Synthesis and Evaluation of Biological Activity of Antimicrobial – Pro-Proliferative Peptide Conjugates

<div><p>Skin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in <i>in vitro</i> tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.</p></div

Effect of the peptides on HaCaT keratinocytes and fibroblasts cell migration in the <i>in vitro</i> scratch test.

<p>HaCaT cells and fibroblasts were grown in 6-well plates until the 100% confluence was reached, then cells were starved for the next 12 hours in DMEM without 10% FCS and scratched once vertically with a 200μL pipette tip. After being washed two times with sterile PBS, a fresh portion of DMEM medium was added and cells were treated with the tested peptides applied at their optimal doses (25 μg/mL). Migration was analyzed after 24 hours by means of Zeiss Observer D1 microscope, wounding areas were analyzed with AxioVision software and expressed as the percentage of the wound width in comparison to the control sample (cells incubated in DMEM without FCS). Control+ FCS corresponds to the sample with cells incubated in the medium containing 10% FCS and was treated as the additional positive control. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.</p

Stimulating effect of DAL-PEG-KSLW peptide on the migration of keratinocytes and fibroblasts after 24 hours of incubation.

<p>A—control probe for keratinocytes, B—migration of keratinocytes in the presence of the peptide (25 μg/mL), C—the control probe for fibroblasts, D- migration of fibroblasts in the presence of the peptide (25 μg/mL). HaCaT cells and fibroblasts were grown on the 6-well plates to confluence. Then cells were serum-starved for the next 12 hours. After that, the medium was changed and cells were stimulated with the tested peptides applied at concentration of 25 μg/mL. Incubation was continued for the next 24 hours, then cells were fixed, dyed with 0.05% of crystal violet and analyzed using Zeiss Observer D1 microscope.</p

Antimicrobial activity of the peptides.

<p>^a)NI—no inhibitory activity</p><p>Antimicrobial activity of the peptides.</p

Primary structures of the peptides with their basic physicochemical characteristics.

<p>^a) Alpha-helical content was calculated according to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140377#pone.0140377.e001" target="_blank">Eq 1</a> on the basis of the results obtained for peptides dissolved in 30 mM SDS</p><p>^b) Mean hydrophobicity values were calculated by means of ProtParam program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140377#pone.0140377.ref016" target="_blank">16</a>] as the sum of hydropathy values of all the amino acids, divided by the number of residues in the sequence (PEG fragment was excluded from the calculations)</p><p>^c) ND—not determined</p><p>D-analogues of the amino acids are marked as italic type</p><p>Primary structures of the peptides with their basic physicochemical characteristics.</p

Like other canonical inhibitors, SFTI-1 interacts with a cognate enzyme (i.e. with trypsin) <i>via</i> its solvent-exposed binding loop between Cys³ and Cys¹¹.

<p>The central peptide bond between Lys⁵ (P₁) and Ser⁶ (P₁′) is known as a reactive site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Luckett1" target="_blank">[17]</a>. The adjacent residues placed on the left side of this bond are marked with non-prime P (P₂, P₃, … P_n etc), whereas, on the right side, with prime P (P₂′, P₃′, … P_n etc) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Schechter1" target="_blank">[18]</a>. Corresponding enzyme substrate binding pockets are called non-prime (S₁, S₂, S₃, …, S_n) and prime (S₁′, S₂′, S₃′, …,S_n′) sites, respectively.</p

Inhibition of chymotrypsin-like (ChT-L) and caspase-like (C–L) activities of SDS-activated human 20S ([E] = 2.7 nm) by BPTI and Z-LLL-CHO (A).

<p>Inhibition of trypsin-like (T-L) activity of latent human 20S ([E] = 2.7 nm) (<b>B</b>). Incubation time was about 30 min. at 37°C.</p

Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).

<p>Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).</p

Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.

<p>Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.</p

The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).

<p>The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).</p