In our laboratory, we focus on the design, synthesis, and evaluation of fluorinated compounds that could be used in the tretament of diseases caused by the Epstein-Barr virus (EBV) and virus vesicular stomatitis (VVS). EBV is a DNA-containing virus. It is characterized by an acute form of infection as well as a latent form, which may lead to cell transformation and to cancer (lymphoma, carcinoma). VVS is a RNA-containing virus.

For this purpose, in a first step, we used the PASS software by which we could identify potential antiviral candidates among which a fluorinated derivative of uracil (G27), a trifluoromethyl-substituted derivative of a thiosugar (SBIO6), a bisphosphonic acid (10s19) and several derivatives of alanin and glucose (10s20 - 10s28).

Our in vitro studies revealed an antiviral activity for a few compounds. Three compounds appeared to be effective against EBV: G27 (EC₅₀ = 100 µg/ml), 10s20 (EC₅₀ =  µg/ml), and 10s25 (EC₅₀ <62 µg/ml, replication of EBV was suppressed at 100 % at a concentration of 62 µg/ml). The ability to inhibit reproduction of VVS was showed for compound 10s19 (EC₅₀ = 19 µg/ml) and for compound 10s24 (EC₅₀ = 29 µg/ml). It was established that the index of selectivity for these compounds ranged from 10 to 100.

As Epstein-Barr virus (EBV) is the cause of several lymphoproliferative diseases, we studied the potency of G27 and SBIO6 compounds to make an apoptosis induction. Apoptotic cells were detected using a flow cytometry. Addition of G27 led to the observation of two peaks in the histogram, what may indicate a break of the cell cycle by this compound. It was also established that by addition of SBIO6 the percentage of apoptotic cells was significantly increased when compared to the control and reached 70 - 90 percent. 

Heat shock proteins (Hsps) are important biological targets in th next generation of cancer treatments. Hsps play vital roles in protein hemostasis pathways (proper folding and stabilization of nascent proteins, inhibition of protein aggregation, degradation of aggregated proteins, signal transduction and protein translocation) in eukaryotic and prokaryotic cells. Hsps are found in different cellular compartments and their expression level is increased in response to cellular and external stress factors (tumorogenesis, UV light, hypoxia, oxidative, infection, stress, fever, temperature variation) [1]. Therefore, pathogenesis of diseases is related with expression level of Hsps. Hsps are over-expressed in cancer cells, and especially, Hsp27, Hsp70 and Hsp90 are involved in all phases of tumorogenesis (apoptosis, metastases, angiogenesis, invasion, and cell differentiation). Hsp27, Hsp70 and Hsp90 ensure stabilization, activation and proper folding of the oncogenic proteins in cancer cells. Therefore, inhibition of Hsps emerged as a significant therapeutic strategy for targeted cancer treatments. Inhibition of Hsp90 chaperone activity has been significant drug- targeted for the past 30 years in cancer treatment. Now, approximately 20 different compounds are in clinical phase studies [2,3]. Clinical and pre-clinical studies demonstrated that inhibition of Hsp90 activity is not enough by itself. Inhibition of Hsp90 triggers expression of Hsp70 and complements inhibited Hsp90 chaperone activity. Moreover, Hsp27 controls and regulates key points of the apoptotic pathway in cancer cells. Therefore, in addition to Hsp90 inhibition, blocking of Hsp70 and Hsp27 chaperone activities is a remarkable therapeutic strategy for cancer treatment [4].  

In our lab, we designed and synthesized novel pyrimidine and coumarin derivatives as Hsp90 inhibitors. Pyrimidine analogs interrupted Hsp90 ATP hydrolysis process through disrupting N terminal domain (NTD) conformational change [5]. Coumarin derivatives inhibited C terminal domain (CTD) of Hsp90, and blocked dimerization process [6]. 

ACKNOWLEDGEMENTS

 The authors gratefully acknowledge the financial support received from the Scientific and Technological Research Council of Turkey, TÜBİTAK (Grant # 114Z365).

