Introduction: Nucleoside analogues have a special place among the most effective antiviral drugs and the study of fluorinated nucleoside sugars has led to the development of novel promising chemotherapeutic agents. Our work is dedicated to the determination of cytotoxicity and antiviral activity towards HSV-1, HSV-2 and HAdV5 of such modified nucleosides.

Methodology: Cells and viruses: cell culture MDBK (bovine kidney), adenovirus serotype 5 (HAdV5), herpes simplex virus 1 and 2 (HSV-1/US, HSV-2/BH) were used.

Tested substances: analogs of nucleoside based on 5-tosyl-6-(trifluoromethyl)uracile (G26), namely 2-N-substituted-4-tosyl-5-(perfluoroalkyl)-1,2,3-triazoles (G8 and G9); polyfluoro-substituted thiopeptide analogs, namely t-butyl(tetrafluoropropanethioyl)-L-alaninate (10S-20), methyl(tetrafluoropropanethioyl)-L-phenylalaninate (10S-21), methyl(tetrafluoropropanethioyl)-D-tryptophanate (10S-22), sodium (polyfluoroalkanethioyl)-L-phenylalaninate (10S-23 and 10S-24).

Cytotoxicity of the compounds was determined by MTT-test or neutral red dye (NR) according to standard protocols. Antiviral effect was determined using PCR, MTT-test and cytomorphological methods. This method was used to identify adenovirus infected cells containing specific virus inclusion. Cells were treated with the compounds in the growth medium after virus adsorption at non-toxic concentrations.

Results: According to the MTT test results, CC₅₀ values of compounds G8 and G9 were 887 and 990 mg/ml, respectively. Other compounds demonstrated the following CC₅₀ values, G26: 532 mg/ml (MTT) and 985 mg/ml (NR); 10S-22: 290 mg/ml (MTT) and 554 mg/ml (NR). Compound 10S-23 showed CC₅₀ value of >1000 mg/ml (MTT, NR). CC₅₀ values obtained using NR were higher than the previous results for the compounds 10S-20, 10S-21, 10S-24 (2022, 1585, and 2519 mg/ml, respectively). CC₅₀ values assessed via MTT test for these compounds were lower – 653, 394 and >1000 mg/ml, respectively. Inhibition of HAdV5 reproduction was demonstrated for three compounds G8, G26 and 10S-23. PCR revealed inhibition of virus reproduction (13 %) for compound G8 at a concentration 62 mg/ml, 95-100 % for G26 at concentrations 31-62 mg/ml and 31 % for 10S-23 at a concentration of 36 mg/ml. The results of cytomorphologycal method showed the following values of EC₅₀ for the compounds: 16, 120, 90 mg/ml, respectively. Using MTT-test it was shown that compounds G8, G9, 10S-20, 10S-23 and 10S-24 suppressed HSV-1/US reproduction by 50 % at concentrations of 50, 59, 62, 71, 35 mg/ml, respectively. Compounds 10S-21 and 10S-22 had lower efficiency and suppressed virus reproduction by 37% and 22%, respectively. Compounds G8, G9, G26 suppressed HSV-2/BH reproduction by 50% at the concentrations of 56, 71 and 44 mg/ml, respectively. 10S-23 suppressed only 12% of the virus at the concentration of 62 mg/ml. Therefore, two compounds G8 and 10S-23 inhibited the reproduction of all viruses with different efficiency. The compound G9 was active only in relation to both types of herpes. G26 suppressed reproduction of HSV-2/BH and HAdV5, whereas compounds 10S-20 and 10S-24 were active only in relation to reproduction HSV-1/US.

Conclusion: The tested compounds emerged as promising hits for further in-depth in vitro and in vivo studies

Casein Kinase 2 (CK2) is an ubiquitous hetrotetrameric eukaryotic serine/threonine protein kinase. CK2 has important roles in different cellular functions. This enzyme enhances cancer phenotype by blocking apoptosis and stimulating cell growth. Thus, inhibition of CK2 can induce the physiological process of apoptosis leading to tumor cell death. Large number of compounds has been developed as kinase inhibitors including CK2 inhibitors. Most of the CK2 inhibitors contain a heterocyclic backbone which fits into the active site of the CK2α subunit and competes with the ATP. Recently we reported on the development of compound 3: di 2,6-Di(furan-3-yl)anthracen-9,10-dion as an active CK2 inhibitor.

With the aim of finding new inhibitors for this target a series of di-substituted 9,10- Dihydrophenanthrene was designed as CK2 inhibitors and docked in crystal structure of the enzyme to evaluate their ability to fit in the active site. The main idea was to explore if the 9,10-Dihydrophenanthrene backbone is more suitable for binding than the backbone of compound 3 and to determine if the introduced functional groups can form hydrogen bonds with the amino acid residue. Some of the designed compounds were synthesized and tested in vitro as well.

