Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7–6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.


Introduction
In recent years, the general misuse of traditional antibiotics has led to a rise in antimicrobial resistance. The strategies being developed to combat antimicrobial resistance include effective policies for monitoring the spread of resistance [1,2], better use of antibiotics, and intensive research and to be an interesting class of molecules because they combine antimicrobial activity with low resistance selection [3][4][5]. In order to overcome the problem of peptide instability while retaining peptide properties and selectivity, researchers have changed peptide structure in various ways, such as by incorporating unnatural amino acids (e.g., D-amino acids), β-peptides or peptoids (N-substituted glycines) [6,7], or by cyclization. Several years ago, branched peptides, such as multiple antigen peptides, which have a peptidyl core of radially branched lysine residues onto which peptide sequences can be added, were synthesized [8]. These peptides have strong resistance to proteolysis which makes them particularly suitable for use in vivo [9][10][11].
The therapeutic potential of antimicrobial peptides is not limited to their antimicrobial activity, since some peptides have antitumor properties [12,13] and others are intrinsic components of innate immunity. The immunomodulatory properties of these antimicrobial peptides are also attracting much attention for the development of new anti-inflammatory drugs [14][15][16]. Inflammation is the first response of the immune system to infection or injury. Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are surface membrane components of Gram-negative and Gram-positive bacteria, respectively, which trigger the inflammatory response. LPS and LTA interact with host immune cells such as macrophages and monocytes through TLR-4 and TLR-2, producing proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [17,18].
The synthetic peptide SET-M33L, produced with L amino acids, was previously reported to have strong antimicrobial activity in vitro and in vivo against major Gram-negative pathogens [19][20][21][22][23]. SET-M33L was synthesized in tetra-branched form for better stability in biological fluids. Its mode of action is based on a two-step mechanism: (1) high affinity binding to LPS [24]; (2) disruption of bacterial membranes [25].
We also studied the tetra-branched peptide when synthesized with D amino acids (SET-M33D) (Figure 1) [26]. This peptide showed 4-16-fold higher activity than the SET-M33L against Grampositive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, thus becoming an interesting candidate for multifunctional drug development. The increased activity shown by SET-M33D against Gram-positives compared to SET-M33L was likely due to its resistance to proteases, such as elastase, produced by Gram-positive bacteria [26].
Here we report the strong antibacterial activity of SET-M33D, and its efficacy in neutralizing LPS and LTA, thus inhibiting expression of inflammatory mediators. The resistance selection, toxicity, and efficacy in vivo of SET-M33D were also studied using an infection model with the highly virulent methicillin-resistant S. aureus (MRSA) strain USA 300.  Here we report the strong antibacterial activity of SET-M33D, and its efficacy in neutralizing LPS and LTA, thus inhibiting expression of inflammatory mediators. The resistance selection, toxicity, and efficacy in vivo of SET-M33D were also studied using an infection model with the highly virulent methicillin-resistant S. aureus (MRSA) strain USA 300.

Antimicrobial Activity In Vitro
Minimum inhibitory concentrations (MICs) were determined against a collection of 41 selected isolates including reference strains and isolates of clinical origin including major Gram-positive and Gram-negative pathogens with relevant resistance genotypes (e.g., carbapenemase genes, acquired linezolid resistance gene cfr and novel transferable colistin resistance gene mcr). Tested isolates included five S. aureus, six coagulase-negative Staphylococci and six Enterococci among Gram positives, and seven P. aeruginosa, three Acinetobacter baumannii, eight E. coli, one Enterobacter cloacae, and five K. pneumoniae among Gram negatives (Table 1). The peptide showed strong activity against Gram-negative and Gram-positive bacteria with MICs ranging from 0.7 to 6.0 µM, and slightly better activity against the Gram positives. This finding confirmed data already available from different strains [26].

