PROJECT CODE PN-II-RU-TE-2014-4-1670
Grant number 342/01/10/2015
Title: Sortase A inhibitors. New therapeutic approach for the management of infections with gram-positive multiresistant cocci

ACRONIM: SIREN
Project Leader: Niţulescu George Mihai
FUNDING AUTHORITY - THE NATIONAL AUTHORITY FOR SCIENTIFIC RESEARCH OF ROMANIA – UEFISCDI
Budget: 517.000 lei
REASERCH PROJECTS for YOUNG RESEARCH TEAMS
(http://uefiscdi.gov.ro/articole/4162/Proiecte-propuse-la-finantare_TE-2014.html)

Project Summary

The project tries to resolve the problem of the limited number of therapeutic resources for the management of gram-positive multiresistant cocci infections. Based on the project leader's expertise in targeted-drug design and in antimicrobial agents’ development, the project has the objectives to synthesize original molecules designed to target sortase A, to validate their effectiveness as anti-enterococcal agents, to assess their safety profile and to extend the research on other pathogens within the group of positive cocci.

The compounds will be subjected to physicochemical and biopharmaceutical analyses, will be tested on susceptible/resistant bacterial strains. Toxicity assessment will use alternative methods including the use of Jurkat cell cultures and invertebrate models, like Artemia salina and Daphnia magna, with the purpose of reducing the number of animals used further. Structure-activity relationship analysis will be used for structural optimization.

The results of the present project can be used to obtain original Romanian drugs with antimicrobial activity and to decrease the morbidity and mortality of affected patients. A special emphasis was put in promoting young, but experienced, researchers in a well-balanced interdisciplinary team.

STAGE I (2015)

Budget: 44.000 lei

The activities planned for this phase of the contract were:
• In silico analysis of the optimal parameters characterizing ligand - sortase A interactions
• Evaluation of biopharmaceutical profile by identifying physico-chemical molecular descriptors, molecular weight, partition coefficient octanol/water, number of hydrogen bonds donors/acceptors

Results 

All activities under the work plan have been completed, obtaining the following results:
• a structural basis of compounds containing 71 sortase inhibitors structures
• assessment of the main biopharmaceutical parameters for the sortase inhibitors using computational methods
• a structural profile of the typical sortase A inhibitor for virtual screening

STAGE II (2016)
Budget: 295.692 lei

The activities planned for this phase of the contract were:

• developing a strategy and methodology for synthesis, determination of optimal parameters for the synthesis process and for the extraction of compounds from vegetable products to provide substantial quantities necessary for microbiology and toxicology subsequent testing
• physico-chemical characterization and assessment of the purity of the synthesized compounds: 1H and 13C NMR, elemental analysis, IR spectra
• microbial susceptibility testing against certain Gram-positive strains using disk diffusion method; quantitative evaluation of the antimicrobial effect using the micro-dilution technique bullion; determination of minimum inhibitory concentration
• assessment of toxicity by bioassays using cell culture, invertebrates species, and rodents
• elaborating articles, communications, posters at conferences, symposia, national and international congresses, patent application

Results 

All activities under the work plan have been completed, obtaining the following results:

• several synthesis methods were elaborated and the synthesis processes were used to obtain 3 series of new compounds
• a large variety of plant products were used to obtain vegetable extracts or essential oils
• both synthetic compounds and the plant products were physico-chemical characterizated using several methods: 1H and 13C NMR, elemental analysis, IR spectra
• microbial susceptibility was determined against certain Gram-positive strains using the disk diffusion method
• the toxicity of both synthetic compounds and the plant products was determined using Daphnia and/or Artemia species
• several article manuscripts were prepared, two of them were published and two are under evaluation
• a number of 16 communications and posters were presented at national and international conferences, symposia, and congresses.

The results of this research stage were capitalized by the following articles published in ISI journals

• Nitulescu, G. M.; Margina, D.; Juzenas, P.; Peng, Q.; Olaru, O. T.; Saloustros, E.; Fenga, C.; Spandidos, D. Α.; Libra, M.; Tsatsakis, A. M. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review). Int. J. Oncol. 2016, 48, 869–85.  (Impact factor: 3.03).
• Nitulescu, G.; Zanfirescu, A.; Olaru, T. O.; Nicorescu, M. I.; Nitulescu, M. G.; Margina, D. Structural Analysis of Sortase A Inhibitors. Mol. 2016, 21. (Impact factor: 2.46).

