Mosquitoes are responsible for the transmission of serious and potentially fatal diseases to humans, such as malaria, dengue fever, West Nile fever, leishmaniasis and more recently Zika virus^[1] infection. According to the World Health Organization, the prevalence of human mortality causing from infected mosquitoes amounts to one million, annually.^[2,3] Nowadays, the increasing resistance of vectors to existing repellents render them as ineffective, creating the need for development of novel repellents with advanced properties to the existing ones in terms of duration of the protection, minimum effective dose, efficacy against a wide variety of insects’ bites and safety. The main goal of the current study is the discovery of novel hit compounds which may evolve as insect repellents by performing a combination of computational and analytical chemistry methodologies. Particularly, a pharmacophore-based virtual screening of natural compound libraries coupled with molecular docking were applied to identify novel hits towards Odorant Binding Protein 1 (OBP1) a molecular target for the most widely used synthetic repellents DEET^[4,5] and Icaridin^[6]. Compounds were selected to bear the appropriate physicochemical properties associated with insect repellency. Six compounds were evaluated against female mosquitoes (Aedes albopictus). Results presented insect repellent activity of 35-57,9% compared to untreated hand which indicate the proposed scaffolds as starting points for further structure optimization.

References

[1] G. S. Rawal et al., J Family Med Prim Care 2016, 5, 523-527.

[2] R. Lozano et al., Lancet 2012, 380, 2095-2128.

[3] World Health Organization. Vector-borne diseases fact sheet N. 387. WHO website http://www.who.int/mediacentre/factsheets/fs387/en/ WHO, 2016

[4] K. E. Tsitsanou et al., Cell Mol Life Sci 2012, 69, 283-297.

[5] S. E. Zografos et al., ''QSAR in Environmental and Health Sciences", 65-99.

[6] C. E. Drakou et al., Cell Mol Life Sci 2017, 74, 319-338.

 Acknowledgements

This study was supported by “IKY FELLOWSHIPS OF EXELLENCE FOR POSTGRADUATE STUDIES IN GREECE-SIEMENS PROGRAM”.

On a global scale, Epstein-Barr virus (EBV) infects over 90% of the adult population and is responsible for ~1% of all human cancers. Fluorine is one of the most abundant elements on earth. However, it occurs extremely rarely in biological compounds. The introduction of the fluorine atom(s) into many biologically active molecules can bring about remarkable and profound changes in their properties. Development of potential drugs is closely related to in silico methods, which include PASS, QSAR, COMPARE-analysis and more. 

The aim of this work was to analyze the potential biological activity and the target of action of derivatives of bisphosphonic acids by using in silico methods and examined received results by in vitro study. For this purpose, PASS software, web-server PharmMapper, PCR, MTT assay, trypan blue and neutral red assay were used.

According to PASS prediction two compounds (10s-20 and 10s-21) may possess antiviral activity, Pa/ Pi was 0,294/0,005 and 0,214/0,084, respectively. Also, all compounds may possess a cytochrome c as substrate. Several targets were identified by using molecular docking (PharmMapper). It was shown that a lot of possible targets are proteins, such as Gag-Pol protein (viral protein) and different kinds of protein kinases. A study in vitro shown anti-EBV activity for all compounds. On the other hand, derivatives of bisphosphonic acids had a high level of cytotoxicity on different lymphoblastoid cell lines.

Therefore, the in silico screening presents a good approach for the development of new anti-EBV agents. Our results showed, that derivatives of bisphosphonic acids may possess  apoptosis modulating properties for treatment of lymphoproliferative diseases.

Salmonella represents one of the major causes of foodborne diseases in humans, in addition to provoking important economic losses in the agri-food sector worldwide. Therefore, the surveillance and control of this human pathogenic bacterium in foodstuffs and biological fluids are necessary in order to prevent and diagnose the disease. Molecular methods based on the detection of DNA sequences specific to pathogenic species are an appealing alternative to traditional culture-based methods that require 5 to 6 days to obtain a definitive result. Among them, and because of its easy miniaturization, electrochemical genosensors are a suitable option for decentralized genetic testing [1-2]; however, they often require a set of sample pretreatment steps before genetic DNA analysis, thus making their implementation at the point of need more difficult.

