TY  - CONF
AU  - Sibinčić, Nikolina
AU  - Krstić-Ristivojević, Maja
AU  - Stojanović, Marijana
AU  - Mladenović Stokanić, Maja
AU  - Vasović, Tamara
AU  - Ćirković-Veličković, Tanja
AU  - Stojadinović, Marija
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6051
AB  - The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluency and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.
PB  - Beograde : Serbian Biochemical Society
C3  - "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
T1  - Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells
SP  - 91
EP  - 91
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6051
ER  - 

@conference{
author = "Sibinčić, Nikolina and Krstić-Ristivojević, Maja and Stojanović, Marijana and Mladenović Stokanić, Maja and Vasović, Tamara and Ćirković-Veličković, Tanja and Stojadinović, Marija",
year = "2023",
abstract = "The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluency and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.",
publisher = "Beograde : Serbian Biochemical Society",
journal = ""Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia",
title = "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells",
pages = "91-91",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6051"
}

Sibinčić, N., Krstić-Ristivojević, M., Stojanović, M., Mladenović Stokanić, M., Vasović, T., Ćirković-Veličković, T.,& Stojadinović, M.. (2023). Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
Beograde : Serbian Biochemical Society., 91-91.
https://hdl.handle.net/21.15107/rcub_cherry_6051

Sibinčić N, Krstić-Ristivojević M, Stojanović M, Mladenović Stokanić M, Vasović T, Ćirković-Veličković T, Stojadinović M. Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia. 2023;:91-91.
https://hdl.handle.net/21.15107/rcub_cherry_6051 .

Sibinčić, Nikolina, Krstić-Ristivojević, Maja, Stojanović, Marijana, Mladenović Stokanić, Maja, Vasović, Tamara, Ćirković-Veličković, Tanja, Stojadinović, Marija, "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells" in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia (2023):91-91,
https://hdl.handle.net/21.15107/rcub_cherry_6051 .

TY  - JOUR
AU  - Sibinčić, Nikolina
AU  - Kalinin, Stanislav
AU  - Sharoyko, Vladimir
AU  - Efimova, Julia
AU  - Gasilina, Olga
AU  - Korsakov, Mikhail
AU  - Gureev, Maxim
AU  - Krasavin, Mikhail
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6049
AB  - Abstract: Aims: To synthesize novel sulfonamide inhibitors of carbonic anhydrase and develop in vitro prioritization workflow to select compounds for in vivo evaluation.
Background: Carbonic anhydrase (CA) inhibitors gain significant attention in the context of drug discovery research for glaucoma, hypoxic malignancies, and bacterial infections. In previous works, we have successfully used direct sulfochlorination approach to develop diverse heterocyclic primary sulfonamides with remarkable activity and selectivity against therapeutically relevant CA isoforms.
Objective: Synthesis and investigation of the CA inhibitory properties of novel trifluoromethylisoxa- zolyl- and trifluoromethylpyrazolyl-substituted (hetero)aromatic sulfonamides.
Methods: Thirteen trifluoromethylisoxazolyl- and thirteen trifluoromethylpyrazolyl-substituted (het-
ero)aromatic sulfonamides were synthesized by direct sulfochlorination of hydroxyisoxazolines and
pyrazoles followed by reaction with ammonia. The compound structures were confirmed by 1H and
13
C NMR as well as element analysis. The obtained compounds were evaluated, using the CA es-
terase activity assay, for their potential to block the catalytic activity of bovine CA (bCA).
Results: Eight most potent compounds selected based on the esterase activity assay data were tested
for direct affinity to the enzyme using the thermal shift assay (TSA). These compounds displayed Kd values (measured by TSA) in the double-digit nanomolar range, thus showing comparable activity to the reference drug acetazolamide.
PB  - Bentham Science Publishers
T2  - Medicinal Chemistry
T1  - A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies
VL  - 19
IS  - 2
SP  - 193
EP  - 210
DO  - 10.2174/1573406418666220831112049
ER  - 

