XIX. WORKSHOP OF BIOPHYSICAL CHEMISTS AND ELECTROCHEMISTS BOOK OF ABSTRACTS. 14 th JUNE 2019 Brno

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1 XIX. WORKSHOP OF BIOPHYSICAL CHEMISTS AND ELECTROCHEMISTS BOOK OF ABSTRACTS 14 th JUNE 2019 Brno

2 XIX. Workshop of Biophysical Chemists and Electrochemists Book of abstracts 14 th June, 2019 Masaryk University Press Brno 2019

3 THE ORGANIZATION HOSTING THE CONFERENCE Masaryk University Faculty of Science Department of Chemistry Kotlářská Brno THE ORGANIZATIONAL SECURITY OF THE CONFERENCE Libuše Trnková libuse@chemi.muni.cz (Department of Chemistry, Faculty of Science, Masaryk University) The publication did not undergo the language control. All contributions are publicated in the form, in which they were delivered by the authors. Authors are also fully responsible for the material and technical accuracy of these contributions Masaryk University ISBN

4 The Workshop of Biophysical Chemists and Electrochemists was supported by research organizations: 4

5 The sponsors of the Workshop of Biophysical Chemists and Electrochemists: The organizers thank a lot to all this year s sponsors for the support, which enabled to organize this traditional conference: Metrohm Czech Republic s.r.o., Institute of Biophysics of the Czech Academy of Sciences in Brno, Eppendorf Czech & Slovakia s.r.o., Oncomed manufacturing a.s., UNIMED Praha, s.r.o., Chromservis, s.r.o., MERCI, s.r.o. and Czech Chemical Society, subdivision Brno. The main sponsor The main sponsor 5

6 An introductory word... On behalf of the Organizing Committee I am honored to welcome you to Brno, for the 19 th Workshop of (Bio)physical Chemists and Electrochemists which is held again in Masaryk University (MU) Campus (Kamenice 5, Bohunice, building A11/room 205). This year s conference will be held to celebrate two events, the 100 th anniversary of Masaryk University and 60 th years of awarding the Nobel Prize to Prof. Jaroslav Heyrovský, Czech physical chemist. During the conference we will remember also Prof. Emil Paleček, the famous Czech electrochemist who worked in Brno and devoted his career to electrochemistry of biologically important substances, especially of nucleic acids. According to the attached program you can see the schedule of plenary lectures, invited lectures as well as oral presentations of young scientists. The conference will also include presentations of posters and companies which supporting the event. Special thanks go to the main sponsor, which, in addition to the Biophysical Institute, financially participates in the Emil Paleček Award. All participants are invited not only to the Young Scientists Session and the Poster Session but also to the gala party on Friday early in the evening ( On the foodbridge ) where three winners of the Young Scientists Session and one winner of the Poster Session will be announced. We are confident that our 19 th Workshop of Biophysical Chemists and Electrochemists will be memorable for its scientific quality, also thanks to your contributions. We do hope that you will welcome the opportunity to present and discuss your scientific results with respect the Heyrovsky s logo: Experimenting to knowledge, learning to progress. Welcome to Brno and enjoy this conference! Libuše Trnková Motto: Experimenting to knowledge, learning to progress Jaroslav Heyrovský 6

7 Remembering Prof. Emil Paleček Na podzim roku 2018 nás navždy opustil významný český vědec, který jako první na světě v roce 1960 objevil způsob, jak zkoumat DNA pomocí elektrochemie. S využitím objevu Jaroslava Heyrovského polarografie výrazně přispěl k pokroku v oblasti chemické reaktivity nukleových kyselin a studia lokálních struktur. Zasloužil se i o rozvoj elektrochemie proteinů. Pomocí elektroanalytických metod studoval chování proteinů a jejich komplexů s DNA na elektricky nabitých površích, zabýval se analýzou glykoproteinů se zvláštním zřetelem na její budoucí uplatnění v lékařství. Jeho celoživotní dílo je významné, mnohé jeho poznatky vešly do učebnic biofyziky a molekulární biologie. Věnujme tichou vzpomínku tomuto významnému českému vědci a držiteli mnoha čestných ocenění. Ocenění vědecké práce profesora Emila Palečka v posledních letech 2009 Čestná medaile Akademie věd České republiky De scientia Et Humanitate Optime Meritis Profesor Jiří Drahoš, předseda Akademie věd ČR, udělil 18. září 2009 Čestnou medaili AV ČR De Scientia et Humanitate Optime Meritis prof. Emilu Palečkovi z Biofyzikálního ústavu AV ČR v Brně. Medaile je udělována za mimořádné aktivity v oblasti vědy a úspěšné zajišťování infrastuktury výzkumu a vývoje. Foto: Zdeněk Tichý, Archiv KNAV 7