 REFERENCES

[1] Tutar, L.; Tutar, Y. Heat shock proteins; an overview. Curr. Pharm.Biotechnol., 2010, 11, 216-222.

[2] Ozgur, A.; Tutar, Y. Heat shock protein 90 inhibitors in oncology. Curr.Proteo., 2014, 11, 2-16.

[3] Ozgur, A.; Tutar, Y. Heat Shock Protein 90 Inhibition in Cancer Drug Discovery: From Chemistry to Futural Clinical Applications. Anticancer Agents Med. Chem., 2016, 16, 280-90.

[4] Tutar, Y. Inhibition of Heat Shock Protein 70 and 90 (Hsp70 And Hsp90) in Target

Specific Cancer Treatment. Adv. Tech. Biol. Med., 2015, 3.

[5] Koca, İ.; Ozgur, A.; Er, M.; Gümüş, M.; Coşkun, K.A.; Tutar, Y. Design and synthesis of pyrimidinyl acyl thioureas as novel Hsp90 inhibitors in invasive ductal breast cancer and its bone metastasis. Eur. J. Med. Chem., 2016, 122, 280-290. 

[6] Koca İ, Gümüş M, Özgür A, Dişli A, Tutar Y. A novel approach to inhibit heat shock response as anticancer strategy by coumarine compounds containing thiazole skeleton. Anticancer Agents Med. Chem., 2015, 15, 916-930.

Antimicrobial peptides (AMPs) are anti-infective agents that may represent a novel and untapped class of biotherapeutics. In the lab of bioinformatics of IBCEB, the Database of Antimicrobial Activity and Structure of Peptides (DBAASPv.2 - accessible at http://dbaasp.org) has been developed. DBAASP provides information and analytical resources to the scientific community in order to develop antimicrobial compounds with high therapeutic index.

Quantitative structure-activity relationship (QSAR) studies for the development of predictive model for AMPs are generally based on discriminative analysis and especially machine learning methods. These methods, as a positive training set, have used a full set of antimicrobial peptide sequences, without taking into account variation in mechanisms of action, structure, mode of interaction with membrane and other differences. Contrary to available approaches, we think that strategy of prediction should be based on the fact that there are at least four kinds of AMPs for which four independent algorithms of prediction have to be developed in order to get high efficacy. We can distinguish linear cationic antimicrobial peptides (LCAP), cationic peptides stabilizing structure by intra-chain covalent bonds, proline and arginine-rich peptides, and anionic antimicrobial peptides.

Simple predictive model which can discriminate AMPs from non-AMPs has been developed for LCAP. Sequences have been taken from DBAASP.  As descriptors the sequence-based physical-chemical characteristics responsible for capability of the peptide to interact with an anionic membrane were considered. On the basis of these characteristics, a new simple algorithm of prediction is developed and in silico evaluation of the efficacy of characteristics is done. The algorithm was based on the clusterization of AMPs by their physicochemical properties. The results show that descriptors relied mainly on hydrophobic and hydrophilic features allow us to predict AMPs with the high accuracy.

The developed predictive model was used to create new amino acid sequences. On the basis  of  designed sequences  the  peptides  have  been  synthesized. Antimicrobial potency of  the new  peptides  has  been  evaluated   by  in vitro testing of peptides activity against  different  pathogenic   bacteria  (including  drug  resistant strains). In vitro  estimation  shows  that  the  accuracy  of  th developed  predictive  model  is higher than 90%.

Introduction. The polypeptide produced by monocyte-macrophage cells and known as tumor necrosis factor (TNF), acts not only as the cytotoxicity effector mediator ensuring destruction of cancer target cells, but also participates in the regulation of various physiological and pathological processes in the body. Research of the biological activity of TNF, especially the mechanisms of its implementation is important for the elaboration of indications and methods for its clinical application not only in the treatment of patients with malignant tumors, but also in other areas of clinical medicine, and for creation of new drugs that affect its production in the body [1].