Frequency of Selection of Resistant Mutants after Exposure to SET-M33D
Selection of SET-M33D and colistin resistant mutants was performed on three reference strains (i.e., E. coli ATCC 25922, K. pneumoniae ATCC 13,833 for both agents and S. aureus ATCC 29,213 for SET-M33D only). SET-M33D MIC values were determined and resulted to be 1.5 µM for the selected E. coli and S. aureus strains and 3 µM for the K. pneumoniae strain, being overall consistent with what previously observed [26].
Colistin-resistant mutants were selected at a frequency of approximately 3 × 10 −8 CFU for both the E. coli and K. pneumoniae strains. When selection using SET-M33D containing medium was attempted, a frequency of selection of approximately 6 × 10 −10 CFU or lower was observed for both strains, suggesting an overall lower propensity of SET-M33D to select resistance with respect to colistin ( Table 2). S. aureus ATCC 29213 1.5 -5.9 × 10 −9 ± 2.8 × 10 −9 -* In these cases, the frequency of mutation was reported as lower than the observed median values since no mutants were selected in the experimental conditions.
Regarding the S. aureus reference strain, SET-M33D-resistant mutants were selected at a frequency 10 times higher than the tested Gram-negative strains ( Table 2). In this case, no comparison of selection frequency with colistin-resistant mutants could be performed, due to the intrinsic resistance to colistin of this species.

In Vivo Antimicrobial Efficacy of SET-M33D
Antimicrobial activity of SET-M33D was evaluated in vivo in an animal model of infection with the highly virulent methicillin-resistant S. aureus (MRSA) strain USA 300, a lineage that has become a dominant cause of community associated MRSA infections in North America ( Figure 2) [27,28]. Mice were infected i.p. with the smallest number of bacteria causing lethal infection, and treated i.p. with the peptides 30 min, 3 and 6 h post-infection. A 100% survival after 4 days was obtained with mice treated with 5 mg/kg of SET-M33D, while mice treated with 2.5 mg/kg of SET-M33D showed a mortality of 10%, confirming the potent in vivo activity of SET-M33D.

LPS and LTA Neutralization
The ability of SET-M33D to neutralize LPS from P. aeruginosa and LTA from S. aureus was evaluated in terms of inhibition of protein production of the proinflammatory cytokines TNF-α and IL-6. RAW 264.7 macrophages were stimulated with 20 ng/mL of LPS for 4 h ( Figure 3A) or with 2 µg/mL of LTA for 24 h ( Figure 3B) and treated with different doses of SET-M33D. Then, the release of TNF-α and IL-6 in the medium was measured by ELISA. SET-M33D inhibited TNF-α and IL-6 with an IC50 of 1.36 and 1.30 µM, respectively, under LPS stimulation, and with an IC50 of 3.64 and 2.78 µM for TNF-α and IL-6, respectively, under LTA stimulation.
SET-M33D was also analyzed for its capacity to inhibit the gene expression of proinflammatory cytokines MIP1, KC, IP10, TNF-α, and IL6 induced by LPS or LTA in macrophages ( Figure 3C-L). Gene expression analysis evaluated by RT-PCR showed that stimulation of RAW264.7 with LPS from E. coli or with LTA from S. aureus induced an increase in gene expression of all cytokines tested. In cells stimulated with LPS and then treated with 10 µM of SET-M33D, expression of proinflammatory cytokines was strongly inhibited ( Figure 3C-G). SET-M33D inhibited 92% of IL6, 80% of KC, 70% of IP10, 65% of MIP1, and 52% of TNF-α. No inhibition of proinflammatory cytokines was observed in cells stimulated with LPS and treated with 1 µM of SET-M33D. When cells were stimulated with LTA and then treated with the peptide 10 µM the cytokine inhibition resulted as follows: >65% for IP10 and MIP1; 58% for IL6; around 40% for TNF-α and KC ( Figure 3H-L). SET-M33D 1 µM produced a lower cytokine inhibition than the peptide 10 µM, still reducing the maximum level provoked by toxin stimulation (dark grey columns Figure 3H-L).

