Browsing by Author "Naydenova E."
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Item Ammonium hydrogen (RS)-[(5-methyl-2-oxo-1,3-oxazolidin-3-yl)methyl]phospho- nate(2010-01-22) Todorov P.; Naydenova E.; Nikolova R.; Shivachev B.In the title compound, NH4+·C 5H9NO5P-, the five-membered methyl-oxazolidin-2-one unit is disordered over two positions, the major component having a site occupancy of 0.832 (9). A three-dimensional network of O-H⋯O and N-H⋯O hydrogen bonds stabilizes the crystal structure.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 Benzimidazoles Containing Piperazine Skeleton at C-2 Position as Promising Tubulin Modulators with Anthelmintic and Antineoplastic Activity(2023-11-01) Anichina K.; Mavrova A.; Vuchev D.; Popova-Daskalova G.; Bassi G.; Rossi A.; Montesi M.; Panseri S.; Fratev F.; Naydenova E.Benzimidazole anthelmintic drugs hold promise for repurposing as cancer treatments due to their interference with tubulin polymerization and depolymerization, manifesting anticancer properties. We explored the potential of benzimidazole compounds with a piperazine fragment at C-2 as tubulin-targeting agents. In particular, we assessed their anthelmintic activity against isolated Trichinella spiralis muscle larvae and their effects on glioblastoma (U-87 MG) and breast cancer (MDA-MB-231) cell lines. Compound 7c demonstrated exceptional anthelmintic efficacy, achieving a 92.7% reduction in parasite activity at 100 μg/mL after 48 hours. In vitro cytotoxicity analysis of MDA-MB 231 and U87 MG cell lines showed that derivatives 7b, 7d, and 7c displayed lower IC50 values compared to albendazole (ABZ), the control. These piperazine benzimidazoles effectively reduced cell migration in both cell lines, with compound 7c exhibiting the most significant reduction, making it a promising candidate for further study. The binding mode of the most promising compound 7c, was determined using the induced fit docking–molecular dynamics (IFD–MD) approach. Regular docking and IFD were also employed for comparison. The IFD–MD analysis revealed that 7c binds to tubulin in a unique binding cavity near that of ABZ, but the benzimidazole ring was fitted much deeper into the binding pocket. Finally, the absolute free energy of perturbation technique was applied to evaluate the 7c binding affinity, further confirming the observed binding mode.Item COMPUTER MODELLING OF ALL TYPES OF SOMATOSTATIN RECEPTORS(2020-01-01) Dzimbova T.; Wesselinova D.; Naydenova E.; Milanov P.The somatostatin receptors (SSTRs) are a very important class of receptors that attract the attention of a great number of scientists. The development of effective and selective ligands of each somatostatin receptor type is a time consuming process, which involves the knowledge and the skills of different researchers: chemists, biologists, medics, pharmacologists, etc. A large number of compounds is synthesized, characterized and biologically tested, but just some of them have the desired efficacy and selectivity to a respective somatostatin receptor type. The aims of the present study are: (1) to choose templates, among the recently published crystallographic structures, for homology modelling of all five types of SSTRs; (2) to evaluate the models by different computational tools (Procheck, MolProbity, docking). The structures of SSTRs are modelled using the Chimera software and their characteristics are evaluated on the ground of different methods. The best models are used for docking with recently synthesized and biologically tested somatostatin analogues selective to certain SSTR. The data, especially from docking, shows that the newly generated model of SSTRs could be further used for in silico experiments providing a faster and a better design of selective and effective ligands of SSTRsItem IMIDAZOLIDINEDIONE DERIVATIVES OF NALIDIXIC ACID: SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL STUDIES(2021-01-01) Marinov M.; Kostova I.; Naydenova E.; Stoyanov N.Imidazolidine-2,4-diones (hydantoins and spirohydantoins) and nalidixic acid (1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid) are heterocyclic compounds whose ring structures include nitrogen atoms. These substances exhibit potent pharmacological activity as antitumor, antibacterial and antifungal agents, as