Synthesis and biological activity of ester derivatives of mycophenolic acid and acridines/acridones as potential immunosuppressive agents

<p>Improved derivatives of mycophenolic acid (MPA) are necessary to reduce the frequency of adverse effects, this drug exerts in treated patients. In this study, MPA was coupled with <i>N</i>-(ω-hydroxyalkyl)-9-acridone-4-carboxamides or <i>N</i>-(ω-hydroxyalkyl)acridine-4-carboxamides to give respective ester conjugates upon Yamaguchi protocol. This esterification required protection of phenol group in MPA. Designed conjugates revealed higher potency <i>in vitro</i> than parent MPA. Acridine derivatives were more active than acridone analogs and length of the alkyl linker between MPA and heterocyclic units influenced the observed cytotoxicity. Derivatives <b>2b, 2d, 3a, 3b</b> displayed the most promising immunosuppressive activity.</p

New conjugates of mycophenolic acid and their antiproliferative activity

<p>The new conjugates of mycophenolic acid (MPA) were obtained in the reaction of <i>N</i>⁶-(ω-aminoalkyl)adenosines with MPA in the presence of EDCI as a coupling reagent. New compounds <b>4a–h</b> were evaluated on leukemia cell line (Jurkat) and PBMC from healthy donors. Length of the linker influenced observed activity. The compound <b>4b</b> possessing 1,3-diamine spacer exhibited the most promising results and can be considered to further investigations.</p

Synthesis and antiproliferative activity of new mycophenolic acid conjugates with adenosine derivatives

<p>New conjugates of mycophenolic acid (MPA) and adenosine derivatives were synthesized and assessed as potential immunosuppressants on Jurkat cell line and peripheral blood mononuclear cells (PBMC) from healthy donors. As compared to MPA, all compounds were found to be more active against Jurkat cell line. The antiproliferative activities were compared with MPA and adenosine, in both 2′,3′-<i>O</i>-isopropylidene protected and free hydroxyl groups possessing forms. The obtained results were also discussed in terms of selectivity index, defined as SI = IC₅₀/EC₅₀.</p

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

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

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

Novel amides of mycophenolic acid and some heterocyclic derivatives as immunosuppressive agents

The group of 18 new amide derivatives of mycophenolic acid (MPA) and selected heterocyclic amines was synthesised as potential immunosuppressive agents functioning as inosine-5′-monophosphate dehydrogenase (IMPDH) uncompetitive inhibitors. The synthesis of 14 of them employed uronium-type activating system (TBTU/HOBt/DIPEA) while 4 of them concerned phosphonic acid anhydride method (T3P/Py) facilitating amides to be obtained in moderate to excellent yields without the need of phenolic group protection. Most of optimised protocols did not require complicated reaction work-ups, including chromatographic, solvent-consuming methods. The biological activity assay was performed on the T-Jurkat cell line and peripheral mononuclear blood cells (PBMCs) which are both dedicated for antiproliferative activity determination. Each of designed derivatives was characterised by reduced cytotoxicity and benzoxazole analogue (A2) revealed the most promising activity. Subsequently, an observed structure-activity relationship was discussed.</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