LPS and LTA Neutralization
The ability of SET-M33D to neutralize LPS from P. aeruginosa and LTA from S. aureus was evaluated in terms of inhibition of protein production of the proinflammatory cytokines TNF-α and IL-6. RAW 264.7 macrophages were stimulated with 20 ng/mL of LPS for 4 h ( Figure 3A) or with 2 µg/mL of LTA for 24 h ( Figure 3B) and treated with different doses of SET-M33D. Then, the release of TNF-α and IL-6 in the medium was measured by ELISA. SET-M33D inhibited TNF-α and IL-6 with an IC50 of 1.36 and 1.30 µM, respectively, under LPS stimulation, and with an IC50 of 3.64 and 2.78 µM for TNF-α and IL-6, respectively, under LTA stimulation.
SET-M33D was also analyzed for its capacity to inhibit the gene expression of proinflammatory cytokines MIP1, KC, IP10, TNF-α, and IL6 induced by LPS or LTA in macrophages ( Figure 3C-L). Gene expression analysis evaluated by RT-PCR showed that stimulation of RAW264.7 with LPS from E. coli or with LTA from S. aureus induced an increase in gene expression of all cytokines tested. In cells stimulated with LPS and then treated with 10 µM of SET-M33D, expression of proinflammatory cytokines was strongly inhibited ( Figure 3C-G). SET-M33D inhibited 92% of IL6, 80% of KC, 70% of IP10, 65% of MIP1, and 52% of TNF-α. No inhibition of proinflammatory cytokines was observed in cells stimulated with LPS and treated with 1 µM of SET-M33D. When cells were stimulated with LTA and then treated with the peptide 10 µM the cytokine inhibition resulted as follows: >65% for IP10 and MIP1; 58% for IL6; around 40% for TNF-α and KC ( Figure 3H-L). SET-M33D 1 µM produced a lower cytokine inhibition than the peptide 10 µM, still reducing the maximum level provoked by toxin stimulation (dark grey columns Figure 3H-L).

Inhibitory Effects of SET-M33D on COX-2 and iNOS Expression and Nitric Oxide Production
The inflammatory response is accompanied by an increase in several inflammatory mediators in addition to cytokines, such as PGE2 and nitric oxide, which are produced by COX-2 and iNOS enzymes. To investigate the possible involvement of COX-2, RAW264.7 cells were treated with LPS from P. aeruginosa (20 ng/mL) alone or in combination with SET-M33D at the concentrations indicated in Figure 4A and COX-2 expression was evaluated by Western blot. When the cells were exposed to LPS alone, a sharp increase in COX-2 expression was observed, while after cell treatment with different doses of the peptide, COX-2 expression dropped in a dose-dependent manner, reaching the minimum at SET-M33D 2 µM (a reduction of 78.4% ± 1.15) ( Figure 4A). Cells were also treated with LTA from S. aureus (2 µg/mL) alone and with SET-M33D (2 µM) for 24 h. LTA induced a considerable increase in COX-2 expression, and the peptide downregulated its expression by 30.1% (±8.9) ( Figure 4B).  Values were determined as ratios between the intensities of protein bands in treated and untreated cells, assigning the value 1 to the control. The data is expressed as mean ± SD of three independent experiments (n = 3). Nitric oxide release (C,D) was measured as described in Section 4. The data is expressed as mean ± SD of four independent experiments (n = 4); * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated using Student's t-test and compared to the LPS group.
The activation of COX-2 protein is closely related to induction of nitric oxide synthase (iNOS) and the resulting production of nitric oxide. RAW 264.7 cells were stimulated with LPS (1 µg/mL) Values were determined as ratios between the intensities of protein bands in treated and untreated cells, assigning the value 1 to the control. The data is expressed as mean ± SD of three independent experiments (n = 3). Nitric oxide release (C,D) was measured as described in Section 4. The data is expressed as mean ± SD of four independent experiments (n = 4); * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated using Student's t-test and compared to the LPS group. The activation of COX-2 protein is closely related to induction of nitric oxide synthase (iNOS) and the resulting production of nitric oxide. RAW 264.7 cells were stimulated with LPS (1 µg/mL) alone and with SET-M33D (0.5-2 µM) for 24 h, and nitric oxide production was measured as described in Section 4. LPS caused a significant release of nitric oxide (8.5-fold over unstimulated cells) which was dose-dependently attenuated by SET-M33D with maximal effect (87.0% ± 1.8 inhibition) at 2 µM peptide ( Figure 4C). Inhibition of nitric oxide release by SET-M33D under LTA (2 µg/mL) stimulation for 24 h was 32.2% (± 6.2) using the concentrated peptide 2 µM ( Figure 4D) (LTA alone produced a 2.8-fold increase over unstimulated cells).
To examine whether inhibition by SET-M33D could be attributed to its modulation of iNOS protein expression, Western blot analysis was carried out. A certain amount of iNOS protein was produced under LPS and LTA stimulation and it was reduced by SET-M33D in a concentration-dependent manner. SET-M33D (2 µM) reduced iNOS expression, restoring expression to basal level when cells were stimulated with LPS ( Figure 4E), and inhibiting protein expression by about 32.6% (± 3.7) when cells were stimulated with LTA ( Figure 4F).