The results of this research stage were capitalized by the following presentations at international of national congresses:

1. Nitulescu, G. M., Draghici, C., Velescu, B. S., Nitulescu, G., Olaru, O. T. Synthesis of new pyrazole derivatives as AKT inhibitors, MÆDICA - a Journal of Clinical Medicine, Volum 11, Supplement, 2016, p. 54, ISSN 2501-6903.
2. Nitulescu, G. M., Olaru, O. T., Socea L. I., Barbuceanu, S. F. Prediction targeted combinatorial library design, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 278
3. Nitulescu, G. M., Olaru, O. T., Nitulescu, G., Margina, D. Synthesis of new 1,5-dimethyl-2-phenylpyrazol-3-one derivatives targeted towards bacterial sortase A, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 279.
4. Barbuceanu, S. F., Nitulescu, G. M., Socea L. I., Barbuceanu, F., Nitulescu, G., Saramet, G., Draghici, C. Synthesis and characterization of some 1,3,4-thiadiazoles derivatives as potential biological agents, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 281.
5. Socea L. I., Draghici, C., Nitulescu, G. M., Olaru, O. T., Nitulescu, G., Barbuceanu, S. F. Synthesis of novel hydrazinecarbothioamides and products of their cyclization, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 282.
6. Olaru, O.; Ivopol, M.; Nitulescu, G.; Dune, A.; Calinescu, I.; Ivopol, G.; Popescu, M.; Nitulescu, G. M. Toxicity of various antimicrobial essential oils assessed using Artemia salina and Daphnia magna. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S68. ISSN: 0378-4274.
7. Ivopol, M.; Olaru, O.; Nitulescu, G. M.; Dune, A.; Popescu, M.; Spinu, C.; Nitulescu, G.; Calinescu, I. Zingiberaceae extracts and essential oils with potential sortase inhibitory effects. Preparation, characterization and toxicity evaluation. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S69. ISSN: 0378-4274.
8. Nitulescu, G.; Olaru, O.; Nitulescu, G. M.; Ungurianu, A.; Margina, D. Toxicity assessment of sortases inhibitors. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S75. ISSN: 0378-4274.
9. Ivopol, M.; Nitulescu, G. M.; Calinescu, I.; Dune, A.; Popescu, M.; Ivopol, G.; Nitulescu, G.; Olaru, O. GC/MS analysis and toxicity assessment of essential oils and extracts with antimicrobial activity obtained from plants belonging to Pinaceae and Cupressaceae. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S68. ISSN: 0378-4274.
10. Nitulescu, G. M.; Nitulescu, G.; Olaru, O.; Margina, D. Structure-based prediction of side effects of sortase A inhibitors. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S121. ISSN: 0378-4274.
11. Nitulescu, G. M.; Drăghici, C.; Nitulescu, G.; Olaru, O. T. Design and synthesis of new pyrazole derivatives as sortases inhibitors.  Congresul Național de Farmacie din România, 28 sept. - 1 oct. 2016, București, România, Volum de rezumate, p. 55, ISBN 2537-2823.
12. Nitulescu, G.; Olaru, O. T.; Nitulescu, G. M.;  Margina, D.; Potolea, I. M.; Pirvu, O. M. Effect of ultrasounds on triticum root elongation. Congresul Național de Farmacie din România, 28 sept. - 1 oct. 2016, București, România, Volum de rezumate, p. 223, ISBN 2537-2823.
13. Nitulescu, G. M.; Draghici, C; Barbuceanu, S. F.; Socea, L. I.; Olaru, O. T. Synthesis of new phenazone derivatives as potential bacterial sortase a inhibitors. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 50, ISSN: 2285-8334.
14. Olaru, O. T.; Guțu, C. M.; Chirita, C.; Iordache, A. A.; Ionita, I. M.; Ilie, M.; Zanfirescu, A.; Nitulescu, G. M. The obtaining and analysis of Salvia glutinosa L. (Lamiaceae) volatile oil. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 51, ISSN: 2285-8334.
15. Guțu, C. M.; Olaru, O. T.; Iordache, A. A.; Chirita, C.; Ionita, I. M.; Velescu, B. S.; Seremet, O. C.; Nitulescu, G. M. Variation in composition of essential oil from Tanacetum vulgare L. Flowers from different locations. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 40, ISSN: 2285-8334.
16. Barbuceanu, S. F., Socea, L. I., Nitulescu, G. M., Apostol, T. V., Barbuceanu, F., Olaru, O. T. Cytotoxicity evaluation of some compounds from triazole and acylthiosemicarbazide class with potential antimicrobial activity. International Congress of the Medical Sciences, Behavioral Sciences, Education and Physical Rehabilitation from the perspective of Quality of Life, 27-29 mai 2016, Craiova, Volum de rezumate, p. 104-106, ISSN: 2457-2772

STAGE III (2017)
Budget: 177.308 lei

The activities planned for this phase of the contract were:

• assessment of the sortase A inhibiting capacity of the selected compounds
• evaluation of the capacity of newly synthesized antimicrobial compounds to increase the susceptibility of enterococci to aminoglycosides and cephalosporins. Quantitative assessment of the antibacterial effect of associations by assessing the minimum inhibitory concentration using the micro-dilution technique in the broth
• establishing correlations between molecular descriptors, antimicrobial activity and toxic effects in order to optimize the compounds synthesized in the first stages in order to obtain molecules with superior antimicrobial effect and low toxicity
• elaboration of articles, communications / posters at national / international conferences / symposiums / congresses