Herein, we report the integration of a nucleic acid-based sensor and an isothermal DNA amplification technique, helicase-dependent amplification or HDA, onto indium tin oxide (ITO) surfaces for the detection of a DNA sequence specific for the typA gene of Salmonella. DNA amplification process occurs at 65 ºC with short oligonucleotides flanking the target sequence, which act as primers. The reversed primer is covalently bound to the ITO surface through a thiol group present at its 5’ terminus, whereas forward fluorescein-tagged primer is incorporated in solution. As a result of the isothermal elongation step, fluorescein-tagged DNA duplexes are attached to the ITO surface and their enzymatic labelling is achieved via Fab fragments directed against fluorescein, conjugated with the redox enzyme alkaline phosphatase. Then, α-naphthyl phosphate is enzymatically dephosphorylated into an electroactive derivate α-naphthol whose amount, directly related to the Salmonella present in the sample, is measured by differential pulse voltammetry. This developed integrated sensing platform allows the detection of Salmonella down to 10 genomes in just over 2 hours [3], the same detection limit as that achieved by real-time PCR but without need of high-end benchtop instrumentation. Furthermore, the sensing layer built onto ITO surfaces maintains its performance even after 9 months storage, and possesses a great potential to be extended to the in-situ, fast and reliable detection of other pathogens.

References:

[1] D. Mabey, R.W. Peeling, A. Ustianowski and M.D. Perkins, Nat. Rev Microbiol., 2004, 2, 231-240.

[2] A.S. Patterson, K. Hsieh, H.T. Soh and K.W. Plaxco, Trends Biotechnol., 2013, 31, 704-712.

[3] S. Barreda-García, R. Miranda-Castro, N. de-los-Santos-Álvarez, A.J. Miranda-Ordieres, M.J. Lobo-Castañón, Chem. Comm., 2017, 53, 9721-9724.

Acknowledgments: This work has been supported by the Spanish Ministerio de Economía y Competitividad (CTQ2015-63567-R), the Principado de Asturias government (FC-15-GRUPIN14-025), and co-financed by FEDER funds.

Tuberculosis (TB) is an infectious disease caused by the bacterium, Mycobacterium tuberculosis (Mtb). The last surveys conducted by the World Health Organization (WHO) have described TB as the infectious disease responsible for the highest number of deaths worldwide. Furthermore, an increasing number of multidrug-resistant (MDR) and extensively-drug resistant (XDR) strains have complicated the scenario. In this regard, various N-oxide derivatives, such as furoxan, benzofuroxan, and quinoxaline 1,4-di-N-oxide, have been previously described as promising scaffolds with a potential to be explored as novel antitubercular drugs. Herein, 22 new N-oxide-containing compounds were synthesized followed by in vitro evaluation of their antitubercular potential against Mtb. Moreover, their safety and primary mechanism of action were also explored. The compounds demonstrated MIC₉₀ values ranging from 0.40 to 62 μM. Among the different heterocyclic compounds containing N-oxide, the benzofuroxan derivative 8 was found to be the most promising compound, with MIC₉₀ values of 1.10 and 6.62 μM against active and non-replicating Mtb, respectively. Cytotoxicity tests using MRC–5 cell line demonstrated an IC₅₀ value of 519.2 μM. Compound 8 was also active against monoresistant strains. Microarray-based initial studies on the mechanism of action revealed an upregulation of a number of transcripts encoding proteins belonging to both small and large subunits of the ribosome, suggesting that compound 8 blocked the process of translation. Altogether, these results indicated benzofuroxan derivative 8 to be a promising lead compound for the development of a novel chemical class of antitubercular drugs.

Mixed viral infection is one of the current and unexplored issues of human infectious diseases. A special place in the development of these pathologies is occupied by adeno- and herpes viruses that are able to persist for a long time in a latent condition in the body. There is a huge lack of knowledge about antiviral activities of specific drugs during the mixed infections. The study of know drugs and discovery of new compounds using not only standard mono-infections but also with created mixed infections is a topical and a new direction in antivirus screening. Previously in our department, the models of adeno-herpetic infections in cells of different origins were created and the features of the development of viral infections in these systems were studied.

     The model of simultaneous adeno-herpetic infection of MDBK cells was used for the analysis of the antiherpetic drug acyclovir (ACV) and for research of new fluorine-containing derivatives of L-phenylalanine (10S-23 and 10S-24, synthesized in Institute of Organic Chemistry of the NAS of Ukraine). Determination of the antiviral activity accessed via real-time PCR and infectious virus yield reduction assay demonstrated the inhibitory effect of these compounds on the late stage of the HSV-1 and HAdV5 reproduction. With the presence of 10S-24 and ACV in the conditions of mono-infection, HSV-1 DNA replication was inhibited on 39 and 100%, respectively. Furthermore, the significant delays of HSV-1 reproductions were observed, the titer of virus obtained de novo reduces on >99%. It was shown that for adenovirus infection of the cells, all compounds reduced the titer of the virus on 34-96%.