well as aldose reductase inhibitors. This article presents the synthesis of novel heterocyclic compounds, based on the interaction of imidazolidinediones with nalidixic acid. The imidazolidinedione derivatives obtained were characterized by physicochemical parameters, elemental analysis, IR, 1H and 13C NMR spectral data. The biological activity of the synthesized products was evaluated against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Salmonella abony, the yeasts Candida albicans and Saccharomyces cerevisiae and the mold Aspergillus brasiliensis. All compounds showed activity against the Gram-positive and Gram-negative bacteria tested.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 Involvement of the opioidergic and nociceptinergic systems in the analgesic effects of novel nociceptin analogues after acute and chronic immobilization stress(2022-01-01) Himcheva I.; Stavreva G.T.; Naydenova E.; Bocheva A.Stress is known to exert an influence on neuroendocrine, autonomic, hormonal functioning. Various stress models have been reported to induce analgesia. This is a phenomenon, referred to as stress-induced analgesia (SIA). Nociceptin/Orphanin FQ(N/OFQ) is a heptadecapeptide that has been found to play a direct role on pain perception. This study aimed to investigate the effects of novel nociceptin analogues on nociception after acute and chronic immobilization stress (CIS) and the involvement of the opioid and nociceptinergic systems in analgesic effects. Analgesic effects were examined by paw-pressure (PP) and hot-plate (HP) tests. Our data showed that acute immobilization stress induced hypoalgesia. The analgesic effect was more pronounced in pain caused by a mechanical stimulus than by a thermal one. CIS attenuated the hyperalgesic effect of naloxone and JTC-801 for mechanical and thermal stimulation. The effects of the opioid system are more pronounced in acute immobilization stress, while the nociceptin mechanisms predominate after chronic stress.Item New C2-and N3-Modified Thieno[2,3-d]Pyrimidine Conjugates with Cytotoxicity in the Nanomolar Range(2022-04-01) Mavrova A.T.; Dimov S.; Yancheva D.; Rangelov M.; Wesselinova D.; Naydenova E.Aims: The aim of the current study was to develop and explore a series of new cytotoxic agents based on the conjugation between the thieno[2,3-d]pyrimidine moiety and a second pharmacophore at the C2 or N3 position. Background: As the thieno[2,3-d]pyrimidine core is a bioisostere of the 4-anilinoquinazoline, various new thienopyrimidine derivatives were synthesized by modifying the structure of the clinically used anticancer quinazoline EGFR inhibitors of the first generation – gefitinib, and second-generation – dacomitinib and canertinib. It was reported that some thieno[2,3-d]pyrimidine derivatives showed improved EGFR inhibitory activity. On the other hand, the benzimidazole heterocycle is present as a pharmacophore unit in the structure of many clinically used chemotherapeutic agents. Some 2-aminobenzimidazole derivatives, possessing anticancer activity, demonstrated EGFR inhibition and the benzimidazole derivative EGF816 is currently in the second phase of clinical trials. Objective: The objectives of the study were the design of a novel series thieno[2,3-d]pyrimidines, synthesis of the compounds and investigation of their effects towards human cancer HT-29, MDA-MB-231, HeLa, HepG2 and to normal human Lep3 cell lines. (American Type Culture Collection, ATCC, Rockville, MD, USA). Methods: The synthetic protocol implemented cyclocondensation of 2-amino-thiophenes and nitriles in an inert medium, azaMichael addition to benzimidazole derivatives and nucleophylic substitution at the N3 place. MTS test was used in order to establish the cytotoxicity of the tested compounds. SAR analysis and in silico assessment of the inhibitory potential towards human oncogenicV599EB-Raf were performed using Molinspiration tool and Molecular Operating environment software. Results: The MTS test data showed that almost all studied thieno[2,3-d]pyirimidines (9-13, 21-22 and 25) manifest high inhibitory effect on cell proliferation at nanomolar concentrations, whereas compounds 9 (IC50 = 130 nM) and 10 (IC50 = 261 nM) containing amino acid moiety, and 21 (IC50 = 108 nM) possessing two thienopyrimidine moieties attached