Results

• a chemically diverse set of 56 compounds were tested for their ability to inhibit SrtA activity, as potential anti-virulence agents
• myricetin, palmatine, and esculetin were identified as SrtA inhibitors, with promising IC50 values
• microbial susceptibility was determined against certain Gram-positive strains using the disk diffusion method
• using molecular docking and structure-activity relationship analyses, several lead inhibitors were identified
• the toxicity of both synthetic compounds and the plant products was determined using Daphnia and/or Artemia species
• several article manuscripts were prepared, three of them were published and one is under evaluation
• a number of 6 communications and posters were presented at national and international conferences, symposia, and congresses.
• a national patent application is underwork

The results of the research were capitalized by the publication of four articles in ISI journals, with a cumulative impact factor of 9.24. An article has been published, two have been accepted for publication, and will be published in issue 12 of 2017 and 9 in 2018, and a fourth is in the evaluation stage.

1. Georgiana Nitulescu, Isabela Madalina Nicorescu, Octavian Tudorel Olaru, Anca Ungurianu, Dragos Paul Mihai, Anca Zanfirescu, George Mihai Nitulescu,* and Denisa Margina, Molecular Docking and Screening Studies of New Natural Sortase A Inhibitors, Int J Mol Sci. 2017 Oct; 18(10): 2217. doi: 10.3390/ijms18102217. Impact factor: 3.23.
2. Socea Laura Ileana, Barbuceanu Stefania Felicia, Drăghici Constrantin, Nitulescu George Mihai, Saramet Gabriel, Socea Bogdan, Apostol Theodora Venera, New heterocyclic compounds from 1,2,4-triazoles and 1,3,4-oxadiazoles class containing 5H-dibenzo[a,d][7]annulene moiety, Revista de Chimie, 68 (12), 2017. Impact factor: 1.23.
3. Barbuceanu Stefania Felicia, Olaru Octavian Tudorel, Nitulescu George Mihai, Drăghici Constantin, Socea Laura Ileana, Barbuceanu Florica, Enache Cristian, Saramet Gabriel, Synthesis and Structural Characterization of Some Potential Anti-Virulence 1,2,4-Triazoles and 1,3,4-Thiadiazoles, Revista de Chimie, 69, 2018. Impact factor: 1.23.
4. Zanfirescu Anca, Cristea Aurelia Nicoleta, Nitulescu George Mihai, Velescu Bruno Stefan, Grădinaru Daniela, Monosodium Glutamate Chronic Administration Induced Hyperalgesia in Mice, Nutrients. (manuscris nutrients-233026 of 27.09.2017). Impact factor: 3.55.

The results of this research stage were capitalized by the following presentations at international of national congresses:

1. Nitulescu, G., Olaru, O. T., Nitulescu, G. M., Synthesis and acute toxicity assesment of new pyrazolinone derivatives as antivirulence agents, 6th FIP Pharmaceutical Sciences World Congress 2017, 21-24 mai 2017, Stockholm, Suedia, Book of Abstracts.
2. Olaru Octavian Tudorel, Isabela Madalina Nicorescu, Georgiana Nitulescu, Oana Cristina Seremet, George Mihai Nitulescu, Antimicrobial screening of some extracts from Fallopia Adans. species (Polygonaceae), Congresul UMF Carol Davila, editia a 5-a, 29-31 mai 2017, Bucureşti, Maedica a Journal of Clinical Medicine, 12 (Supp. 2017), pag 42.
3. Georgiana Nițulescu, Anca Ungurianu, George Mihai Nițulescu, Isabela Mădălina Nicorescu, Octavian Tudorel Olaru, Denisa Margină, Screening natural scaffolds as antivirulence agents, Prioritatile Chimiei pentru o Dezvoltare Durabila - PRIOCHEM XIII – 2017, 25-27 octombrie 2017, Bucureşti, Volume of summaries, pag 73, ISSN 2285 – 8334.
4. Georgiana Nițulescu, Octavian Tudorel Olaru, George Mihai Nițulescu, Evaluarea toxicității acute  a  unor  compuși  naturali, Congresul National de Toxicologie, 03-04 octombrie 2017, Bucureşti, pag 74, ISSN 2601-1077.
5. George Mihai Nițulescu, Predictive Methods in Toxicology. Challenges and Perspectives, Congresul National de Toxicologie, 03-04 octombrie 2017, Bucureşti, pag 45, ISSN 2601-1077.
6. George Mihai Nitulescu, Perspective complementare şi interdisciplinare în descoperirea de medicamente anticanceroase (Complementary and interdisciplinary perspectives in anticancer drug discovery), Congresul UMF Carol Davila, editia a 5-a, 29-31 mai 2017, Bucureşti, Maedica a Journal of Clinical Medicine, 12 (Supp. 2017).