Modifications of the substance of the target recombinant protein usually lead to improving the biological properties, increase activity and reduce the toxicity of TNF [2]. In our previous studies, it was shown that combining the nanoparticles of cerium dioxide (CND) with interferon [3] or vaccines [4] ensures the growth of their biological activity.  One of the possible mechanisms for implementation of the above-mentioned effect involves the nano-biocomposite formation. Based on the preliminary data, we have explored the process of interaction CND with TNF.

Materials and methods. The interaction of CND with TNF and the formation of nano-biocomposite lead to an increase in peptide hydrodynamic diameter (HD). HD was monitored via dynamic light scattering (DLS) method using the two angle particles and molecular size analyzer Zetasizer Nano ZS (Malvern, UK).  As a model object, the Human Recombinant Tumor Necrosis Factor-α (rhTNF-α) was used (Promega Corporation Part# 9PIG524). The rhTNF-α concentration was 3.8•10⁴ U/ml (1µg/ml). The CND (size ~5.8 [5,65-5,85] nm, ζ-potential ~ +12 mV) was synthesized by O.S. Ivanova (IGIC RAS) as an aqueous 0.01 M sol [5]. CND was added to the rhTNF-α in the weight ratio of 1:3 and analyzed ex tempore. Interaction of rhTNF-α with CND-sol was analyzed in dynamics. The measurements of the HD were carried out for 17 hours at intervals of 3-4 minutes. All measurements were performed at a constant temperature (25°C) and neutral media (pH=7.2). The obtained data were analyzed using methods of mathematical statistics and were presented as the median and interquartile range.

Results and discussion. The initial HD of rhTNF-α was ~0.62 [0.56-0.71] µm. Adding CND to rhTNF-α caused an increase of HD. Four hours after introduction of CND, the HD of solute was twofold increased. By the tenth hour, the size of the objects was ~1.57 [1.46-1.74] µm. In this time interval, the growth of the H|D of the particles was almost linear. In the range of 10 to 17 hours of observation, the CND-rhTNF-α nano-biocomposites size was stabilized. By the end of his observations, the HD was ~1.69 [1.66-1.74] µm. Thus, the modification of rhTNF-α by CND leads to the formation of stable nano-biocomposites having increased HD. Our preliminary in vitro study [6] showed that such nano-biocomposite of CND-modified rhTNF-α is more active than the pristine rhTNF-α (probably, due to adjuvant-mimic properties of CDN). The findings are the rationale for the creation of new highly efficient nanocomposite drugs.

References.

1) Gough DB, Winstanley FP et al. Regulation of tumor necrosis factor production in healthy humans and in patients with cancer. Arch Surg. – 1992; 127(6):713-717.

2) Pat. RF № 2,386,447 Anticancer drug based on nanoparticles bearing recombinant human tumor necrosis factor alpha. Masycheva VI, Lebedev LR, Danilenko ED. – Publ. 20.04.2010.

3) Zholobak N.M., Krivokchatska L.D., Shcherbakov A.B., et al. The influence of nanoceria dioxide colloid solution on the activity of viferon. Conference “Nanotechnology in oncology – 2010” – Moscow, 30.10.2010; P.44-48.

4) Zholobak N.M., Mironenko A.P., Shcherbakov A.B., et al. Cerium dioxide nanoparticles increase immunogenicity of the influenza vaccine. Antiviral Research 2016; 127; P.1-9.

5) Ivanov V.K., Polezhaeva O.S., Shcherbakov A.B., et al. Microwavehydrothermal synthesis of stable nanocrystalline ceria sols for biomedical uses. Russian Journal of Inorganic Chemistry, 2010; 55:1-5

6) Shydlovska O.A, Kharchenko E, Zholobak N.M., et al. Nanoparticles of cerium dioxide increase activity of recombinant tumor necrosis factor-alpha. XIV International Scientific Conference of Students, PhD Students & Young Scientists: Shevchenkivska vesna: Biology – 2016; P.23–24.