to a 1,3-disubstituted benzimidazole linker, revealed many times lower toxicity against Lep3 cells compared to the cancer cells. Thienopyrimidines 11-13 possessed high selectivity against HeLa cells. Compound 13 showed high inhibitory activity against MDA-MB-231 and HepG2, with IC50 1.44 nM and 1.11 nM respectively. To outline the possible biological target of the studied coumpounds, their potential to interact with human oncogenicV599EB-Raf was explored by a docking study. As a result, it was suggested that the benzimidazolyl and glycyl fragments could enhance the binding ability of the new compounds by increasing the number of hydrogen bond acceptors and by stabilizing the inactive form of the enzyme. Conclusion: The thienopyrimidines tested in vitro for human cancer HT-29, MDA-MB-231, HeLa, HepG2 and normal human Lep3 cell lines demonstrated cytotoxicity in the nanomolar range. It was established that compounds 9, 10 and 21 showed many times lower toxicity against normal Lep3 cells that can provide a high selectivity towards all four cancer cell lines at small concentrations. Based on the analysis of the structure-activity relationship, the observed trends in the cytotoxicity could be related to the lipophilicity and the topological polar surface area of the tested compounds. The docking study on the potential of the new thieno[2,3-d]pyrimidine-4-ones to interact with mutantV599EB-Raf showed that the compounds might be able to stabilize the enzyme in its inactive form.Item Peptide-based targeted cancer therapeutics: Design, synthesis and biological evaluation(2022-09-01) Iwanov I.; Rossi A.; Montesi M.; Doytchinova I.; Sargsyan A.; Momekov G.; Panseri S.; Naydenova E.Cancer is the leading cause for human mortality together with cardiovascular diseases. Abl (Abelson) tyrosine kinases play a fundamental role in transducing various signals that control proliferation, survival, migration and invasion in several cancers such as Chronic Myeloid Leukemia (CML), breast cancer and brain cancer. For these reasons Abl tyrosine kinases are considered important biological targets in drug discovery. In this study a series of lysine-based oligopeptides with expected Abl inhibitory activity were designed resembling the binding of FDA-approved drugs (i.e. of Imatinib and Nilotinib), synthesized, purified by High Performance Liquid Chromatography (HPLC), analyzed by mass spectrometry (MS) and biologically tested in vitro in CML (AR-230 and K-562), breast cancers (MDA-MB 231 and MDA-MB 468) and glioblastoma cell lines (U87 and U118). The solid-phase peptide synthesis (SPPS) by Fmoc (9-fluorenylmethoxycarbonyl) chemistry was used to synthesize target compounds. AutoDock Vina was applied for simulation binding to Abl. The biological activities were measured evaluating cytotoxic effect, induction of apoptosis and inhibition of cancer cells migration. The new peptides exhibited different concentration-dependent antiproliferative effect against the tumor cell lines after 72 h treatment. The most promising results were obtained with the U87 glioblastoma cell line where a significant reduction of the migration ability was detected with one compound (H-Lys1-Lys2-Lys3-NH2).Item Structure, conformation and hydrogen bonding of two amino-cycloalkanespiro-5-hydantoins(2009-01-09) Todorov P.; Petrova R.; Naydenova E.; Shivachev B.The crystal structures of 3-amino-cycloheptanespiro-4′- imidazolidine-2′,5′-dione (I) {systematic name: 3-amino-1,3-diazaspiro[4.6] undecane-2,4-dione} and 3-amino-cyclooctanespiro-4′- imidazolidine-2′,5′-dione (II) {systematic name: 3-amino-1,3-diazaspiro[4.7] dodecane-2,4-dione}, have been determined. In both compounds the polar hydantoin groups cause molecules to aggregate via N-H⋯O and N-H⋯N interactions, forming a layer structure, in which the cycloalkane rings project outwards from the central, more polar, region. The observed molecular structure is compared with that calculated by density functional theory methods. © Versita Warsaw and Springer-Verlag Berlin Heidelberg 2009.Item STUDY OF EFFECTS OF NEWLY SYNTHESISED NOCICEPTIN ANALOGUES ON THE ENDOCANNABINOID SYSTEM AND PAIN AFTER CHRONIC IMMOBILIZATION STRESS(2023-01-01) Himcheva I.; Stavreva G.; Naydenova E.; Bocheva A.Stress provokes stress-induced analgesia (SIA), which depends on