Effect of SET-M33D on NF-κB Nuclear Translocation
NF-kB/p65, a member of the NF-kB protein family, is transferred to the nucleus in response to stimulation with LPS or LTA. NF-kB/p65 translocations into macrophages were analyzed by immunofluorescence after LPS or LTA stimulation and SET-M33D treatment. NF-kB/p65 was located in the cytoplasm in the control group ( Figure 5A, sharp green signal), while in LPS-or LTA-stimulated cells, it translocated to the nucleus ( Figure 5B,C), as shown by merging of colors. Treatment with SET-M33D clearly restored the NF-kB/p65 to the cytoplasm ( Figure 5E,F). SET-M33D alone did not produce any change in NF-kB/p65 translocation ( Figure 5D). Antibiotics 2020, 9, x FOR PEER REVIEW 9 of 18 alone and with SET-M33D (0.5-2 µM) for 24 h, and nitric oxide production was measured as described in Section 4. LPS caused a significant release of nitric oxide (8.5-fold over unstimulated cells) which was dose-dependently attenuated by SET-M33D with maximal effect (87.0% ± 1.8 inhibition) at 2 µM peptide ( Figure 4C). Inhibition of nitric oxide release by SET-M33D under LTA (2 µg/mL) stimulation for 24 h was 32.2% (± 6.2) using the concentrated peptide 2 µM ( Figure 4D) (LTA alone produced a 2.8-fold increase over unstimulated cells). To examine whether inhibition by SET-M33D could be attributed to its modulation of iNOS protein expression, Western blot analysis was carried out. A certain amount of iNOS protein was produced under LPS and LTA stimulation and it was reduced by SET-M33D in a concentrationdependent manner. SET-M33D (2 µM) reduced iNOS expression, restoring expression to basal level when cells were stimulated with LPS ( Figure 4E), and inhibiting protein expression by about 32.6% (± 3.7) when cells were stimulated with LTA ( Figure 4F).

Effect of SET-M33D on NF-κB Nuclear Translocation
NF-kB/p65, a member of the NF-kB protein family, is transferred to the nucleus in response to stimulation with LPS or LTA. NF-kB/p65 translocations into macrophages were analyzed by immunofluorescence after LPS or LTA stimulation and SET-M33D treatment. NF-kB/p65 was located in the cytoplasm in the control group ( Figure 5A, sharp green signal), while in LPS-or LTAstimulated cells, it translocated to the nucleus ( Figure 5B,C), as shown by merging of colors. Treatment with SET-M33D clearly restored the NF-kB/p65 to the cytoplasm ( Figure 5E,F). SET-M33D alone did not produce any change in NF-kB/p65 translocation ( Figure 5D).
SET-M33D was also analyzed for its capacity to damage red blood cells ( Figure 6B). The peptide did not cause more than 25% hemolysis, even at a concentration of 340 µM, which is more than 55 times the highest MIC reported in Table 1.

Acute Toxicity In Vivo
CD-1 mice were treated i.v. with SET-M33D 30, 25, or 20 mg/kg in a single dose (Figure 7), and were monitored for 4 days. No signs of toxicity were observed at 20 and 25 mg/kg in any animal. At 30 mg/kg, SET-M33D caused 10% mortality after 24 h. All the mild signs of toxicity recorded immediately after inoculation disappeared in live animals within 24 h. No significant variation in body weight was detected (not shown). SET-M33D was also analyzed for its capacity to damage red blood cells ( Figure 6B). The peptide did not cause more than 25% hemolysis, even at a concentration of 340 µM, which is more than 55 times the highest MIC reported in Table 1.

