(1) Background: (KLAKLAK)₂ is a representative of the antimicrobial peptide group which also shows good anticancer properties. (2) Methods: Herein, we report synthesis using SPPS and characterization by HPLC/MS of a series of shortened analogues of (KLAKLAK)₂. They contain single sequence KLAKLAK as C-terminal amides. In addition, substitution of some natural amino acids with unnatural β-Ala and nor-Leu is realized. In addition, these structures are conjugated with second pharmacophore with well proven anticancer properties 1,8-naphthalimide or caffeic acid. Cytotoxicity, antiproliferative effect and antimicrobial activity of newly synthesized structures were studied. (3) Results: The obtained experimental results reveal significant selective index for substances with common chemical structure KLβAKLβAK-NH₂. The antibacterial properties of newly synthesized analogues at two different concentrations 10 μM and 20 μM, were tested against Gram-negative microorganisms Escherichia coli K12 407. Only two of the studied compounds KLAKLAK-NH₂ and the one conjugated with second pharmacophore 1,8-naphthalimide and unnatural amino acid nor-Leu showed moderate activity against tested strains at concentration of 20 μM. (4) Conclusions: The obtained results reveal that the introducing of 1,8-naphthalimideGly- and Caf- increase the cytotoxicity and antiproliferative activity of the peptides but not their selectivity. Only two compounds KLAKLAK-NH₂ and 1,8-naphthalimideGKnLAKnLAK-NH₂ show moderate activity against Escherichia coli K12 at low concentration of 20 μM.

(1) Background: Peptides are good candidates for anticancer drugs due to their natural existence in the body and lack of secondary effects. (KLAKLAK)₂ is an antimicrobial peptide that also shows good anticancer properties. (2) Methods: The Solid Phase Peptide Synthesis (Fmoc-strategy) was used for the synthesis of target molecules, analogs of (KLAKLAK)₂-NH₂. The purity of all compounds was monitored by HPLC, and their structures were proven using mass spectrometry. Cytotoxicity and antiproliferative effects were studied using 3T3 NRU and MTT tests, respectively. For determination of antimicrobial activity, the disc-diffusion method was used. Hydrolytic stability at three pH values, which mimic the physiological pH in the body, was investigated by means of the HPLC technique. (3) Results: A good selective index against MCF-7 tumor cell lines, combined with good cytotoxicity and antiproliferative properties, was revealed for conjugates NphtG-(KLAKLAK)₂-NH₂ and Caf-(KLAKLAK)₂-NH₂. The same compounds showed very good antifungal properties and complete hydrolytic stability for 72 h. The compound Caf-(KLβ-AKLβ-AK)₂-NH₂ containing β-Ala in its structures exhibited good antimicrobial activity against Escherichia coli K12 407 and Bacillus subtilis 3562, in combination with very good antiproliferative and cytotoxic properties, as well as hydrolytic stability. (4) Conclusions: The obtained results reveal that all synthesized conjugates could be useful for medical practice as anticancer or antimicrobial agents.

Peptides are a promising alternative of conventional medical drugs for the treatment of different diseases because they have no or have very few side effects owing to the natural mechanisms for their elimination. There are a lot of examples of drugs on the pharmaceutical market based on modified amino acids and peptides. Herein, we report the synthesis and studies on the antimicrobial peptide (KLAKLAK)₂-NH₂ where Leu is replaced by the unnatural amino acid nor-Leu. In addition, a second pharmacophore with well proven anticancer properties is introduced to the peptide moiety. All structures were synthesized by conventional solid phase peptide synthesis. The antiproliferative and antimicrobial activities were studied using MTT-dye reduction assay and disk-diffusion test, respectively. Biological activity assays showed that the introduction of nor-Leu in the primary structure of the parent compound does not lead to an increase in the antiproliferative activity. However, the combination with the second pharmacophore 1,8-naphtalimide in a hybrid structure 1,8-NphtG-(KNleAKNleAK)₂-NH₂ leads to a significant increase in the antiproliferative properties. The antimicrobial tests showed that all tested compounds exhibit antimicrobial activity. The peptide and the second pharmacophore had a synergistic effect. In combination with complete hydrolytic stability for 72 h in model systems, the compound 1,8-NphtG-(KNleAKNleAK)₂-NH₂ is the best candidate for a medical drug in the treatment of mammary gland type A adenocarcinoma (MCF-7) in combination with antimicrobial properties.

