans
Gu4, and Gu5 clinical isolates [26
] were gifts from Joachim Morschhäuser, Würzburg, Germany. The yeast strains were maintained on YPD agar plates (1% (w
) yeast extract, 2% (w
) peptone, 2% (w
) glucose, % (w
) agar) in 30 °C and bacterial strains on LA (Luria-Bertani agar; 1% (w
) tryptone, 1 % (w
) NaCl, 0.% (w
) yeast extract, 2% agar) plates in 37 °C for 16–24 h.
3.5. Stability Testing
Cell culture of S. aureus ATCC 25,923 was refreshed on solid LA overnight (37 °C). The next day, cell suspensions were prepared in LB liquid medium, and after about 2 to 3 h (logarithmic growth phase), CIP-S-S-TP10-NH2 (3) was added at concentrations of 16 μgmL−1 in a volume of 1 mL. The cultures were continued overnight (37 °C, 180 rpm). The next day, 1 mL of cell cultures was transferred to 2-mL tubes and centrifuged (12,000 rpm, 1 min, 4 °C) (from this moment, the samples were kept on ice). The supernatant was discarded and the pellet was suspended in 0.5 mL of PBS. Zirconia beads (A&A Biotechnology, Gdynia, Poland) and 350 μL of lysis buffer (Bioline) were added to the samples. The tubes were shaken on a BeadBeater device to break the cell wall (3 × 20 s). The suspension was transferred to new tubes and 8.5 μL of Protease Inhibitor Cocktail (100 × concentrated, Fermentas, Waltham, MA, USA) was added. The tubes were centrifuged (12,000 rpm, 20 min, 4°C). The supernatant was transferred to new tubes and stored at −20 °C until the LC-MS analysis. Before MS analyses, the samples were desalted using ZipTip® C18 Pipette Tips (Merck Millipore Ltd., Burlington, MA, USA) according to the manufacturer’s protocol. LC-MS experiments were performed on a Qtof lcms9030 spectrometer (Shimadzu, Japan) equipped with an electropress ion source, LC Nexera X2 module with autosampler. Samples were dissolved in water and analysed on an XB C18 Aeris Peptide column (Phenomenex) o (100 mm × 2.1 mm); 3.6 μm bead diameter. The LC system was operated with mobile phase: solvent A: 0.1% formic acid in H2O and solvent B: 0.1% formic acid in MeCN. Samples were separated with a linear gradient (optimised for the best separation of the analysed samples), maintained at a flow rate of 0.2 mLmin−1. The injection volume was between 0.1 and 0.5 μL.
3.6. Antibacterial Activity Assay
The antibacterial activity (bacteriostatic: MIC and bactericidal: MBC) of the tested compounds was determined in MHB (Mueller Hinton Broth, Sigma Aldrich, St. Louis, MO, USA) using a serial two-fold dilution method in 96-well microtiter plates according to CLSI recommendations, described in M07-A10 document. The exact conditions have been described previously [9
]. All experiments were performed in three replicates. The MIC90
values were defined as the concentrations of compounds tested that caused a 90% or 50% reduction of growth, respectively, comparing to the drug-free control.
3.7. Determination of Resistance Induction Potential
ATCC 25,922 or S. aureus
ATCC 29,213 cells were taken from −80 °C and freshly grown on Mueller Hinton Agar (MHA) plates. The MIC values for CIP and CIP-S-S-TP10-NH2
) were determined. The cells (108
CFU) were grown on MHA plates containing one of the tested compounds at several concentrations equal or slightly higher than the MIC values determined as described in Section 2.7
. The highest concentration at which the cells were able to grow (MIC-108
) was selected for the rest of the experiment. Cells (108
cells per plate) were incubated on MHA plates in the presence or absence (control) of the tested compound at MIC-108
and passaged 10 times at 1- to 3-day intervals. MHA plates with the addition of the tested compound were freshly prepared every time before the passage. After the 5th and the 10th passage, cells from colonies grown on the plates were collected for determination of MIC values.