Acute Toxicity In Vivo
CD-1 mice were treated i.v. with SET-M33D 30, 25, or 20 mg/kg in a single dose (Figure 7), and were monitored for 4 days. No signs of toxicity were observed at 20 and 25 mg/kg in any animal. At 30 mg/kg, SET-M33D caused 10% mortality after 24 h. All the mild signs of toxicity recorded immediately after inoculation disappeared in live animals within 24 h. No significant variation in body weight was detected (not shown).

Discussion
Antimicrobial peptides cannot be considered a complete alternative to traditional antibiotics, because with respect to the latter, they generally have lower activity, poor stability, and sometimes production difficulties. However, they can play a very important role in the difficult fight against bacteria because they are often active against bacteria resistant to traditional antibiotics, and may not lead to selection of resistant bacteria [29,30]. Furthermore, some have a multifactorial mechanism of action: they kill bacteria while neutralizing bacterial toxins, thus strongly reducing the inflammatory process triggered by living or dead bacteria [31,32].
The peptide SET-M33D is a synthetic tetrameric molecule synthesized with D amino acids. It is derived from a previous peptide synthesized with L amino acids [20,22], which only showed activity against Gram-negative bacteria. SET-M33D also proved to be active against Gram-positive pathogens, by virtue of its stability to the proteases of these bacteria [26]. Here we described its strong activity against a panel of Gram-positive and Gram-negative bacteria, including clinical isolates with multidrug-resistant phenotypes, as well as its low ability to cause selection of resistant mutants, its lack of hemolytic action, and its anti-inflammatory activity due to neutralization of LPS and LTA derived from pathogens of clinical interest. We demonstrated that the neutralization of bacterial toxins provoked a strong decrease in cytokines TNF-α, IL-6, MIP-1, KC, IP10, and in enzymes iNOS and COX-2, which are considered crucial agents for triggering and fostering inflammatory processes [33]. Its anti-inflammatory property linked to LPS and LTA neutralization was further confirmed by inhibition of NF-kB translocation into the cell nucleus. NF-kB activation and translocation is due to a series of cell events related to pathogen-associated molecular patterns, and LTA and primarily LPS have long been considered to be the prototypical class of such patterns [34,35].
By virtue of its dual activity, namely killing bacteria and restraining inflammation, this molecule could be very suitable for treating lung infections in cystic fibrosis patients, where bacterial growth and uncontrolled inflammation together play a crucial role in progression of lung damage and evolution of the disease. It is noteworthy that S. aureus and P. aeruginosa, the main pathogens involved in lung infections in cystic fibrosis patients [36], are among the most susceptible species to SET-M33D.

Discussion
Antimicrobial peptides cannot be considered a complete alternative to traditional antibiotics, because with respect to the latter, they generally have lower activity, poor stability, and sometimes production difficulties. However, they can play a very important role in the difficult fight against bacteria because they are often active against bacteria resistant to traditional antibiotics, and may not lead to selection of resistant bacteria [29,30]. Furthermore, some have a multifactorial mechanism of action: they kill bacteria while neutralizing bacterial toxins, thus strongly reducing the inflammatory process triggered by living or dead bacteria [31,32].
The peptide SET-M33D is a synthetic tetrameric molecule synthesized with D amino acids. It is derived from a previous peptide synthesized with L amino acids [20,22], which only showed activity against Gram-negative bacteria. SET-M33D also proved to be active against Gram-positive pathogens, by virtue of its stability to the proteases of these bacteria [26]. Here we described its strong activity against a panel of Gram-positive and Gram-negative bacteria, including clinical isolates with multidrug-resistant phenotypes, as well as its low ability to cause selection of resistant mutants, its lack of hemolytic action, and its anti-inflammatory activity due to neutralization of LPS and LTA derived from pathogens of clinical interest. We demonstrated that the neutralization of bacterial toxins provoked a strong decrease in cytokines TNF-α, IL-6, MIP-1, KC, IP10, and in enzymes iNOS and COX-2, which are considered crucial agents for triggering and fostering inflammatory processes [33]. Its anti-inflammatory property linked to LPS and LTA neutralization was further confirmed by inhibition of NF-kB translocation into the cell nucleus. NF-kB activation and translocation is due to a series of cell events related to pathogen-associated molecular patterns, and LTA and primarily LPS have long been considered to be the prototypical class of such patterns [34,35].
By virtue of its dual activity, namely killing bacteria and restraining inflammation, this molecule could be very suitable for treating lung infections in cystic fibrosis patients, where bacterial growth and uncontrolled inflammation together play a crucial role in progression of lung damage and evolution of the disease. It is noteworthy that S. aureus and P. aeruginosa, the main pathogens involved in lung infections in cystic fibrosis patients [36], are among the most susceptible species to SET-M33D.
Furthermore, the promising in vivo efficacy and low in vivo toxicity reported here allow us to predict a favorable therapeutic index for eventual clinical use of this peptide.