Increased protein kinase CK2 activity is involved in many human diseases such as cancer (1). In consequence CK2 is an emerging major target for drug design. Several indeno[1,2-b]indole-9,10-dione derivatives containing N⁵-isopropyl substitutions on the C-Ring were synthesized and have been reported as potent ATP-competitive CK2 inhibitors (2,3).

Here we report on the evaluation of these inhibitors, containing different substituents in the A- and D-rings, for their effects on various tumor cell lines: breast cancer cells MCF-7, lung carcinoma cells A427 and epidermal cancer cell line A431.

The most potent CK2 inhibitor contains an O-prenyl residue R₁ and exhibits an IC₅₀ value of 0,025 µM. Treatment of MCF-7 cells with 20 µM of that compound for 24 h results in a reduction of the total cell number by 90%. Most of the remaining cells exhibited apoptotic morphology and showed nearly none proliferating activity. In contrast treatment of A431 cells and A427 cells caused only a moderate decrease of cell proliferation by 30% for all tested compounds.

This study shows that potent CK2 inhibitors as tested can exhibit distinct effects on different tumor cell lines. Compound containing an O-prenyl residue R₁ appears to be an antiproliferating agent with high activity toward MCF7 cells.

 References

1. Guisiano, S. et al: Eur. J. Cancer 2011, 47, 792-801
2. Alchab, F. et al : Pharmaceuticals 2015, 8, 279-302
3. Gozzi, et al: J. Med. Chem 2015, 58, 265-277

Human milk protein α_S1-casein (CSN1S1) was shown to be overexpressed in autoimmune diseases (osteoarthritisbenign prostatic hyperplasia, multiple sclerosis) as well as in cancer. CSN1S1 displays opioid-like activity and modulates the innate immune response of intestinal cells. Recently, it was demonstrated, that CSN1S1 induces the expression of proinflammatory cytokines (IL‑1β and IL‑6) in monocytic cells via MAPK-p38 signaling [1, 2].

In this study the human TLR4 receptor, a receptor of the innate immune system, was identified as interaction partner of human CSN1S1 inducing expression of cytokines IL‑1β, IL‑6 and IL‑8 in human monocytic cells concentration- and time-dependently [3]. In transfected HEK293 cells expressing TLR4, human CSN1S1 (purified from Escherichia coli) induced secretion of chemokine IL-8. In vitro flow cytometric assays confirmed CSN1S1 - TLR4 receptor interaction. Chemokine secretion as well as binding was not detected for CSN1S1 phosphorylated by protein kinase CK2 as well as for heat denaturated CSN1S1. This supports the hypothesis, that CSN1S1 is a ligand of the TLR4 receptor exerting proinflammatory properties in a phosphorylation-dependent manner.

Literature:

[1] S. Vordenbäumen, et al. BMC Immunol, 2013, 14, 46.

[2] S. Vordenbäumen, et al. J Immunol, 2011, 186, 592.

[3] S. Vordenbäumen, T. Saenger et al. Mol Nutr Food Res, 2016, 60, 1079.

Searching for new enantiomerically pure chiral derivatives of xanthones (CDXs) with potential pharmacological properties has remained an area of interest of our group, namely those with anti-inflammatory activity [1,2]. Herein, we describe in silico studies and in vitro inhibitory assays of different enantiomeric pairs of CDXs. The evaluation of the inhibition of cyclooxygenases (COX-1 and COX-2) activities was performed by using the COX Inhibitor Screening Assay Kit. Docking simulations between the small molecules (CDXs, known ligands and decoys) and the enzyme targets were undertaken with AutoDock Vina embedded in PyRx – Virtual Screening Tool software. All the CDXs evaluated exhibited COX-1 and COX-2 inhibition potential as predicted.

Considering that the (S)-(-)-enantiomer of the nonsteroidal anti-inflammatory drug Ketoprofen preferentially binds to albumin, resulting in lower free plasma concentration than (R)-(+)-enantiomer [3], protein binding affinity for CDXs was also evaluated by spectrofluorimetry. For some CDXs enantioselectivity was observed.

[1] Fernandes, C., et al. Bioorg Med Chem. 2014, 22(3), 1049-1062.

[2] Fernandes, C., et al. Eur. J. Med. Chem. 2012, 55, 1-11.

[3] Evans, S. E. et al. Trends in Environmental Analytical Chemistry, 2014, 1(0), e34-e51.