Background: Specifically designed peptide mimetics offer higher selectivity regarding their toxicity to mammalian cells. In addition to the α-helix conformation, the specific activity is related to the peptide’s ability to penetrate the cell membrane. The alterations in lipid membrane properties were addressed in the presence of the peptide KLAKLAK-NH₂ and analogs containing β-alanine, strengthening the antibacterial activity and/or naphtalimide with proven anticancer properties. Methods: The molecular interactions of the peptide mimetics with POPC bilayers were studied using FTIR-ATR spectroscopy. The thermal shape fluctuation analysis of quasispherical unilamellar vesicles was applied to probe the membrane bending elasticity. The impedance characteristics of bilayer lipid membranes were measured using fast Fourier-transform electrochemical impedance spectroscopy. Results: A lateral peptide association with the membrane is reported for β-alanine-containing peptides. The most pronounced membrane softening is found for the NphtG-KLβAKLβAK-NH₂ analog containing both active groups that corroborate with the indications for 1,8-naphthalimide penetration in the lipid hydrophobic area obtained from the FTIR-ATR spectra analysis. The β-alanine substitution induces strong membrane-rigidifying properties even at very low concentrations of both β-alanine-containing peptides. Conclusions: The reported results are expected to advance the progress in tailoring the pharmacokinetic properties of antimicrobial peptides with strengthened stability towards enzymatic degradation. The investigation of the nonspecific interactions of peptides with model lipid membranes is featured as a useful tool to assess the antitumor and antimicrobial potential of new peptide mimetics.

Using supplements in different sports is a common practice for many athletes. Unfortunately, the growth of this market has entailed some speculations, such as the addition of excessive doses of potentially toxic ingredients. Sometimes the doses listed on the package do not correspond to the real content. Some nutritional supplements on sale may contain undeclared ingredients. Some of those supplements are prohibited substances according to different regulations. Herein, a simple HPLC/DAD procedure that is easy to apply in conventional laboratory practice was developed for the simultaneous determination of 13 substances with steroid structure in nutritional supplements for sport: testosterone, testosterone propionate, testosterone enanthate, methyltestosterone, nandrolone, nandrolone propionate, nandrolone decanoate, methandienone, androstenedione, trenbolone, trenbolone acetate, trenbolone enanthate and boldenone undecylenate. The methodology includes gradient elution with mobile phase A: MeOH:ddH₂O (55:45) and mobile phase B: 100% MeOH in a standard HPLC system containing a Halo 90 Å, C18 (150 x 4.6 mm, 2.7 µm) column, flow rate 0.6 mL/min, UV wavelength of 254 nm, temperature of 40 °C and 20 μL injection volume. The developed methodology was validated according to the corresponding official documents. The key parameters used for the selection of the optimal HPLC conditions were the ability of the mobile phase and solvents to be used with both an HPLC/MS and a GC/MS chromatographic system. The obtained total run time, the reproducibility of the retention times, the separation of all peaks and peak characteristics meet all requirements.

Peptide mimetics imitate natural peptides' structure but they could be specifically designed to be more selective concerning their toxicity to mam-malian cells. In most cases this specificity is due to their ability to form alpha-helix in amphipathic environment. In addition, the specific activity depends on the ability of final structure to penetrate cell membrane. Being responsible for the cell integrity and compartmentalization, biomembranes also play a major role in cellular processes, in which the membrane deformations are important. In the present study we probe peptide-membrane interactions for a shortened amino acid sequence KLAKLAK-NH2 of an antimicrobial peptide with apop-totic effect. The bending rigidity of model lipid bilayers is measured by flicker spectroscopy of quasispherical unilamellar vesicles monitored and analyzed in phase contrast light microscopy. At high peptide concentrations similar to 30 mu mol/L and peptide-to-lipid total molar ratios similar to 0.90 bilayer stacking formation is ob-served. A reduction of the bending constant is reported at peptide-to-lipid total molar ratio similar to 0.80. The membrane softening indicates peripheral peptide orientation at the lipid bilayer, which is considered a prerequisite for channel formation. Based on KLAKLAK-NH2 effect on the membrane bending elastic-ity we provide an evaluation of the peptide partition coefficient characterizing its affinity to POPC bilayers. The acquired results might be helpful in efforts to further tailor the pharmacokinetic properties of antimicrobial peptides in combination with strengthened stability towards enzymatic degradation.