Susceptibility Testing
SET-M33D minimum inhibitory concentrations (MICs) were determined in triplicate on a panel of reference and clinical strains using a reference microdilution assay, performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, M07, 11th ed) as previously described [26]. Briefly, strains were grown on Mueller-Hinton agar (MHA) plates and a single colony for each strain was picked using a sterile cotton swab, streaked in sterile cation-supplemented Mueller-Hinton broth

Selection of Resistant Mutants
Selection of resistant mutants was carried out on reference strains using an MHB-based selection medium containing 1% low electro-osmosis agarose as solidifying agent and 12 µM SET-M33D. Strains were grown on MHA plates and a single colony for each strain was picked using a sterile cotton swab, streaked in sterile cation-supplemented MHB and grown at 37 • C to OD 600 0.5. Up to 3 × 10 9 colony forming units (CFU) were spread on Petri dishes containing 15 mL of the selection medium. The same selection medium containing colistin in equimolar concentration with respect to SET-M33D was used as control for the selection of colistin-resistant mutants of the E. coli and K. pneumoniae strains. Plates were incubated for 16-18 h at 37 • C and colonies grown on the SET-M33D and colistin selection media were counted. Viable cell counts of the used bacterial suspension were obtained by plating appropriate serial dilution on nonselective MHA plates. Three replicates using distinct cultures of each strain were performed. The mutation frequency was calculated as the number of mutants divided by the viable cell count.

In Vivo Efficacy
Balb-c mice (20 g) were infected i.p. with a lethal amount of methicillin-resistant S. aureus (MRSA) strains USA 300 (1 × 10 6 CFU/mouse in 500 µL PBS with 7% mucin; mucin from porcine stomach, type II, Sigma-Aldrich). The mice were treated three times with i.p. injection of SET-M33D, diluted in 0.9% NaCl solution, at 5 and 2.5 mg/kg, 0, 3, and 6 h post-infection. Control animals received only vehicle (PBS). Groups consisted of 10 animals each. Moribund animals were killed humanely to avoid unnecessary distress.

Cell Culture
Murine macrophage cell line RAW 264.7 was from ECACC (European Collection of Cell Cultures, Salisbury, UK) and was maintained in DMEM supplemented with 10% FBS, antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin) and L-glutamine (2 mM) at 37 • C in a humidified incubator under a 5% CO 2 atmosphere. 16HBE14o -(human bronchial epithelial cells) and CFBE41o -(cystic fibrosis bronchial epithelial cells with DF508 mutation in the CFTR gene) were provided by Dr. Dieter Gruenert (California Pacific Medical Center Research Institute, San Francisco, CA, USA) and maintained in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS, 0.1 mM nonessential amino acids (NEAA), 2 mM L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin G, at 37 • C in a 5% CO 2 incubator.