This research was partially supported by the Structured Program of R&D&I INNOVMAR –Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF) and by Foundation for Science and Technology (FCT) and COMPETE under the projects PTDC/MAR-BIO/4694/2014 (POCI-01-0145-FEDER-016790) and COXANT–CESPU- 2016.

A library of 69 synthetic benzenesulfonyl derivatives of heterocycles, with drug-like properties, was assayed for in vitro antiparasitic activity and the results were added to our previous reports for a comprehensive SAR discussion. Seven compounds showed an IC₅₀ between 0.25-3μM against L. donovani and low cytotoxicity. Compound 1-(2,3,5,6-tetramethylphenylsulfonyl)-2-methylindoline (G16), was particularly interesting with an IC₅₀ similar to the reference drug miltefosine. In addition, seven compounds showed an IC₅₀ below 6µM against T. cruzi, and three of them (E3, E9 and G3) were identified as lead scaffolds for further optimization based on their activity-toxicity profile. Furthermore, two promising structures (B15 and G15) have shown moderate inhibitory activity against P. falciparum. In general, the presence of a benzenesulfonyl moiety improves the antiparasitic activity of the heterocycles included in this study (with exception of T. b. rhodesiense) validating the criteria used in the selection of the fragment-based drug design approach. Finally, from the SAR analysis it could be concluded that the presence of electron withdrawing and lipophilic groups were favorable for the antiparasitic activity.

Inhibition of virus-cell interactions, which are required for virus replication, constitutes an exciting area of research for the discovery of novel antiviral agents. RNA based drugs are in clinical use for treatment of infectious diseases as immunomodulators, anti-inflammatory and antiviral agents. Nuclex, a low molecular weight exogenous yeast RNA, consists of a pure, homogeneous 25-membered oligonucleotide. The preventive and therapeutic effects of Nuclex were demonstrated against pandemic strains of influenza virus, parainfluenza virus, Hepatitis C, Herpes simplex virus types 1 and 2, cytomegalovirus, and HIV on experimental models in vitro and in vivo. The mechanism of the antiviral action of the drug relies on its ability to change conformations of cell regulatory proteins and viral coat proteins. An antiviral effect of Nuclex against Epstein-Barr virus (EBV) etiological agent of infectious mononucleosis associated with a number of lymphoproliferative and autoimmune diseases was studied in this work.

The study was performed with EBV-positive human B-cells (Raji) – latently infected with EBV, and EBV-producing tamarin (Saguinus oedipus) B-cell line (B95-8). The drug cytotoxicity was assessed by MTT-method. As a result of investigations it was found that the drug has low toxicity relative to both lymphoblastoid cell cultures. The inhibitory concentrations suppressing 10% of cell viability (IC₁₀) were 2000 µg/ml and IC₅₀s (50% inhibited cell viability) were 5000 µg/ml for both cell lines. An antiviral activity of Nuclex was measured by PCR in EBV infected cells Raji, as a model of acute infection and in non-induced B95-8 cells as a model of chronic EBV infection after 48 hours incubation. The drug effectively inhibited viral replication in the studied concentration range from 10 to 500 µg/ml in EBV-infected Raji cells and B95-8 cells. Effective concentration of Nuclex that inhibited 90% level of the viral DNA accumulation (EC₉₀) was 50 µg/ml for in both cell lines. A virucidal activity of the drug was investigated considering the ability of Nuclex to influence the conformation of the viral surface proteins. The levels of inhibition of the EBV DNA accumulation were 30% after incubation of the drug at the concentration 100 µg/ml with the EBV for the 30 and 60 min exposures and the subsequent infection of Raji cells incubated for 48 hours, while 50% inhibition of viral DNA accumulation was observed at the drug concentration 200 µg/ml under 30 min exposure and 30% at 60 min exposure.

Thus Nuclex showed a strong antiviral effect against EBV. Considering an antiviral activity of the drug (EC₉₀) at 50 µg/ml, and a virucidal action EC₅₀ at 200 µg/ml, determination of its mechanism of action, which could be related to siRNA properties, will constitute the next stage of our research.

