Browsing by Author "Jaber S."
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Item Antimicrobial activity of (KLAKLAK)–NH2 analogs against pathogenic microbial strains(2024-01-01) Jaber S.; Evstatieva Y.; Nemska V.; Nikolova D.; Naydenova E.; Georgieva N.; Danalev D.Many microorganisms pose a threat to human health due to the ever-increasing bacterial resistance to conventional drugs. Nowadays, searching for new alternatives to conventional antibiotics to fight bacterial resistance is a main task. Thus, natural molecules such as amino acids and peptides arise as possible solutions to the problem. The antimicrobial activity of targeted compounds was studied by the agar-diffusion method, using the prepared working solutions of the targeted peptides with the corresponding concentrations. The results of the antimicrobial activity against different test pathogens show specificity, as antimicrobial activity against the used test microorganisms was not found in the investigated short-chain synthetic peptides Si6, Si3 and Si13. Antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, and the yeasts Malassezia furfur and Candida albicans was established for the long-chain synthetic peptides Si1, Si5 and Si16, except Si5 which does not show activity against pathogenic fungal strain C. albicans. The compound Si16 where natural Leu in (KLAKLAK)2-NH2 is replaced by unnatural Nle is the best candidate for medical drug due to the combined antibacterial and antiproliferative effect as well as long hydrolytic stability.Item BENDING ELASTICITY OF PHOSPHOLIPID BILAYERS CONTAINING AN AMPHIPATHIC PEPTIDE WITH LOW MAMMALIAN CYTOTOXICITY(2022-01-01) Vitkova V.; Stoyanova-Ivanova A.; Jaber S.; Naydenova E.; Danalev D.Peptide mimetics imitate natural peptides’ structure but they could be specifically designed to be more selective concerning their toxicity to mammalian cells. In most cases this specificity is due to their ability to form α-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 apoptotic 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 ∼ 30 µmol/L and peptide-to-lipid total molar ratios ∼ 0.90 bilayer stacking formation is observed. A reduction of the bending constant is reported at peptide-to-lipid total molar ratio ∼ 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 elasticity 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.Item Development and validation of HPLC-DAD methodology for simultaneous qualitative and quantitative determination of thirteen substances with a steroid structure(2023-01-01) Zaharieva Z.; Atanasova S.; Danalev D.; Jaber S.; Foteva T.; Nemska V.; Tanev D.; Georgieva N.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.Item Interaction of KLAKLAK-NH2 and Analogs with Biomimetic Membrane Models(2024-03-01) Vitkova V.; Antonova K.; Petkov O.; Stoyanova-Ivanova A.; Jaber S.; Ivanova V.; Naydenova E.; Danalev D.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.Item STUDY OF MODEL REACTION OF SYNTHESIS OF n-BUTYL ACETATE USING IR SPECTROSCOPY IN SOLUTION(2023-01-01) Jaber S.; Nedkova-Shtipska M.FT-IR spectroscopy is an absorption method, used to obtain the infrared spectrum of a chemical compound in a sample in order to monitor specific functional groups in organic and inorganic compounds. Herein, the ability of this technique to be used for on-time monitoring of a model chemical reaction is demonstrated. Every chemical reaction is characterized by specific kinetic profile, especially when a reversible reaction as esterification has to be realized. The selection of an appropriate, easy to be applied tracking technique is an important moment in the industry. N-butyl acetate can be obtained by the esterification of n-butan-1-ol and acetic acid. The main purpose of the presented work is to study the kinetics of the model reaction of n-butyl acetate synthesis showing the ability of FT-IR technique in solution to be used. Process of on-time monitoring can help to study the external factors which influence on the both right and reversed reaction in order to obtain the best yield of aimed product. The obtained results showed that FT-IR technique could be used as an easy to be applied for on-time monitoring of esterification reaction technique and the reaction reaches equilibrium in the 90th minute. Otherwise, a yield of 100 % is observed after one hour and half since the beginning of the reactionItem STUDY OF TRANSFORMATION OF p-AMINOPHENOL IN LIQUID PHASE WITH INFRARED SCPECTROSCOPY(2022-01-01) Nedkova-Shtipska M.; Jaber S.; Petrin S.; Karadjova V.; Danalev D.Determining the rate, mechanism and dynamic of chemical reactions, including reactions between organic and inorganic substances, biotransformations involving enzymes, catalytic processes, etc., is key to setting the most appropriate technological parameters in various industries. The conversion of a substrate into a final product can go through various stages, some of which are speed-determining, while others require specific conditions - pressure, temperature, presence of catalyst and others. Therefore, clarifying the exact mechanism and dynamics of the transformation process is extremely important for achieving good yields and purity of the final products. It is also important to ensure the overall course of the transformation reaction, while minimizing the loss of time, as these factors directly affect the budget of the final product. The aim of the present study was to apply the features of IR spectroscopy to follow the dynamics of model acetylation reaction of transformations of p-aminophenol to paracetamol. Finally, we demonstrated the possibility IR spectroscopy in solution to be used as a technique for monitoring of specific transformation reaction in solution. The main reaction was successfully followed at time by preliminary selecting of the most suitable solvent, which is compatible with the requirements of the technique used and the monitored reaction as well as the right IR bands characteristic for the process of transformation.Item Synthesis and biological studies on (KLAKLAK)2-NH2 analog containing unnatural amino acid β-ala and conjugates with second pharmacophore(2021-12-01) Jaber S.; Nemska V.; Iliev I.; Ivanova E.; Foteva T.; Georgieva N.; Givechev I.; Naydenova E.; Karadjova V.; Danalev D.(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.Item Synthesis, antiproliferative and antimicrobial activities of (KLAKLAK)2-NH2 analogue containing nor-Leu and its conjugates with a second pharmacophore(2023-01-01) Jaber S.; Nemska V.; Iliev I.; Ivanova E.; Foteva T.; Georgieva N.; Givechev I.; Tanev D.; Naydenova E.; Danalev D.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.Item Synthesis, antitumor and antibacterial studies of new shortened analogues of (KLAKLAK)2-NH2 and their conjugates containing unnatural amino acids(2021-02-02) Jaber S.; Iliev I.; Angelova T.; Nemska V.; Sulikovska I.; Naydenova E.; Georgieva N.; Givechev I.; Grabchev I.; Danalev D.(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-naphthalimideGlyand 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.