A series of 22 synthetic drug-like hydantoin derivatives, including aryl or alkenyl 5,5-disubstituted hydantoins, spirohydantoins and annulated bicyclic and tricyclic hydantoins, was assayed for in vitro antibacterial and antifungal activity. The antimicrobial activity was tested by determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) using microdilution method. The tested hydantoin derivatives showed moderate antibacterial and weak antifungal activity. The intensity of acting varied depending on the structure and concentration of the test substances and the type of test organisms. The bicyclic benzeneselenenyl derivatives of hydantoin have shown the highest inhibitory activity. The tested compounds appeared as promising for a fragment-based drug design approach and further bioactivity studies.

Aromatic compounds (aminopurine, pyridine, quinoline, fluorene…) are excellent building blocks for the development of new drugs. Fluorenone and Fluorene containing natural products display diverse structural features due to their various ring junctions and chiral centers. Together with potential bioactivities, this family of natural products aroused considerable attention from synthetic chemists. They have interesting physicochemical properties and could be used as fluorescent probes¹. They display also high therapeutic activities as anti-oxidant, anti-bacterial, anti-cancer, anti-malarial, anti-myocardial or anti-Alzheimer^2-7.

For example, Dendroflorin isolated from Dendrobium densiflorum, displays a high antioxidant activity. This compound is cytotoxic at 125.8 µg/mL and have an antimigratory effect at 1.0 µg/mL in 24h for H 460 cells (lungs cancer)³. Some 1,6-disubstituted fluorenones can also induce apoptosis and inhibited the tubulin polymerization in cells. They are active in the range of 0.15 to 0.29 µM for many cancer cells (T47D (breast), HCT 116 (colon), SNU 398 (carcinoma)) and represent thus interesting pharmacophore for the research of new anticancer drugs⁴. Isolated from the radix of Caulophyllum robustum Maxim, Caulophine has an anti-myocardial ischemia activity as a calcium antagonist⁵. Interestingly, some fluorenes have also shown a capacity to reduce the amyloid burden which induces severe neurodegeneration and cognitive deficits in Alzheimer’s disease⁶.

Thus, fluorenes are attractive scaffolds for the design of new therapeutic agents. In our laboratory, we focus in the design and the preparation of novel asymmetric 2,7,9-trisusbtituted fluorenes. Herein, we describe the first steps of a convergent synthesis able to lead to a new library of fluorene enantiopurs with high potential bioactivities.

References

1. Kucherak O. A., Didier P., Mély Y., Klymchenko A. S., J. Phys. Chem. Lett. , 1, 2010, 616–620.
2. Shi Y., Gao S., Tetrahedron 72, 2016, 1717-1735.
3. Klongkumnuankarn P., Busaranon K., Chanvorachote P., Sritularak B., Jongbunpraset V., Likhitwitayawuid K., Hindawi Publishing Corporation, 2015, 1-10.
4. Kemnitzer W., Sirisoma N., Jiang S., Kasibhatla S., Crogan-Grundy C., Tseng B., Drewe J., Xiong Cai S., Bioorg. & Med. Chem. Lett. 20, 2010, 1288-1292.
5. Si K. W., Liu J. T., He L. C., Li X. K., Liu C. H. Li X. Q., Basic & Clinical Pharmacology & Toxicology, 107, 2010, 976-981.
6. Hong H. S., Maezawa I., Budamagunta M., Rana S., Shi A., Vassar R., Liu R., Lam K. S., Cheng R. H., Hua D. H., Voss J. C., Jin L. W., Neurobiology of Aging 31, 2010, 1690-1699.
7. Makanga M., Malaria Journal, 2014, 13:291.

The method of electrospinning was used to create micro-fibers made of cellulose acetate (CA) and essential oils (EOs). CA polymer at 15% w/v was dissolved in acetone and then 1 or 5% v/v of EOs were added to the polymer solution. The essential oils chosen were oregano and rosemary oils. Then the CA-EOs in acetone solution was electrospun creating fibers with approximately 1.0 – 2.0 μm in diameter. Scanning electron microscopy was used to study the morphology, topography and dimensions of the micro-fibers. Raman spectroscopy was used to detect attachment of the EOs in the CA electrospun micro-fibers. The formed CA-EOs micro-fibers found to have good antimicrobial properties against three common bacteria strains; S. aureus, E. coli and C. albicans. The more concentrated with EOs micro-fibers showed the best antimicrobial effects. This work describes an effective and simple method to prepare CA-EOs micro-fibers and open new ways for many applications of such electrospun fibers such as wound dressings, anti-biofilm surfaces, sensors, packaging, cosmetics etc.