     Use of the ACV under mixed infection led to the 46% loss of the drug activity against HSV-1. The application of 10S-23 and 10S-24 at mixed infection induced a decrease of effectiveness of the compounds relatively herpes simplex virus by 58-73% and 16-57%, respectively. It was shown that compounds have been not effective against HAdV5 under conditions of co-infection of cells.

     An abnormal action of drugs in co-infected cells indicates the need for further study of the mechanisms and targets of antiviral activity of compounds in such conditions, because using these compounds can be ineffective in medical practice.

Acknowledgements

This work was supported by a President’s of Ukraine grant for competitive projects F70 of the State Fund for Fundamental Research.

The decline or leveling of the output of the R&D programs of the pharmaceutical companies may suffered recent changes when compared to earlier years of the 21st century. Although a major responsible for this increase is the immunopharmacology-based treatments, small molecules still play an important role. ¹ Medicinal chemistry approaches to find a small molecule lead compound, which shows the desired pharmacological activity, continue to use as sources natural products, synthesis, and existing drugs.

In this communication, we will give examples of antitumor small molecules lead compounds obtained in our research group that arise from two existing drugs, lucanthone and mitoxantrone (MTX). One aim in medicinal chemistry is the study of drug metabolites, and very recently we engaged a project that intend to understand the influence of metabolites in the cardiotoxicity of an antitumor drug, MTX, approved in 1987 as an antitumor drug and in 2002 for use in multiple sclerosis. Although the main human MTX metabolites have been identified, their putative cardiotoxicity was not yet assessed.

Herein, we also exemplify MTX drug metabolites as potential sources of new drugs. Initially, the MTX-naphthoquinoxaline metabolite (NAPHT) was synthetized and studies on NAPHT cardiotoxicity revealed that the parent drug, MTX, caused a higher disruption in the energetic pathways in a cardiac model in vitro, whereas autophagy is involved in the toxicity of both compounds; ² therefore, this metabolite should be regarded as a good option for a safer anticancer therapy since it is less cardiotoxic than MTX. Moreover, previous data has shown that NAPHT can have a potential role on MTX’s anticancer effects. The case studies presented herein are expected to contribute to a recent trend in drug discovery, with the involvement of old pharmaceuticals. To us, existing drugs will continue to provide a fruitful solution in drug discovery and development.

Acknowledgements: We thank FCT/MCTES and ERDF through the COMPETE–POFC programme, under the Strategic Funding UID/Multi/04423/2013, the project PTDC/MAR-BIO/4694/2014 (POCI-01-0145-FEDER-016790; 3599-PPCDT) and PTDC/DTP-FTO/1489/2014 (POCI-01-0145-FEDER-016790) in the framework of PT2020, to INNOVMAR (NORTE-01-0145-FEDER-000035, NOVELMAR), supported by NORTE 2020, under PORTUGAL 2020, through ERDF.

References:

1. Newman D. J.; Cragg G. M. J. Nat. Prod. 2016, 79, 629.
2. Reis-Mendes A.; Gomes A.S.; Carvalho R. A.; Carvalho F.; Remião, F.; Pinto M.; Bastos M. L.; Sousa E.; Costa V. M. Arch Toxicol. 2017,91(4), 1871.

Epstein-Barr virus (EBV) belongs to the human herpes virus family that infects more than 90% of the population. EBV is associated with a number of lymphoproliferative and autoimmune diseases. Use of the drugs, which would not only inhibit the reproduction of the virus, but also would stimulate the elimination of tumor cells, is important for the treatment of virus-associated tumors. In the current research the antiviral effects of the herbal extracts Proteflazid and Neoflazid were studied on the models of latent, acute and chronic EBV infections in Raji and B95-8 lymphoblastoid cells. Neoflazid was more toxic towards Raji cells than Proteflazid: the CC₅₀ indices were 8 µg/ml and 36 µg/ml respectively. Toxicities of these compounds in B95-8 cells were almost the same and their CC₅₀ indices were close to 25 µg/ml. Both drugs showed high antiviral activity against EBV lytic infection in Raji cells and EC₅₀ was 0.02 and 0.083 µg/ml for Proteflazid and Neoflazid, respectively, and selectivity indinces were 1800 and 96. They were less effective in B95-8 cells and even at a concentration of 10 µg/ml these compounds inhibited virus replication by only 10-19%.