8 2011 Cena ministra školství, mládeže a tělovýchovy Cena ministra školství, mládeže a tělovýchovy za mimořádné výsledky výzkumu, experimentálního vývoje a inovací byla v roce 2011 udělena Prof. RNDr. Emilu Palečkovi, DrSc. za výsledky výzkumu v oblasti elektrochemických metod, vhodných k analýze změn ve struktuře bílkovin a jejich aplikace v biomedicíně (například při výzkumu Parkinsonovy choroby a rakoviny) a také za dlouholetý významný přínos k rozvoji elektrochemie nukleových kyselin, která má široké uplatnění při decentralizované analýze DNA Plaketa Johanna Gregora Mendela Za mimořádné zásluhy o rozvoj vědy a vzdělání v oblasti biologie, molekulární biologie a genetiky byl prof. Emil Paleček spolu s Dr. Michaelem Heyrovským poctěn plaketou Johanna Gregora Mendela při příležitosti konání XI. Pracovního setkání chemiků a elektrochemiků na MENDELU v Brně Cena firmy Metrohm Prestižní cenu firmy Metrohm za celoživotní přínos k rozvoji elektroanalytické chemie získal za rok 2013 prof. RNDr. Emil Paleček, DrSc. z Biofyzikálního ústavu AV ČR v.v.i. v Brně. Založil v 60. letech minulého století zcela novou oblast elektrochemii nukleových kyselin. V poslední době se zabýval i výzkumem bílkovin, které mají souvislost se vznikem rakoviny Národní cena vlády Česká hlava Národní cenu vlády Česká hlava za rok 2014 vyhlásil na tiskové konferenci 27. listopadu 2014 místopředseda vlády pro vědu, výzkum a inovace Pavel Bělobrádek. Oceněným pro tento rok byl prof. RNDr. Emil Paleček, DrSc. přední český vědec v oblasti přírodních věd. Svými pracemi položil základ pro obor elektrochemie nukleových kyselin. Na slavnostním Galavečeru v Národním domě na Vinohradech v Praze pak cenu předal premiér vlády České republiky Bohuslav Sobotka. 8

9 2017 Cena Neuron za přínos světové vědě Profesor Emil Paleček z Brna se stal prvním vědcem na světě, který ukázal, že DNA lze analyzovat elektrochemicky, a založil nové vědecké pole, na kterém dnes pracují stovky laboratoří ve světě. Patřil a ještě patří díky více než třem stovkám vydaných vědeckých prací mezi nejcitovanější vědce v zemi. Zaujal tuzemskou odbornou veřejnost, byl hojně citován i v zahraničí. Jeho dílo vešlo do učebnic biofyziky a molekulární biologie. 9

