Synthesis, molecular docking, electrochemical and fluorimetric analysis of new caffeic and cinnamic acid-conjugated hemorphin derivatives designed as potential anticonvulsant and antinociceptive agents.

Based on the pharmacophore model of opioid receptors, our team recently synthesized a series of short-chain hemorphin peptide analogs containing non-natural amino acids. They demonstrated anticonvulsant and antinociceptive activity with low neurotoxicity. In the present study, a series of novel bioconjugates of N-modified hemorphin analogs containing second pharmacophore cinnamic acids (CA) or caffeic (KA) were synthesized by a traditional solid-phase Fmoc chemistry method for peptide synthesis. Electrochemical and fluorimetric analysis, in vivo anticonvulsant and antinociceptive activity in mice were conducted on the compounds. The three CA acid- (H4-CA, H5-CA, and H7-CA) and three KA acid- (H4-KA, H5-KA, and H7-KA) conjugated hemorphin derivatives exhibited potency at the highest doses of 2 µg/5 µl, administered by intracerebroventricular (icv) mode, against seizure spread in the maximal electroshock test (MES) in mice. The KA-conjugated H5-KA derivate, at the lowest dose, was the only compound that suppressed clonic seizures in the subcutaneous pentylenetetrazol (scPTZ) test. Except for the H5-CA, all tested CA acid- and KA acid-conjugated peptide derivates had the potency to increase the latency for clonic seizures in a dose-dependent mode. The activity against the psychomotor seizures in the 6-Hz test was detected only for the H4-CA (0.5 µg) and H4-KA (0.5 µg and 1 µg), respectively. All investigated peptides showed a more pronounced antinociceptive effect in the "intraplantar formalin" test compared to the "hot plate" test. Shorter chain analogs showed a better antinociceptive profile against tonic pain. The data suggest a DOR and KOR-mediated mechanism of action. According to the docking analysis, H7-CA showed a different antinociceptive profile than other investigated peptides. The novel peptide derivates did not exhibit neurotoxicity in the rotarod test. Our findings suggest that conjugated CA and KA morphine peptides can be used to develop novel morphine-related analogs with anticonvulsant and antinociceptive activity.

Additive Anticonvulsant Profile and Molecular Docking Analysis of 5,5'-Diphenylhydantoin Schiff Bases and Phenytoin.

Four 5,5'-diphenylhydantoin Schiff bases possessing different aromatic species (SB1-SB4) were recently synthesized and characterized using spectroscopic and electrochemical tools. The present study aimed to ascertain the anticonvulsant activity of the novel phenytoin derivatives SB1-Ph, SB2-Ph, SB3-Ph, and SB4-Ph, containing different electron-donor and electron-acceptor groups, and their possible mechanism of action. The SB2-Ph exhibited the highest potency to suppress the seizure spread with ED50 = 8.29 mg/kg, comparable to phenytoin (ED50 = 5.96 mg/kg). While SB2-Ph did not produce neurotoxicity and sedation, it decreased locomotion and stereotypy compared to control. When administered in combination, the four Schiff bases decreased the phenytoin ED50 by more than 2× and raised the protective index by more than 7× (phenytoin+SB2-Ph). The strongest correlation between in-vivo and docking study results was found for ligands' interaction energies with kappa and delta receptors. These data, combined with the worst interaction energies of our ligands with the mu receptor, suggest that the primary mechanism of their action involves the kappa and delta receptors, where the selectivity to the kappa receptor leads to higher biological effects. Our findings suggest that the four Schiff bases might be promising candidates with potential applications as a safe and effective adjuvant in epilepsy.

Design, Synthesis, In Silico Studies and In Vitro Evaluation of New Indole- and/or Donepezil-like Hybrids as Multitarget-Directed Agents for Alzheimer's Disease.