We checked the ability of Proteflazid to induce apoptosis and found that the drug stimulated the apoptotic cell death in latent and lytic EBV infections at cytotoxic concentrations (30 µg/ml). The non-toxic concentration (5 µg/ml) induced apoptosis more actively (by 10%) during EBV lytic infection in cells B95-8 than in the case of latent infection in Raji cells.

Urea and thiourea scaffolds have been successfully used in drug design in recent years ⁽¹⁾. The formation of thiazolidines by reaction of propargyl amine with isothiocyanates under harsh conditions has been previously reported. Also, these new molecules have attracted great attention because their biological activity ⁽²⁾.

The coordination with different metals like gold or silver seem to show a better biological activity ⁽³⁾, hence, these metal centers were coordinated to the thiourea and to the heterocyclic compounds.

The reactions between propargylamines and isothiocyanates (Scheme 1), when the stoichiometry was 2:1, showed the formation the thiourea-thiazolidine compounds. These new molecules have coordination atoms such as sulfur and nitrogen where metal atoms can be linked, improving their biological activity. On the other hand, these atoms could work recognizing different target cells leading to higher selectivity for these compounds.

Scheme 1

Thiourea-thiazolidine compounds were coordinated to gold and silver atoms with 1:1 or 2:1 stoichiometry (Scheme 2).

Scheme 2

Finally, these compounds were tested with HeLa cells through the MTT assay. The results had not been as successfully as expected. Only silver compounds showed good cytotoxic values as anticancer compounds. Gold compounds have lower IC₅₀ valuables than their respective organic ligands, but more studies should be perform to improve the complexes in order to developed better candidates for the treatment of cancer.

Graphic 1

References

1. Yao, J.; Chen, J.; He, Z.; Sun, W.; Xu, W. Bioorg. Med. Chem. 2012, 20, 2923–2929.
2. Liu, Y.; Jing, F.; Xu, Y.; Xie, Y.; Shi, F.; Fang, H.; Li, M.; Xu, W. Biorg. Med. Chem. 2011, 19, 2342-2348.
3. O. Rackham, S.J. Nichols, P.J. Leedman, S.J. Berners-Price, A. Filipovska. From Biochemical Pharmacology. 2007, 74(7), 992-1002.

NUC013 (5-aza-2′,2′-difluroro-deoxycytidine) is preclinically safer and more effective than decitabine (Pharmaceuticals 2017, 10, 65).  5-Azacytidines are hydrolyzed at the cytosine’s 6-position, but in vivo, the short half-life is governed by deamination.  For decitabine, attempts have been made to address these issues with continuous infusion, but use of such a regimen is limited by inconvenience and toxicity.

A hydrophobic prodrug was developed for packaging in a hydrophobic matrix to protect NUC013 from hydrolysis and deamination.  In an aqueous environment, the hydrophobic moieties are readily hydrolyzed with release of NUC013 .  This was achieved by conjugating NUC013 with trimethylsilyl (TMS) at the 3’ and 5’ position (NUC041). 

The half-life of NUC013 administered IV in mice is 20.1 minutes.  Below, PK following administration of a dose of 3mg of NUC041 IM in a PEG-phospholipid-depot to mice:

Analyte       C_max (ng/mL)        T_max (hr)        t½ (hr)         AUC_INF (hr·ng/mL)          MRT_INF (hr)

NUC041             4210                       0.5                1.7                          6261                               2.6

NUC013             1333                       1.0                3.4                          5813                               5.1

In an ongoing study, NUC041 was administered at a dose of 3mg qwk to nude mice with a NSCLC H-460 xenograft.  After 3 days of treatment (n=8), tumor starting volume had decreased by 4%.  However, toxicity, likely from vehicle, was also observed at this dose.

.

Novel luminescent squaramide monoesters functionalised with different fluorophore groups have been synthesized and explored in cell imaging for the first time.

Cytotoxicity studies performed in HeLa cervical cancer cells revealed high activity for some of these novel structures, highlighting the importance of the fluorescent fragment in the efficiency of these promising anticancer agents.

In addition, fluorescence cell microscopy disclosed the different biodistribution behaviour depending on the fluorescent moiety, and the possibility of nuclear localisation of chiral non planar squaramide monoesters.