3.8. Antifungal Activity Assay
The antifungal activity of the tested compounds was assessed in 96-well microtiter plates, in buffered RPMI-1640 medium according to the CLSI recommendations (M27-A3 document, Clinical Laboratory Standards Institute 2008). The lowest concentration that prevented the growth of microorganisms was assigned as MIC, while the lowest drug concentration that caused 90% or 50% inhibition of growth compared to the drug-free control was assigned as MIC50 and MIC50, respectively. All experiments were performed in three replicates.
3.9. Cytotoxicity Assay
Cytotoxicity was assessed against three mammalian cell lines: LLC-PK1, Hep G2, and HEK 293. Multiwell (96–well) plates were seeded at 7000 cells/well in Medium 199 supplemented with 7.5% FBS, MEM Eagle’s medium supplemented with 10% FBS, or DMEM medium supplemented with 10% FBS, respectively. All media were supplemented with L-glutamine and antibiotics (penicillin/streptomycin). Cells were allowed to attach overnight. Drugs were dissolved in cell culture medium and added to wells in 100-µL aliquots of 2× concentrated solutions, in triplicates. To control wells, 100 µL of the medium were added. Cells were incubated with the studied compounds for 72 h at 37 °C and 95%/5% or 90%/10% (HEK 293 cells) CO2 atmosphere. Then, 20-µL aliquots of MTT solution in PBS (4 mg/mL) were added to all wells and plates were incubated further for 3 h in 37 °C. Formazan crystals formed were dissolved in 100 µL of DMSO and the absorbance of the solutions was measured using a multiwell plate reader (Spark, Tecan, Männedorf, Switzerland) at λ = 540 nm. Cytotoxicity was determined compared to the drug-free control. All experiments were performed in triplicates.
3.10. Measurement of ROS Generation
ROS generation activity was evaluated as described previously [37
]. Briefly, HEK 293 cells were seeded on 35-mm Petri dishes in the amount of 15,000 cells per plate for 24 h in DMEM medium. Compounds were added at final concentrations: CIP-TP10-NH2
) 5 µM, and CIP 500 µM. HL-60 cells were seeded on 35-mm Petri dishes in the amount of 25,000 cells/5 mL in RPMI 1640 medium and compounds were added at final concentrations: CIP-TP10-NH2
) 2.5 μM, and CIP 100 µM. Positive controls contained 250 µM H2
, while negative controls were without any additions. After 0.5, 2.5, 5.5, and 24 h of incubation at 37 °C, the CM-H2DCFDA molecular probe (Thermo Fischer Scientific, Waltham, USA) was added to the plates and incubation was continued for another 0.5 h. The HEK 293 cells were detached with 0.05% trypsin solution in PBS and HBSS (Hank’s Balanced Salt Solution) and suspended in fresh DMEM medium. All samples were additionally stained with 7-AAD (7-aminoactinomycin D, 0.8 µg/mL) and immediately analysed with a Guava easyCyte flow cytometer (Merc, Burlington, USA). The determinations were performed in triplicates.
3.11. Determination of the Haemolytic Potential
Blood samples were kindly provided by the Regional Center for Blood Donation and Blood Treatment in Gdańsk. Erythrocytes were prepared as described previously [38
]. The hemolysis assays were performed in 96-well plates. The tested compounds were serially diluted in PBS in the concentration range 200–3.125 μg/mL and 100-μL aliquots were poured into wells. 1% Triton X-100 solution, 100 μL, was used as a positive control and 100 μL of the PBS solution served as a negative control. Erythrocyte suspensions in PBS (100 μL) were added to the wells, and plates were incubated at 37 °C for 1 h and then centrifuged (500× g
, 5 min). Supernatants collected (100 μL) were transferred to new microtitration plates. Absorbance at each well was measured at 540 nm. All experiments were performed in biological triplicates.