@article{
author = "Sibinčić, Nikolina and Kalinin, Stanislav and Sharoyko, Vladimir and Efimova, Julia and Gasilina, Olga and Korsakov, Mikhail and Gureev, Maxim and Krasavin, Mikhail",
year = "2023",
abstract = "Abstract: Aims: To synthesize novel sulfonamide inhibitors of carbonic anhydrase and develop in vitro prioritization workflow to select compounds for in vivo evaluation.
Background: Carbonic anhydrase (CA) inhibitors gain significant attention in the context of drug discovery research for glaucoma, hypoxic malignancies, and bacterial infections. In previous works, we have successfully used direct sulfochlorination approach to develop diverse heterocyclic primary sulfonamides with remarkable activity and selectivity against therapeutically relevant CA isoforms.
Objective: Synthesis and investigation of the CA inhibitory properties of novel trifluoromethylisoxa- zolyl- and trifluoromethylpyrazolyl-substituted (hetero)aromatic sulfonamides.
Methods: Thirteen trifluoromethylisoxazolyl- and thirteen trifluoromethylpyrazolyl-substituted (het-
ero)aromatic sulfonamides were synthesized by direct sulfochlorination of hydroxyisoxazolines and
pyrazoles followed by reaction with ammonia. The compound structures were confirmed by 1H and
13
C NMR as well as element analysis. The obtained compounds were evaluated, using the CA es-
terase activity assay, for their potential to block the catalytic activity of bovine CA (bCA).
Results: Eight most potent compounds selected based on the esterase activity assay data were tested
for direct affinity to the enzyme using the thermal shift assay (TSA). These compounds displayed Kd values (measured by TSA) in the double-digit nanomolar range, thus showing comparable activity to the reference drug acetazolamide.",
publisher = "Bentham Science Publishers",
journal = "Medicinal Chemistry",
title = "A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies",
volume = "19",
number = "2",
pages = "193-210",
doi = "10.2174/1573406418666220831112049"
}

Sibinčić, N., Kalinin, S., Sharoyko, V., Efimova, J., Gasilina, O., Korsakov, M., Gureev, M.,& Krasavin, M.. (2023). A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies. in Medicinal Chemistry
Bentham Science Publishers., 19(2), 193-210.
https://doi.org/10.2174/1573406418666220831112049

Sibinčić N, Kalinin S, Sharoyko V, Efimova J, Gasilina O, Korsakov M, Gureev M, Krasavin M. A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies. in Medicinal Chemistry. 2023;19(2):193-210.
doi:10.2174/1573406418666220831112049 .

Sibinčić, Nikolina, Kalinin, Stanislav, Sharoyko, Vladimir, Efimova, Julia, Gasilina, Olga, Korsakov, Mikhail, Gureev, Maxim, Krasavin, Mikhail, "A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies" in Medicinal Chemistry, 19, no. 2 (2023):193-210,
https://doi.org/10.2174/1573406418666220831112049 . .

TY  - JOUR
AU  - Shetnev, Anton
AU  - Tarasenko, Marina
AU  - Kotlyarova, Valentina
AU  - Baykov, Sergey
AU  - Geyl, Kirill
AU  - Kasatkina, Svetlana
AU  - Sibinčić, Nikolina
AU  - Sharoyko, Vladimir
AU  - Rogacheva, Elizaveta
AU  - Kraeva, Liudmila
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6050
AB  - A new route to 5-amino-1,2,4-thiadiazole derivatives via reaction of N-chloroamidines with isothiocyanates has been proposed. The advantages of this method are high product yields (up to 93%), the column chromatography-free workup procedure, scalability and the absence of additive oxidizing agents or transition metal catalysts. The 28 examples of 5-amino-1,2,4thiadiazole derivatives obtaining via the proposing protocol were evaluated in vitro against ESKAPE pathogens strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae). It was found that compounds 5ba, 5bd, 6a, 6d and 6c have potent antibacterial activity (MIC values 0.09–1.5 μg mL−1), which is superior to the activity of commercial antibiotics such as pefloxacin (MIC 4–8 μg  mL−1) and streptomycin (MIC 2–32 μg  mL−1). The additional cytotoxic assay of hit compounds on PANC-1 cell line demonstrated the low or non-cytotoxicity activity at the same level of concentrations. Thus, these 5 compounds are promising starting point for further antimicrobial drug development.
PB  - Springer
T2  - Molecular Diversity
T1  - External oxidant‐free and transition metal‐free synthesis of 5‐amino‐1,2,4‐thiadiazoles as promising antibacterials against ESKAPE pathogen strains
VL  - 27
SP  - 651
EP  - 666
DO  - 10.1007/s11030-022-10445-1
ER  - 