Coumarins are a large class of naturally occurring phenolic substances consisting in fused benzene and α-pyrone rings (benzo-α-pyrones). Coumarins are extremely variable in structure, due to different types of substituents in their scaffold, which can influence their biological activity. Coumarin-derivatives possessing a 4-hydroxyl group have been therapeutically used for their orally anticoagulant activity (e.g. warfarin). Nevertheless, being vitamin K antagonists, coumarins have a delayed onset and offset of action and several interactions with many drugs and food. In opposite, heparin, which is a polysulfated polysaccharide, has a short onset and offset of action, due to an effective mechanism of action, but is only active by parenteral route. As a result, it is important to develop effective orally active antithrombotic agents. In this work, a hybridization strategy was planned joining a coumarin scaffold with a heparin-like sugar sulfated moiety. With this approach it is expected to mimetize the sulfated polysaccharide anticoagulants, while adding some hydrophobic character to the resulting molecule to achieve oral bioavailability.

Five persulfated triazole and non-triazole linked coumarin glucosides were obtained by microwave irradiation with triethylamine-sulfur trioxide adduct, and their structure elucidation was established by IR and NMR for the first time. A purification procedure involving dialysis with a cellulose membrane was successfully applied to remove water soluble impurities. The anticoagulant activity was measured by the classical clotting times - activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). The most active compound exhibited an APTT₂ of 22×10^-5 M. In the future, oral bioavailability of this innovative coumarin hybrid will be evaluated.

The synthesis of novel pyridazinone derivatives and investigation of their biological properties have gained more importance in recent decades. In particular, the pharmacological activity of 4,5-dihydro-6-phenyl-3(2H)-pyridazinones has been extensively studied and such substances are known for their cardiovascular effects. In this field, several compounds such as zardaverine or imazodan have been developed as PDE III inhibitors in the search for new antiplatelet or cardiotonic agents. A number of 6-aryl-2-(4-aryl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-4,5-dihydropyridazin-3(2H)-ones have been designed and synthesized by a sequence of reactions starting from the respective aryl hydrocarbons. The final products (4a–4u) were evaluated for their antihypertensive activity by a non-invasive protocol using the tail-cuff method. Compounds 4e, 4i, and 4k showed appreciable properties comparable with that of  hydralazine and propranolol.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. Natural and synthetic oligoribonucleotides (ORNs) have a wide range of biological activities and can be used in antiviral treatments since they play a key role in antiviral activity and can change a conformation of some proteins. In our previous study it was shown that complexes of ORNs with D-mannitol (ORNs-D-mannitol) have higher antiviral activity against influenza A viruses than ORNs. However, the mechanism of ORNs-D-mannitol antiviral activity is still not clear. So the effects of the ORNs-D-mannitol on HA-glycan interactions were studied. Also interactions between the ORNs-D-mannitol and HA were determined.

It was shown that ORNs-D-mannitol have ability to interfere with HA-glycan interactions. A decrease of HA activity of influenza A (A/FM/1/47(H1N1)) virus by a factor 4 was observed after incubation of virus with ORNs-D-mannitol in comparison to the virus control.

Reduction of the fluorescence intensity of HA of flu virus in the presence of the ORNs-D-mannitol was observed. This effect may indicate that interactions between  HA and ORNs-D-mannitol are responsible for conformation changes of HA. For further verification of this assumption we used fluorescence data to calculate dissociation constants that appeared to be relatively weak (micromolar) in our case (k_d = 4.91μM).

 Our research demonstrates that ORNs-D-mannitol complexes bind to HA of flu virus and in this manner inhibit HA-glycan interactions. It allows us to assume that changes of HA conformations could explain the antiviral activity of those ORNs-D-mannitol complexes.