Alzheimer's disease (AD) is considered a complex neurodegenerative condition which warrants the development of multitargeted drugs to tackle the key pathogenetic mechanisms of the disease. In this study, two novel series of melatonin- and donepezil-based hybrid molecules with hydrazone (3a-r) or sulfonyl hydrazone (5a-l) fragments were designed, synthesized, and evaluated as multifunctional ligands against AD-related neurodegenerative mechanisms. Two lead compounds (3c and 3d) exhibited a well-balanced multifunctional profile, demonstrating intriguing acetylcholinesterase (AChE) inhibition, promising antioxidant activity assessed by DPPH, ABTS, and FRAP methods, as well as the inhibition of lipid peroxidation in the linoleic acid system. Compound 3n, possessing two indole scaffolds, showed the highest activity against butyrylcholinesterase (BChE) and a high selectivity index (SI = 47.34), as well as a pronounced protective effect in H2O2-induced oxidative stress in SH-SY5Y cells. Moreover, compounds 3c, 3d, and 3n showed low neurotoxicity against malignant neuroblastoma cell lines of human (SH-SY5Y) and murine (Neuro-2a) origin, as well as normal murine fibroblast cells (CCL-1) that indicate the in vitro biocompatibility of the experimental compounds. Furthermore, compounds 3c, 3d, and 3n were capable of penetrating the blood-brain barrier (BBB) in the experimental PAMPA-BBB study. The molecular docking showed that compound 3c could act as a ligand to both MT1 and MT2 receptors, as well as to AchE and BchE enzymes. Taken together, those results outline compounds 3c, 3d, and 3n as promising prototypes in the search of innovative compounds for the treatment of AD-associated neurodegeneration with oxidative stress. This study demonstrates that hydrazone derivatives with melatonin and donepezil are appropriate for further development of new AChE/BChE inhibitory agents.

Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells.

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Insights into the SARS-CoV-2 ORF6 Mechanism of Action.

ORF6 is responsible for suppressing the immune response of cells infected by the SARS-CoV-2 virus. It is also the most toxic protein of SARS-CoV-2, and its actions are associated with the viral pathogenicity. Here, we study in silico and in vitro the structure of the protein, its interaction with RAE1 and the mechanism of action behind its high toxicity. We show both computationally and experimentally that SARS-CoV-2 ORF6, embedded in the cytoplasmic membranes, binds to RAE1 and sequesters it in the cytoplasm, thus depleting its availability in the nucleus and impairing nucleocytoplasmic mRNA transport. This negatively affects the cellular genome stability by compromising the cell cycle progression into the S-phase and by promoting the accumulation of RNA-DNA hybrids. Understanding the multiple ways in which ORF6 affects DNA replication may also have important implications for elucidating the pathogenicity of SARS-CoV-2 and developing therapeutic strategies to mitigate its deleterious effects on host cells.

New Indole-3-Propionic Acid and 5-Methoxy-Indole Carboxylic Acid Derived Hydrazone Hybrids as Multifunctional Neuroprotectors.

In light of the known neuroprotective properties of indole compounds and the promising potential of hydrazone derivatives, two series of aldehyde-heterocyclic hybrids combining those pharmacophores were synthesized as new multifunctional neuroprotectors. The obtained derivatives of indole-3-propionic acid (IPA) and 5-methoxy-indole carboxylic acid (5MICA) had good safety profiles: Hemolytic effects < 5% (200 µM) and IC50 > 150 µM were found in the majority of the SH-SY5Y and bEnd3 cell lines. The 2,3-dihydroxy, 2-hydroxy-4-methoxy, and syringaldehyde derivatives of 5MICA exhibited the strongest neuroprotection against H2O2-induced oxidative stress in SH-SY5Y cells and 6-OHDA-induced neurotoxicity in rat-brain synaptosomes. All the compounds suppressed the iron-induced lipid peroxidation. The hydroxyl derivatives were also the most active in terms of deoxyribose-degradation inhibition, whereas the 3,4-dihydroxy derivatives were able to decrease the superoxide-anion generation. Both series of compounds showed an increased inhibition of hMAO-B, with greater expression detected in the 5MICA hybrids. The in vitro BBB model with the bEnd3 cell line showed that some compounds increased the permeability of the endothelial monolayer while maintaining the tight junctions. The combined results demonstrated that the derivatives of IPA and 5MICA showed strong neuroprotective, antioxidant, MAO-B inhibitory activity and could be considered as prospective multifunctional compounds for the treatment of neurodegenerative disorders.