TNF-α and IL-6 Quantification
RAW 264.7 cells were seeded in 24-well plates (2 × 10 5 cells/well) and incubated overnight at 37 • C. They were then treated with (i) LPS from P. aeruginosa 20 ng/mL alone or together with SET-M33D (0.12-2 µM) for 4 h; (ii) LTA from S. aureus (2 µg/mL) alone or together with SET-M33D (0.25-4 µM) for 24 h. TNF-α and IL-6 levels were determined in the medium using commercially available ELISA kits: Peprotech (Rocky Hill, NJ, USA) for TNF-α and Biolegend (San Diego, CA, USA) for IL-6. Cytokine levels in cells treated only with LPS and LTA, were taken as 100 and the data was expressed as mean percentage inhibition. IC50 values were calculated by GraphPad Prism version 5.03 for Windows. and permeabilized with PBS containing 0.3% TritonX-100 and 5% normal bovine serum for 1 h. Cells were incubated with rabbit anti-NF-κB p65 mAb diluted 1:250 in PBS with 1% BSA and 0.3% Triton X-100 overnight at 4 • C. After washing with PBS, antibody-bound cells were incubated with Alexa Fluor 488 conjugated anti-rabbit IgG secondary antibodies (1:2000) for 30 min at room temperature. Cell nuclei were counterstained with DAPI (0.5 µg/mL) for 10 min at room temperature. Coverslips were mounted on slides and examined with a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).

Cell Viability Assay
16HBE14oand CFBE41owere plated at a density of 2.5 × 10 4 per well in 96-well microplates, previously incubated with coating solution (88% LHC basal medium, 10% bovine serum albumin, 30 g/mL bovine collagen type I and 1% human fibronectin), while RAW 264.7 cells were seeded in 96-well plates (5 × 10 3 per well) and incubated for 24 h at 37 • C in a 5% CO 2 atmosphere. Cells were treated with 100 µL fresh medium containing SET-M33D at different concentrations for 48 h. Then 20 µl MTT (5 mg/mL) was added to each well and the plate was incubated at 37 • C for 3 h. Finally, 120 µL HCl 4 mM in isopropanol was added to each well to solubilize the formazan crystals. Optical density was measured with a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. Cell viability was calculated comparing the values of treated groups with those of untreated cells. IC50 were calculated using GraphPad Prism 5.03 software.

Hemolytic Activity
Whole human blood in EDTA was centrifuged (1100 g) for 10 min. Red blood cells diluted 1:100 in physiological solution (0.9% NaCl) were incubated for 24 h at 37 • C with serial dilution of SET-M33D from 1.25 to 340 µM. The absorbance of the supernatants was determined in a 96-well plate at 490 nm with a micro plate reader. Data for 100% hemolysis was obtained by adding 0.1% TritonX-100 in water to cells. The negative control was physiological solution. The hemolysis rates of the peptides were calculated with the following equation: (%) = (A peptide-A physiological solution)/(A triton-A physiological solution) × 100%; where A = absorbance.

Acute Toxicity In Vivo
Animal procedures were approved by the Italian Ministry of Health, 14th January 2016, protocol 34/2016-PR. Eight-week-old BALB/c female mice (Charles River) were used in all experiments. The animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes) and internal guidelines.
CD-1 mice were treated by a single i.v. administration of different amounts (30,25, and 20 mg/kg) of SET-M33D, diluted in physiological solution (0.9% NaCl). Groups consisted of 10 animals, five females and five males each. Signs of toxicity were monitored four times a day by visual inspection. A toxicity score was assigned for the following signs: wiry coat and poor motility = mild signs; very wiry coat, abundant lachrymation and poor motility even under stimulation = manifest signs. Animals were observed for 4 days after inoculation of the peptide. Mice were weighed every day from arrival to the last day of the experiment. Moribund animals were killed humanely to avoid unnecessary distress.

Statistical Analysis
Quantitative data was expressed as the mean ± standard deviation (S.D.) of three to five separate experiments. Statistical analysis was performed using Student's one-tailed t-test. Probability values of p < 0.05 were considered statistically significant.

Conclusions
This study is a further development of SET-M33D as an active drug in eradicating Gram-negative and Gram-positive bacteria. Earlier studies showed that SET-M33D could eradicate biofilms of Gram-negative and Gram-positive bacteria and that the all-D configuration was the key for the wider spectrum of activity compared to the parent compound SET-M33L which was only active against Gram-negative [26].
The next step of development, before filing for starting clinical trials, will be the preclinical characterization in animals aimed at the evaluation of safety pharmacology and dose range finding. Chemistry, Manufacturing, and Control evaluations on SET-M33D production are ongoing.
All these features make the peptide SET-M33D a strong candidate for the full drug development phase of a new broad spectrum antibacterial agent.