@article{
author = "Shetnev, Anton and Tarasenko, Marina and Kotlyarova, Valentina and Baykov, Sergey and Geyl, Kirill and Kasatkina, Svetlana and Sibinčić, Nikolina and Sharoyko, Vladimir and Rogacheva, Elizaveta and Kraeva, Liudmila",
year = "2022",
abstract = "A new route to 5-amino-1,2,4-thiadiazole derivatives via reaction of N-chloroamidines with isothiocyanates has been proposed. The advantages of this method are high product yields (up to 93%), the column chromatography-free workup procedure, scalability and the absence of additive oxidizing agents or transition metal catalysts. The 28 examples of 5-amino-1,2,4thiadiazole derivatives obtaining via the proposing protocol were evaluated in vitro against ESKAPE pathogens strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae). It was found that compounds 5ba, 5bd, 6a, 6d and 6c have potent antibacterial activity (MIC values 0.09–1.5 μg mL−1), which is superior to the activity of commercial antibiotics such as pefloxacin (MIC 4–8 μg  mL−1) and streptomycin (MIC 2–32 μg  mL−1). The additional cytotoxic assay of hit compounds on PANC-1 cell line demonstrated the low or non-cytotoxicity activity at the same level of concentrations. Thus, these 5 compounds are promising starting point for further antimicrobial drug development.",
publisher = "Springer",
journal = "Molecular Diversity",
title = "External oxidant‐free and transition metal‐free synthesis of 5‐amino‐1,2,4‐thiadiazoles as promising antibacterials against ESKAPE pathogen strains",
volume = "27",
pages = "651-666",
doi = "10.1007/s11030-022-10445-1"
}

Shetnev, A., Tarasenko, M., Kotlyarova, V., Baykov, S., Geyl, K., Kasatkina, S., Sibinčić, N., Sharoyko, V., Rogacheva, E.,& Kraeva, L.. (2022). External oxidant‐free and transition metal‐free synthesis of 5‐amino‐1,2,4‐thiadiazoles as promising antibacterials against ESKAPE pathogen strains. in Molecular Diversity
Springer., 27, 651-666.
https://doi.org/10.1007/s11030-022-10445-1

Shetnev A, Tarasenko M, Kotlyarova V, Baykov S, Geyl K, Kasatkina S, Sibinčić N, Sharoyko V, Rogacheva E, Kraeva L. External oxidant‐free and transition metal‐free synthesis of 5‐amino‐1,2,4‐thiadiazoles as promising antibacterials against ESKAPE pathogen strains. in Molecular Diversity. 2022;27:651-666.
doi:10.1007/s11030-022-10445-1 .

Shetnev, Anton, Tarasenko, Marina, Kotlyarova, Valentina, Baykov, Sergey, Geyl, Kirill, Kasatkina, Svetlana, Sibinčić, Nikolina, Sharoyko, Vladimir, Rogacheva, Elizaveta, Kraeva, Liudmila, "External oxidant‐free and transition metal‐free synthesis of 5‐amino‐1,2,4‐thiadiazoles as promising antibacterials against ESKAPE pathogen strains" in Molecular Diversity, 27 (2022):651-666,
https://doi.org/10.1007/s11030-022-10445-1 . .