Asymmetric Monomethine Cyanine Dyes with Hydrophobic Functionalities for Fluorescent Intercalator Displacement Assay.

A new green procedure has been applied for the synthesis and purification of asymmetric monomethine cyanine dyes. The photophysical properties of the newly synthesized compounds have been examined by combined application of spectroscopic and theoretical methods. The structural characteristics of the molecules and dimer formation were characterized by quantum chemical computation and juxtaposed to the aggregachromism in UV/Vis spectra. The applicability of the dyes as fluorogenic nucleic acid probes has been proven by fluorescence titration, and their binding constants have been calculated. The mode of ligand-dsDNA/RNA interaction was rationalized by means of CD spectroscopy, molecular docking analysis, and fluorescent intercalator displacement experiments.

Chemical characterization, antioxidant activity, -amylase and acetylcholinesterase inhibitory potential of Angelica pancicii Vandas ex Velen / Caracterización química, actividad antioxidante, potencial inhibidor de -amilasa y acetilcolinesterasa de Angelica pancicii Vandas ex Velen

Abstract: Comparative study GC - FID /M S of essential oils of fruits, leaves and roots of the endemic plant Angelica pancicii Vandas ex Velen. revealed a significant difference in their chemical composition. The enantiomeric purity of the main component in the fruit oil (+) - ß - phellandrene was a lso confirmed. In addition, imperatorin, isoimperatorin, oxypeucedanin, oxypeucedanin hydrate, angeloylpangelin and umbelliprenin were isolated from the fruit hexane extract. The content of these coumarins in the hexane extracts from different plant parts was further determined by HPLC. The essential oils and hexane extracts were assessed for their antioxidant potential and inhibitory effect towards  - amylase and acetylcholinesterase enzymes. The fruit and leaf essential oils (> 80%) as well as the fruit he xane extract (> 62%) significantly inhibited acetylcholinesterase enzyme. Distinguish free radical scavenging properties were detected for the leaf (Inh. 95.0 ± 2.2 %) and the root (Inh. 66.0 ± 2.4 %) extracts.

Resumen: Estudio comparativo GC - FID / MS de aceites esenciales de frutas, hojas y raíces de la planta endémica Angelica pancicii Vandas ex Velen revelaron una dife rencia significativa en su composición química. También se confirmó la pureza enantiomérica del componente principal del aceite de fruta (+) - ß - felandreno. Además, se aislaron imperatorina, isoimperatorina, oxipeucedanina, hidrato de oxipeucedanina, angeloi lpangelina y umbeliprenina del extracto de hexano del fruto. El contenido de estas cumarinas en los extractos de hexano de diferentes partes de la planta se determinó adicionalmente mediante HPLC. Los aceites esenciales y extractos de hexano se evaluaron p or su potencial antioxidante efecto inhibidor de las enzimas -  - amilasa y acetilcolinesterasa. Los aceites esenciales de frutas y hojas (> 80%), así como el extracto de hexano de frutas (> 62%) inhibieron significativamente la enzima acetilcolinesterasa. Se detectaron propiedades de captación de radicales libres diferenciadas para los extractos de hoja (Inh. 95,0 ± 2,2%) y de raíz (Inh. 66,0 ± 2,4%).

Study on the Neuroprotective, Radical-Scavenging and MAO-B Inhibiting Properties of New Benzimidazole Arylhydrazones as Potential Multi-Target Drugs for the Treatment of Parkinson's Disease.

Oxidative stress is a key contributing factor in the complex degenerating cascade in Parkinson's disease. The inhibition of MAO-B affords higher dopamine bioavailability and stops ROS formation. The incorporation of hydroxy and methoxy groups in the arylhydrazone moiety of a new series of 1,3-disubstituted benzimidazole-2-thiones could increase the neuroprotective activity. In vitro safety evaluation on SH-SY5Y cells and rat brain synaptosomes showed a strong safety profile. Antioxidant and neuroprotective effects were evaluated in H2O2-induced oxidative stress on SH-SY5Y cells and in a model of 6-OHDA-induced neurotoxicity in rat brain synaptosomes, where the dihydroxy compounds 3h and 3i demonstrated the most robust neuroprotective and antioxidant activity, more pronounced than the reference melatonin and rasagiline. Statistically significant MAO-B inhibitory effects were exerted by some of the compounds where again the catecholic compound 3h was the most potent inhibitor similar to selegiline and rasagiline. The most potent antioxidant effect in the ferrous iron induced lipid peroxidation assay was observed for the three catechols-3h and 3j, 3q. The catecholic compound 3h showed scavenging capability against superoxide radicals and antioxidant effect in the iron/deoxyribose system. The study outlines a perspective multifunctional compound with the best safety profile, neuroprotective, antioxidant and MAO-B inhibiting properties.

