Aim: To evaluate relationship between histamine (HIST), serotonin (5-HT), dopamine (DA), gamma-amino-butyric acid (GABA) and IGF-1 in 20 GH-deficient boys.                                  

Research design and methods: This study included 20 boys (5-14 years) with GH deficit clinically established and a 10 matched normal group with no endocrine dysfunction. All of GH deficient patients underwent GH replacement therapy. In 2017, all subjects were tested by analytical methods for blood: HIST,GABA,DA,5-HT,IGF-1.                                                           

Results: We divided this study group into a low HIST lot 1 (10 subjects): HIST median: 3.48 nM/L and a high HIST lot 2 (10 subjects): HIST median: 11nM/L. Median parameters in lot 1 vs. lot 2 were: 5-HT: 212.5 vs. 370ng/mL, DA: 30 vs. 45pg/ml, GABA: 30 vs. 56.5ng/mL, IGF-1: 373.5 vs. 200ng/mL. Median values in normal subjects were as it follows: HIST: 5.55nM/L; 5-HT: 235.5ng/mL; DA: 31.5pg/mL; GABA: 81ng /mL. T-Test revealed a statistical significance between HIST in lot 1 vs. lot 2 (P<0.001), HIST in lot 1 vs. normal group (P<0.01) or HIST in lot 2 vs. normal group (P<0.01).We can also underline a statistical significance between 5-HT in lot 1 vs. lot 2 (P<0.05) or in lot 2 vs. normal group (P=0.01).                                                                

Conclusion: Our study underlined a HIST/5-HT positive relationship in low HIST group vs. a negative relationship HIST/5-HT in high HIST group, with small IGF-1 increments under r-GH therapy.

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.

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”.

The human milk protein α_S1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4)¹. In this study, the binding site of α_S1-casein to TLR4 was identified by selective inhibition of the intracellular binding domain and extracellular ecto-domain of TLR4.

For this, Interleukin 8 (IL-8) secretion was monitored after stimulation of TLR4/MD2 (myeloid differentiation factor 2)/CD14 (cluster of differentiation 14)-transfected HEK293 cells (TLR4⁺) and Mono Mac 6 cells (MM6) with recombinant α_S1-casein, or lipopolysaccharide (LPS) as control. The α_S1-casein-induced IL-8 secretion was inhibited by TAK-242, an antagonist of the intracellular binding site and mianserine, an antagonist of the extracellular binding domain. TAK-242 inhibited α_S1-casein-induced IL-8 secretion with an IC₅₀ of 259 nM and LPS-induced IL-8 secretion with an IC₅₀ of 23 nM. Mianserine was found as moderate inhibitor of the α_S1-casein-induced IL-8 secretion with an IC₅₀-range between 10-51 µM. Therefore, we suggested α_S1-casein as an inhibitor of the extracellular binding site of TLR4. These findings were supported by binding experiments using microscale thermophoresis (MST). Human α_S1-casein bound to the purified extracellular TLR4/MD2-complex with a K_D of 2.2 µM in comparison to LPS binding TLR/MD2 with a K_D of 8.7 µM. Furthermore α_S1-casein showed binding to MD2 with a K_D of 0.3 µM and CD14 with a K_D of 2.7 µM. In addition, human α_S1-casein induced IL-8 secretion via TLR4 was inhibited by inhibitory anti-CD14-IgA.

Human α_S1-casein induced proinflammatory effects by binding to the ecto-domain of TLR4 and CD14 is required as cofactor. Hence human α_S1-casein activates TLR4 in a different manner than LPS.

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.