3.12. Monitoring of the Cellular Uptake
Overnight cultures of C. albicans ATCC 10,231 cells in YBG medium were centrifuged at 5000 rpm for 3 min, and the cells were rinsed with phosphate-buffered saline (PBS). Cells were suspended in PBS and incubated with fluorescein-labelled CIP, TP10-NH2, or conjugate (7) (20 µg/mL) at 30 °C for 15 min. If appropriate, one drop of nuclear-staining dye Hoechst 33,342 solution (1 µg/mL) was added to the sample. Cell suspensions were centrifuged (5000 rpm, 3 min, room temperature), rinsed three times with PBS buffer, and cells were suspended in PBS buffer. Cellular fluorescence was visualised using a lens 63×, at λex/λem = 485 nm/520 nm for fluorescein and λex/λem = 350 nm/461 nm for Hoechst 33342, using an Olympus BX60 epifluorescence microscope (Olympus, Tokyo, Japan) or LSM 800 T-PMT confocal microscope (Carl Zeiss AG, Oberkochen, Germany) with a CCD camera. Images were acquired and processed with ZEN Blue software.
3.13. Inhibition of DNA Relaxation Mediated by DNA Topoisomerase II
A yeast DNA topoisomerase II Relaxation Assay Kit was purchased from Inspiralis (Norwich, UK) and assays were performed according to the manufacturer’s procedure. Briefly, the reaction mixture contained 500 ng of pBR322 DNA in reaction buffer (10 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 100 mM KCl, 2% (v/v) glycerol, 1 mM ATP) as well as the studied compounds dissolved and diluted in dd H2O at the indicated concentrations. The reaction was initiated by the addition of topoisomerase II and allowed to proceed at 30 °C for 30 min. Reactions were terminated by the addition of 5 µL of the loading buffer (NEB; #B7024). The studied compounds were extracted from the reaction mixtures on vortex (for 30 s) with 30 µL of chloroform/isoamyl alcohol solution (24:1; v/v). After centrifugation (3 min, 20,000× g), half of the upper aqueous phases were separated in 1% agarose gels at 90 V for 4 h in TBE buffer (90 mM Tris-base, 70 mM boric acid, 1 mM EDTA, pH 8). Gels were stained with 1 μg/mL ethidium bromide (EtBr) for 15 min to visualise DNA and unbound EtBr was removed by washing gel in 1 mM MgSO4 solution in dd H2O for 15 min. Gels were photographed under UV illumination with a ESSENTIAL V6 (UVITEC, Cambridge, UK).
3.14. Bacterial Viability Assay
Overnight bacteria cultures at 37 °C were diluted and grown up to OD600 0.2. Cultures were centrifuged at 7000 rpm for 3 min, and the cells were washed with 0.9% NaCl and suspended in 0.9% NaCl. The tested compound was added at a concentration equal to the MBC value. Equal aliquots of 3.34 mM Syto 9 and 20 mM propidium iodide were mixed, and 1 μL was added to 300 μL of cell suspension. Then, 3–5 μL of the samples were spotted on 0.8% agarose pads in 0.9% NaCl. Microphotographs were acquired using a Zeiss Axio Observer microscope with a CCD camera. Images were processed in ZEN Blue 2.6 software.
Conjugation of CIP and LVX with TP10-NH2
markedly changed the biological properties of these drugs. Particularly, unlike the mother fluoroquinolones, their conjugates with the cell-penetrating peptide exhibited antifungal activity and mammalian cytotoxicity. The latter seems to be at least in part due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2
, which was demonstrated in hemolytic studies. The earlier reports concerning the mammalian cytotoxicity of TP10 were confusing since no toxicity of this CPP against HeLa cells was observed [19
]. In contrast, in another study, substantial membrane toxicity (measured by the lactate dehydrogenase leakage) against three cell lines and some hemolytic effect was noted [29
]. On the other hand, the antifungal growth inhibitory effect of conjugates observed by us was clearly related to their facilitated uptake by sensitive yeast cells. All CIP, LVX, TP10-NH2
, and their conjugates were found to be inhibitors of yeast type II DNA topoisomerase. CIP and LVX appeared as much weaker inhibitors of this enzyme than their conjugates with TP10-NH2
. However, no similar relationship was found for the antifungal activity of cleavable (3
, and 6
, possibly releasing a free drug intracellularly) and non-cleavable (2
) conjugates. Therefore, topoisomerase II may not be a primary target of these compounds.