New C2- and N3-Modified Thieno[2,3-d]Pyrimidine Conjugates with Cytotoxicity in the Nanomolar Range.

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, aza- Michael 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 oncogenic V599EB-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 oncogenic V599EB-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 mutant V599EB-Raf showed that the compounds might be able to stabilize the enzyme in its inactive form.

Modulation Effect on Tubulin Polymerization, Cytotoxicity and Antioxidant Activity of 1H-Benzimidazole-2-Yl Hydrazones.

1H-benzimidazol-2-yl hydrazones with varying hydroxy and methoxy phenyl moieties were designed. Their effect on tubulin polymerization was evaluated in vitro on porcine tubulin. The compounds elongated the nucleation phase and slowed down the tubulin polymerization comparably to nocodazole. The possible binding modes of the hydrazones with tubulin were explored by molecular docking at the colchicine binding site. The anticancer activity was evaluated against human malignant cell lines MCF-7 and AR-230, as well as against normal fibroblast cells 3T3 and CCL-1. The compounds demonstrated a marked antineoplastic activity in low micromolar concentrations in both screened in vitro tumor models. The most active were the trimethoxy substituted derivative 1i and the positional isomers 1j and 1k, containing hydroxy and methoxy substituents: they showed IC50 similar to the reference podophyllotoxin in both tumor cell lines, accompanied with high selectivity towards the malignantly transformed cells. The compounds exerted moderate to high ability to scavenge peroxyl radicals and certain derivatives-1l containing metha-hydroxy and para-methoxy group, and 1b-e with di/trihydroxy phenyl moiety, revealed HORAC values high or comparable to those of well-known phenolic antioxidants. Thus the 1H-benisimidazol-2-yl hydrazones with hydroxy/methoxy phenyl fragments were recognized as new agents exhibiting promising combined antioxidant and antineoplastic action.

Molecular Mechanism of the Anti-Inflammatory Action of Heparin.

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Anti-Idiotype scFv Localizes an Autoepitope in the Globular Domain of C1q.

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.

Evaluation of the combined activity of benzimidazole arylhydrazones as new anti-Parkinsonian agents: monoamine oxidase-B inhibition, neuroprotection and oxidative stress modulation.