an opioid and non-opioid components. The non-opioid one comprises several systems among which are endocannabinoid (ECS), adrenergic, and nitricoxidergic participating in the descending antinociceptive system of the body. The ECS system has a well-established role in the modulation of pain perception and behavioral responses after stress. Nociceptin/Orphanin FQ(N/OFQ) is a heptadecapeptide that has been found to play a role in pain perception. This study aimed to investigate the effects of novel nociceptin N/OFQ(1-13)NH2 analogues on nociception after chronic immobilization stress (CIS) and the involvement of the ECS in analgesic effects. The experiments were carried out on male Wistar rats. The animals were immobilized in a tube for 3 hours daily for 4 days. Analgesic effects were examined by the paw-pressure (PP) test. All novel analogues of N/OFQ(1-13)NH2, the cannabinoid receptor type 1 (СВ1-receptor) agonist N-arachidonoylethanolamide (AEA), and the СВ1-receptors antagonist N-(Piperidin-1-yl)-5-(4-iodophenyl)-1- (2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AМ251) were administered intraperitoneally (i.p) dissolved in Dimethyl sulfoxide (DMSO). Statistical analysis was performed using one-way ANOVA. The results showed that nociceptin and analogues administered after CIS decreased the pain threshold significantly compared to a group that underwent chronic stress only. The administration of AEA immediately after the end of stress led to a significantly increased pain threshold, while administration of AM251 significantly decreased the pain threshold versus the both control and group that underwent chronic stress only. Nociceptin and analogues co-administered with СВ1-receptor agonist (AEA) or antagonist (AM251) after the end of stress decreased immobilization SIA. Our study gives us reason to assume the participation of ECS in the analgesic effects of the novel nociceptin analogues after chronic immobilization stress.Item Synthesis and antimicrobial activity of 1,8-naphthalimide derivatives of nalidixic acid(2019-01-01) Marinov M.; Kostova I.; Naydenova E.; Stoyanov N.Various naphthalimides are synthesized and their biological activity is studied. The target compounds are prepared by the interaction of different substituted 2-amino-1H-benzo[de]isoquinoline-1,3(2H)-diones with nalidixic acid (1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid). The structures of all synthesized products are verified via their physicochemical parameters, an elemental analysis, IR, 1H and 13C NMR spectroscopy. The antimicrobial activity of the compounds obtained is determined against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Essherichia coli, Pseudomonas aeruginosa and Salmonella abony, the yeasts Candida albicans and Saccharomyces cerevisiae and the molds Penicillium chrysogenum and Aspergillus niger. It is found that N-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxamide, N-(6-1H-indol-3-yl-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxamide and N-(6-piperidin-1-yl-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl)-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxamide are active against the Gram-positive and Gram-negative bacteria tested.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 AND CHARACTERIZATION OF N3-ACETYL DERIVATIVES OF SPIROHYDANTOINS(2022-01-01) Marinov M.; Frenkeva M.; Naydenova E.; Penchev P.; Stoyanov N.The following article describes the synthesis of N3-acetyl derivatives of spirohydantoins. The compounds are derived by applying the following technique: the Bucherer-Lieb method (α-series) and the Strecker method (β-series). The synthesized products are characterized by physicochemical parameters, elemental analysis, FTIR, 1H NMR, 13C NMR and 13C DEPT135 spectral data.Item Synthesis and Cytotoxicity of Platinum(II) Complexes of 3-Aminocyclopentanespiro-5-hydantoin and 3-Aminocycloheptanespiro-5-hydantoin(2003-01-01) Kushev D.; Maneva L.; Grancharov K.; Spassovska N.; Naydenova E.; Popova J.Four new platinum(II) complexes of 3-aminocyclopentanespiro-5-hydantoin (acpsh) and 3-aminocycloheptanespiro-5-hydantoin (achpsh) were synthesized and characterized by elemental analysis, IR and 1NMR spectra. The spectral analyses indicated a cis-square planar structure of the complexes with ligands