Tuberculosis remains a serious health problem responsible to cause millions of deaths annually. The scenario becomes alarming when it is evaluated that the number of new drugs does not increase proportionally to the emergence of resistance to the current therapy. Furoxan derivatives, known as nitric oxide donors, have been described to exhibit a wide range of biological activities, including antitubercular. Herein, a novel series of twelve hybrid furoxanyl (1,2,5-oxadiazole 2-N-oxide) derivatives was designed, synthesized and evaluated in vitro against Mycobacterium tuberculosis H37Rv using the Resazurin Microtiter Assay (REMA) method. The furoxan derivatives have exhibited MIC₉₀ values ranging from 1.03 to 62 µM. For most active compounds, the selectivity index ranged from 3.78 – 52.74 (MRC-5) and 1.25 – 34.78 (J774A.1). Furthermore, these most active compounds were also evaluated against a clinically isolated multi-drug resistant strain (isoniazid, rifampicin, streptomycin and etambutol) and exhibited MIC₉₀ values ranging from 1.44 to 5.63 µM. The MIC₉₀ values of the four selected compounds were greater than several first and second line antitubercular drugs, such as ethambutol (2.4 µM), pyrazinamide (>48 µM), cycloserine (245 µM) and kanamycin (3.4 µM). In addition for those compounds, values of logP_o/w vary between 2.1 – 2.9, reaching the optimal range for oral administration. The amount of nitric oxide (NO) released was indirectly detected by Griess reaction through the measurement of nitrites in the medium. All compounds were able to release NO at levels ranging from 0.16 – 44.23%. Among the series, the phenylsulfonylfuroxan derivatives were the best NO-donor with the lowest MIC₉₀ values. The most active compound was also stable at different pHs in a chemical stability study (5.0 and 7.4). In conclusion, furoxan derivatives were identified as new promising compounds useful to treat sensitive and resistant tuberculosis. Currently, metabolomics and transcriptome analysis are underway in order to elucidate the possible mechanism of action of these compounds as well as an antitubercular activity study in vivo for the most potent furoxan derivative.

The interest in determining the influence of new metal complexes on microorganisms is increasing due to the growing pathogenic resistance. This investigation showed influence of 5 new Pt(IV) complexes on 16 strains of bacteria. Antibacterial activity was tested using microdilution method with resazurin while antibiofilm activity was observed by tissue culture plate method and antibiotic doxycycline was used as positive control. The results were expressed as minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm inhibitory concentration (BIC). The complexes were labeled as: C1 for Pt(S-bz-thiosal)₃, C2 for Pt(S-met-thiosal)₃, C3 for Pt(S-et-thiosal)₃, C4 for Pt(S-pr-thiosal)₃ and C5 for Pt(S-bu-thiosal)₃. The best result on Gram positive bacteria was obtained with C1 and MIC on Staphylococcus aureus ATCC 25923 was Ë‚7.81 μg/ml. Bifidobacterium animalis subsp. lactis (probiotic) was sensitive to C2 (MIC at 15.625 μg/ml). The best sensitivity on Gram negative bacteria was observed on Escherichia coli ATCC 25922 with C1, C2, C3 and C4, on Proteus mirabilis ATCC 12453 with C1, and on Pseudomonas aeruginosa with C2, C3 and C5 (all MICs at 250 μg/ml). The tested complexes were more efficient as antibiofilm agents and the best results were obtained with C2 acting against S. aureus and S. aureus ATCC 25923 biofilm.

In conclusion, we noticed that the tested compounds exhibited promising properties as antibacterial agents and antibiofilm agents, 

Introduction. Tilorone (amixin, dihydrochloride 2,7-bis[2-(diethylamino)ethoxy] fluorenone-9) is a low molecular interferon inducer that is effective against a wide range of viral infections including herpes viruses. The mechanism of tilorone antiviral action is associated with inhibition of virus-specific peptides translation in infected cells resulting in the suppression of virus replication. An appropriate therapy of the viral infections with external rash besides the systemic preparations must include topical drugs that will reduce clinical signs of infection, improve skin epithelialization, and decrease time of virus elimination. Considering this, we developed a new topical antiviral 2% tilorone preparation in the form of ointment.

The aim of investigation was to study the parameters of acute toxicity of the 2 % tilorone ointment.

Results and Discussion. In accordance with the requirements of the European Pharmacopoeia 8.0 the acute toxicity of the developed ointment was studied using two methods – intragastric administration and cutaneous application. All animals were treated in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [Council of Europe, Strasbourg, 2006].

Acute intragastric toxicity was carried out in two species of rodents [54 white outbred mice (20-25 g) and 27 white Vistar rats (200-220 g)] that were given intragastrically an aqueous solution of ointment at a dose of 0,5g/kg. Acute dermal toxicity was studied in 27 white outbred mice (20-25 g); the application area was 5,5-6,0 cm².

The animals were monitored daily for clinical signs (breathing, physical activity, convulsions, analgesia, muscle tone, ophthalmic, cardiovascular and gastrointestinal symptoms, diuresis, skin signs) in the course of 14 days. All the animals remained alive and active; the distinctive symptoms of intoxication were absent. It was also determined that an effective dose of 2% test ointment does not cause toxic effects on human body is DL₀≤0,4 g/kg.