Neuroprotective drugs and selective monoamine oxidase inhibitors can slow down the progression and improve symptoms of Parkinson's disease (PD). Since there is an implication of oxidative stress in the pathophysiological mechanisms of the disease, the compounds possessing an ability to reduce the oxidative stress are prime candidates for neuroprotection. Thereby our current study is focused on the development of new multi-target PD drugs capable of inhibiting the activity of monoamine oxidase-B while exerting neuroprotective and antioxidant properties. A small series of benzimidazole derivatives containing hydroxy and methoxy arylhydrazone fragments has been synthesized and the neurotoxicity of the compounds has been evaluated in vitro on neuroblastoma SH-SY5Y cells and on isolated rat brain synaptosomes by measuring the cell viability and the levels of reduced glutathione and a good safety profile has been shown. The 2-hydroxy-4-methoxy substituted arylhydrazone 7 was the least toxic on neuronal SH-SY5Y cells and showed the lowest neurotoxicity in rat brain synaptosomes. The neuroprotective properties of the test compounds were further assessed using two models: H2O2-induced oxidative stress on SH-SY5Y cells and 6-hydroxydopamine-induced neurotoxicity in rat brain synaptosomes. Compound 7 showed more pronounced neuroprotective activity on SH-SY5Y cells, compared to the referent melatonin and rasagiline. It also preserved the synaptosomal viability and the reduced glutathione levels; the effects were stronger than those of rasagiline and comparable to melatonin. All the tested compounds were capable to inhibit human monoamine oxidase-B enzyme to a significant extent, however, compound 7 exerted the most prominent inhibitory activity, similar to selegiline and rasagiline. The carried out molecular docking studies revealed that the activity is related to the appropriate molecular structure enabling the ligand to enter deeper in the narrow and highly lipophylic active site pocket of the human monoamine oxidase-B and has a favoring interaction with the key amino acid residues Tyr326 and Cys172. Since much scientific evidence points out the implication of iron dyshomeostasis in PD, the compounds were tested to reduce the ferrous iron induced oxidative molecular damage on biologically important molecules in an in vitro lecithin containing model system. All the investigated compounds denoted protection effect, stronger than the one of the referent melatonin. In order to support the assignments of the significant neuroprotective and antioxidant pharmacological activities, the radical-scavenging mechanisms of the most promising compound 7 were evaluated using DFT methods. It was found that the most probable free radicals scavenging mechanism in nonpolar phase is the hydrogen atom transfer from the amide group of compound 7, while in polar medium the process is expected to occur by a proton transfer. The current study outlines a perspective leading structure, bearing the potential for a new anti-PD drug. All performed procedures were approved by the Institutional Animal Care Committee of the Medical University of Sofia (Bulgarian Agency for Food Safety with Permission № 190, approved on February 6, 2020).

Chemical evolution: from formamide to nucleobases and amino acids without the presence of catalyst.

Abiotic synthesis of nucleobases and amino acids is of critical importance as it sheds light on potential prebiotic chemical reactions. During thermal decomposition of formamide in vacuum conditions, purine, cytosine, adenine, hypoxanthine, uracil, pterin, urea, urocanic acid, glycine, alanine and norvaline were detected. The compounds were obtained without catalyst by heating at 100-180 °C or microwave heating of formamide. Reaction network of self-catalyzed chemical reactions is suggested, showing how from only one parent molecule, nucleobases, urea and the amino acid glycine can be produced. The reaction pathways are theoretically determined using SCS-MP2 calculations.Communicated by Ramaswamy H. Sarma.

Biological activity of quinazoline analogues and molecular modeling of their interactions with G-quadruplexes.

BACKGROUND: Quinazolines 1 to 6, with an aromatic or aryl-vinyl substituent in position 2 are selected with the aim to compare their structures and biological activity. The selection includes a natural alkaloid, schizocommunin, and the synthetic 2-(2'-quinolyl)-3H-quinazolin-4-one, known to interact with guanine-quadruplex dependent enzymes, respectively telomerase and topoisomerase. METHODS: Breast cancer cells of the MDA cell line have been used to study the bioactivity of the tested compounds by the method of Comet Assay and FACS analyses. We model observed effects assuming stacking interactions of studied heterocycles with a naked skeleton of G-quadruplex, consisting of guanine quartet layers and potassium ions. Interaction energies are computed using a dispersion corrected density functional theory method, and an electron-correlated molecular orbital theory method. RESULTS: Selected compounds do not remarkably delay nor change the dynamics of cellular progression through the cell cycle phases, while changing significantly cell morphology. Our computational models quantify structural effects on heterocyclic G4-complex stabilization energies, which directly correlate with observed biological activity. CONCLUSION: Our computational model of G-quadruplexes is an acceptable tool for the study of interaction energies of G-quadruplexes and heterocyclic ligands, predicting, and allowing design of novel structures. GENERAL SIGNIFICANCE: Genotoxicity of quinazolin-4-one analogues on human breast cancer cells is not related to molecular metabolism but rather to their interference with G-quadruplex regulatory mechanisms. Computed stabilization energies of heterocyclic ligand complexes of G-quadruplexes might be useful in the prediction of novel telomerase / helicase, topoisomerase and NA polymerase dependent drugs.

Structure-activity relationship study on new hemorphin-4 analogues containing steric restricted amino acids moiety for evaluation of their anticonvulsant activity.