coordinated via the NH2 group. The complexes were evaluated for in vitro cytotoxicity in murine erythroleukemia (MEL) cells, clone F4N, using cell-growth and macromolecular synthesis assay. The compounds, with exception of [Pt(NH3)(achpsh)Cl2] (IV), exhibited much lower cytotoxicity than that of cisplatin (DDP). Compound IV was nearly as cytotoxic as DDP. The new complexes exerted low antibacterial activity as assessed by seven bacterial strains. © 1946–2014, Verlag der Zeitschrift für Naturforschung. All rights reserved.Item SYNTHESIS OF NOVEL NOCICEPTIN ANALOGUES: INVOLVEMENT IN ANALGESIC EFFECTS OF OPIOID AND NITRIC OXIDEERGIC SYSTEMS AFTER COLD STRESS(2021-01-01) Himcheva I.; Angelova N.; Naydenova E.; Stavreva G.; Krastev D.; Kochev D.; Bocheva A.Stress is known to exert an influence on neuroendocrine, autonomic, hormonal, and immune functioning. Various stress models have been reported to induce analgesia. This is a phenomenon, referred to as stress-induced analgesia. Nociceptin and analogues are neuropeptides, neuromodulators, which are able to inhibit the expression of some forms of SIA. Nociceptin/Orphanin FQ(N/OFQ) is a heptadecapeptide which has been found to play a direct role on pain perception. Nitric oxide (NO) plays an important role in initiation and maintenance of pain. It is also known that acute and chronic stresses induce biochemical changes affecting both pain threshold and behaviour. Thus, endogenous opioid peptides and NO, mediated a wide variety of physiological processes including pain transmission and SIA. The aim of the present study was to investigate the effects of novel analogues of N/OFQ(1-13)NH2, where Lysine (Lys) at position 9 and/or 13 was substituted by L-ornithine (Orn) on nociception after cold stress and the involvement of the opioid and nitric oxideergic systems in these effects. Analgesic activity was examined by nociceptive test - pawpressure (PP). All novel analogues of N/OFQ were injected at a dose of 10 p.g kg-1; naloxone (Nal, 1 mg kg-1), JTC-801 (NOP receptor antagonist, 0,5 mg kg-1), NG-nitro-L-arginine methylester (L-NAME, 10 mg kg-1) and L-arginine (L-Arg, 1mg kg-1). All drugs were dissolved in saline and were injected intraperitoneally (i.p.). The nociceptive tests were performed 10 min after peptide injection. Antinociceptive effects were statistically accessed by ANOVA. In conclusion we suggest that in analgesic effects of the novel analogues of nociceptin were involved opioid-, nociceptin- and nitric- oxideergic systems after cold stress.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, Antiproliferative Effect and In Silico LogP Prediction of BIM-23052 Analogs Containing Tyr Instead of Phe(2023-04-01) Danalev D.; Iliev I.; Dobrev S.; Angelova S.; Petrin S.; Dzimbova T.; Ivanova E.; Borisova D.; Naydenova E.(1) Background: Hydrophobicity (or lipophilicity) is a limiting factor in the ability of molecules to pass through cell membranes and to perform their function. The ability to efficiently access cytosol is especially important when a synthetic compound has the potential to become a drug substance. D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2 (BIM-23052) is a linear analog of somatostatin with established in vitro GH-inhibitory activity in nanomolar (nm) concentrations and high affinity to different somatostatin receptors. (2) Methods: Series of analogs of BIM-23052 were synthesized where Phe residue(s) in the BIM-23052 molecule were replaced with Tyr using standard SPPS, Fmoc/t-Bu strategy. Analyses of target compounds were performed using HPLC/MS technique. Toxicity and antiproliferative activity were studied using in vitro NRU and MTT assays. The values of logP (partition coefficient in octanol/water) for BIM-23052 and its analogs were calculated. (3) Results: The obtained data show the best antiproliferative effect against studied cancer cells for compound D-Phe-Phe-Phe-D-Trp-Lys-Thr-Tyr7-Thr-NH2 (DD8), the most lipophilic compound according to the predicted logP values. (4) Conclusions: Multiple analyses of the obtained data reveal that compound D-Phe-Phe-Phe-D-Trp-Lys-Thr-Tyr7-Thr-NH2 (DD8) where one Phe is replaced by Tyr has the best combination of cytotoxicity, antiproliferative effect and hydrolytic stability.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.