Conjugation with TP10-NH2 mostly lowered the antibacterial activity of the fluoroquinolones tested, although in the case of CIP-S-S-TP10-NH2 (3), this effect was negligible. It should be noted, however, that the conjugates 2, 3, and 5 demonstrated a relatively strong growth inhibitory effect against Pseudomonas aeruginosa, i.e., bacterium that appeared completely resistant to TP10-NH2.
The antifungal activity and mammalian cytotoxicity of redox-sensitive [10
were very similar to that of their redox-resistant analogues 2
. 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
. It seems, therefore, that intracellular CIP release from its conjugate with TP10-NH2
facilitates the interaction of the fluoroquinolone with bacterial gyrase, while in eukaryotic cells, conjugate cleavage is not a prerequisite for effective interaction with a molecular target.
Although the antifungal effect has not been observed for all conjugates and all fungal cell lines, the observed phenomenon is, in our opinion, pretty interesting. Since the antifungal effect was much more robust in conjugates where TP10-NH2
was linked to the fluoroquinolone by a disulfide bond, it seems likely that the antifungal activity may be due to the interaction of parental drugs with DNA and nuclear enzymes, following proteolytic cleavage of the conjugate inside the cell. Additionally, the ligand covalently bound with TP10-NH2
may disrupt the membrane [40
]. Therefore, the modification of fluoroquinolone’s action by linkage with TP10-NH2
is strictly a result of the conjugation. As we were able to show a proof of concept of expanding antibacterial drug action by antifungal activity for clinically established compounds (originally lacking this activity), it suggests a future perspective for the use of cell-penetrating peptides in clinical treatment. Additionally, we found an unusual mechanism of action, including both topoisomerase II inhibition and a membrane effect. Such observations suggest, however, a next step towards acquiring a possible antifungal drug candidate. This is a conjugation of CIP or LVX to a CPP exhibiting optimal parameters of mammalian cell toxicity. Alternatively, it is worth exploring the linking of TP10-NH2
with other antimicrobial drugs.
We were able to show a proof of concept that conjugation of CPP to an established antibacterial drug may result in the expansion of the activity spectrum. In particular, fluoroquinolones CIP and LVX conjugated to TP10-NH2 peptide acquired antifungal activity. Notably, this activity against C. albicans and C. krusei was slightly better than that of the known antifungal drug Fluconazole. Moreover, the clinical strain of C. albicans resistant to FLU due to the FLU-induced overexpression of Cdr1p and Cdr2p drug efflux pumps remained sensitive to conjugates 1–6. This finding suggests that the conjugates are not good substrates for Cdr1p and Cdr2p. The cytotoxic potential of the conjugates, while interesting within the scope of anticancer approaches, reduces their potential for a safe use to address non-neoplastic conditions, e.g., fungal (and other microbial) infections. This actually agrees with previous promising reports on CP–drug conjugates. Their vast majority addresses potential anticancer applications, which is probably related to the fact that there is increasing evidence (also supported by this work) on the cytotoxicity and hemolytic activity of most such conjugates.
Interestingly, during our studies, we discovered the antileukemic activity of the studied conjugates (manuscript in preparation). The antileukemic activity added upon antibacterial, as well as novel antifungal activity, may prove advantageous for leukaemia treatment. Specifically, for patients receiving allogeneic blood marrow transplantation (allo-BMT), autologous blood marrow transplantation (auto-BMT), or peripheral blood stem cell transplants (PBSCT)s, bacterial infection remains one of the leading causes of morbidity and mortality (with an incidence ranging from 18.6% to 43.6%) despite early aggressive antimicrobial therapy [41
]. The gold standard therapy regimens consist of either levofloxacin (LVX) or ciprofloxacin (CIP), exhibiting negligible anticancer activity [43
Future investigations will be focused on the understanding of the fundamental molecular mechanism of action of the tested conjugates. Currently, we are performing computer simulation studies for selected conjugates to examine their interaction with the cell membrane, including the possibility of aggregation and formation of intra-membrane channels as well as interaction with DNA after proteolysis. We would also like to move to ex vivo studies on patient-derived cell lines.