In the present study, several new analogues of hemorphin-4, modified with unnatural conformationally restricted amino acids followed the structure Aaa-Tyr-Xxx-Trp-Thr-NH2, where Aaa is the low-molecular-weight lipophilic adamantyl building block, and Xxx is Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) was synthesized, characterized and investigated for anticonvulsant activity in three seizure tests, the maximal electroshock test (MES), 6-Hz psychomotor seizure test and timed intravenous pentylenetetrazole infusion (ivPTZ) test. The acute neurological toxicity was determined using the rota-rod test. The new synthetic neuropeptide analogues were prepared by solid-phase peptide synthesis-Fmoc chemistry and were evaluated in three doses of 1, 3 and 5 µg, respectively, administered intracerebroventricularly in male ICR mice. The physicochemical properties of these peptide analogues were evaluated as pKa and pI values were calculated using potentiometry. The IR spectrum of the compounds was recorded and the characteristic lines of both adamantane moiety and the peptide backbone were registered in the wavelength range from 4000 to 400 cm-1. The hexapeptide Ang IV was used as a positive control. From the six synthesized peptide analogues, the P4-5 was the most active at doses of 1 and 3 µg in the three seizure tests. The order of potency of other peptides was as follows: P4 > P4-3 = P4-4 > P4-2 > Ang IV in MES, P4-4 ≥ P4-1 > P4-3 > P4-2 > P4 > Ang IV in 6-Hz test and P4-4 = P4-3 > P4-2 = P4 > Ang IV in ivPTZ test. None of the peptides displayed neurotoxicity in the rota-rod test. Docking study results suggest that direct H-bonding and ionic interactions between our synthetic ligands and residues, responsible for coordination of Zn2+ along with hydrophobic interactions between our ligands and IRAP active site are the most important for the ligand binding. The results propose that incorporation of adamantane and cycloalkane building blocks in the peptide chain of the hemorphin-4 scaffold is important for the potential high biological activity.

Caffeoylquinic Acids, Cytotoxic, Antioxidant, Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria.

Chlorogenic (5-CQA), 1,5-, 3,5-, 4,5- and 3,4-dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus-christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess.) DC. and I. germanica L. by HPLC analysis. The amount of 5-CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5-DCQA (4.05-55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5-DCQA, 4,5-DCQA and 3,4-DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 µg/mL toward non-cancer (MDCK II) and cancer (A 549) cells.

Binding of Gold(III) Porphyrin by the Pro-metastatic Regulatory Protein Human Galectin-3.

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.

Synthesis, characterization and anticonvulsant activity of new series of N-modified analogues of VV-hemorphin-5 with aminophosphonate moiety.

A new series of N-modified analogues of the VV-hemorphin-5 with aminophosphonate moiety have been synthesized, characterized and investigated for anticonvulsant activity. The novel peptide analogues were prepared by solid-phase peptide synthesis-Fmoc-strategy and were evaluated in the timed intravenous pentylenetetrazole infusion test (ivPTZ) and 6-Hz psychomotor seizure test in mice. The acute neurological toxicity was determined using the rotarod test. The redox potentials at glass carbonic electrode (GC) and the acid/base properties as pKa values of these peptide analogues were compared with the electrochemical behaviour of tyrosine- and tryptophan-containing peptides using different voltamperometric modes. Among the five tested peptide analogues, V3p was the most active against the ivPTZ test with effect comparable to that of the VV-hemorphin-5 (V1) used as a positive control. Dose-dependent elevation of the seizure threshold for myoclonic twitch and generalized clonic seizures was observed after i.c.v. administration of V2p, V4p and V5p as well as for forelimbs tonus in V4p peptides. The peptide analogues V2p-V5p were able to suppress dose-dependent psychomotor seizures in the 6-Hz test. In contrast, the V6p peptide showed either a pro-convulsant effect in the iv PTZ test or was inactive in the 6-Hz test. No changes in motor coordination were noted with the novel peptides. Docking study results suggest that kappa opioid receptor binding could be the mechanism of action of peptide derivatives with anticonvulsant activity. The results suggest that incorporation of aminophosphonate moiety at position 1 of the VV-hemorphin-5 scaffold deserve further evaluation in models of epilepsy and derivatization.