10 Table of contents POLAROGRAPHY IN CONTEMPORARY MOLECULAR ELECTROCHEMISTRY SUPRAMOLECULAR PUZZLES: HOST-GUEST COMPLEXES IN METALLODRUG RESEARCH SIMPLE AND RAPID ELECTROCHEMICAL ASSAY FOR DETECTION OF HPV IN CLINICAL SAMPLES EFFECT OF THE LENGTH OF CYTOSINE OLIGONUCLEOTIDE BLOCKS ON THEIR ELECTROREDUCTION AT THE MERCURY ELECTRODE CHARGE TRANSPORT IN EXTENDED BIPYRIDINIUM SINGLE MOLECULE JUNCTIONS.. 16 PRINTED CIRCUIT BOARD ELECTRODES AS A PLATFORM FOR DISPOSABLE SENSING PARTS SIMULTANEOUS ACTIVATION AND NANOMATERIAL MODIFICATION OF ELECTRODE SURFACES USING SPARK DISCHARGES STRUCTURAL STUDY OF ζ MONOMERIC MUTANT ELECTROCHEMICAL ASSAY FOR microrna DETECTION USING SPECIFIC ANTIBODY AND HYBRIDIZATION CHAIN REACTION: APPLICATION TO CLINICAL SAMPLES AMPEROMETRIC IMMUNOSENSOR FOR DIAGNOSIS OF EUROPEAN FOULBROOD STUDY OF CHARGE TRANSFER AND CHARGE TRANSPORT MECHANISM IN EXPANDED PYRIDINIUM MOLECULES BIOPHYSICAL ANALYSIS OF SILVER NANOPARTICLES AND USE THEIR ANTIMICROBIAL ACTIVITY IN 3D PRINTING POLYMER NANOSPHERE-ASSEMBLED SURFACE FOR BIOSENSING BASED ON ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY ELECTROCHEMICAL DETERMINATION OF INSULIN ON NiONPS MODIFIED CARBON ELECTRODES AMINOFERROCENE: DETERMINATION OF THE ACIDITY OF UNSTABLE COMPOUNDS.. 34 EFFECT OF NEWLY DESIGNED STABLE CFTR-MESSENGER RNA ON TRANSFECTION OF HUMAN CYSTIC FIBROSIS AIRWAY EPITHELIUM ITP ANALYSIS OF SWEET BEVERAGES AND BEERS APOFERRITIN NANOCAGE AS A PROMISING DOXORUBICIN NANOCARRIER AND ITS EFFECTS ON NEUROBLASTOMA CELL LINES ANALYSIS OF METABOLISM AND DNA ADDUCT FORMATION BY ARISTOLOCHIC ACIDS I AND II IN RATS IN VIVO DIRECT ELECTRODEPOSITION OF SILVER AMALGAM PARTICLES ON SCREEN PRINTED SILVER ELECTRODES USING DOUBLE PULSE CHRONOAMPEROMETRY

11 METABOLISM OF TYROSINE KINASE INHIBITOR CABOZANTINIB BY LIVER MICROSOMES COMPARISON OF EFFICIENCIES OF PEROXIDASES TO OXIDIZE THE ANTICANCER DRUG ELLIPTICINE AND THEIR INFLUENCING BY VANDETANIB, LENVATINIB AND CABOZANTINIB DEVELOPMENT OF INSTRUMENTATION FOR COULOMETRIC TITRATIONS ON ELECTROCHEMISTRY OF 1-PENTYL-3-(1-NAPHTOYL)INDOLE AND 1-PENTYL-3-(2- METHOXYPHENYLACETYL)INDOLE OXIDATION OF A TYROSINE KINASE INHIBITOR VANDETANIB BY RAT ENZYMATIC SYSTEMS IN VITRO APPLICATION OF LOW-FIELD 1 H NMR SPECTROSCOPY IN ANALYTICAL CHEMISTRY STUDY ON ENCAPSULATION OF LENVATINIB AND ELLIPTICINE INTO NANOTRANSPORTERS; EXPERIMENTAL AND THEORETICAL APPROACHES METALLOTHIONEIN AND SELENITE IN BRDIČKA REACTION OXIDATION POTENTIALS OF GUANINE SPECIES APPLICATION OF CD SPECTROSCOPY IN ANALYTICAL CHEMISTRY METABOLISM OF THE TYROSINE KINASE INHIBITOR LENVATINIB BY HUMAN HEPATIC MICROSOMES AND CYTOCHROMES P

12 POLAROGRAPHY IN CONTEMPORARY MOLECULAR ELECTROCHEMISTRY Jiří LUDVÍK J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, Prague, Czech Republic Lecture dedicated to 60th anniversary of Nobel Prize for polarography with thanks to my teachers, prof. Jiří Volke and prof. Petr Zuman Polarography was awarded by the Nobel Prize originally as a new analytical method (by the way the first fully automatic analytical method ever). After years of its theoretical and experimental development in 40th and 50th, in 60th and later it was broadly used not only in chemical research, but also in biology, medicine, pharmacy, archaeology, metallurgy, then since 80th up to now in environmental sciences and in biochemistry. Today in practical applications, polarography in its original form (DME) is often replaced by more fast, more sophisticated (but also more expensive) analytical methods which could be operated by laboratory assistants and which can be easily standardized. However, polarography (and its derived methods based on mercury electrodes) is currently a unique, irreplaceable and indispensable part of a spectrum of electrochemical methods namely for fundamental electrochemical research. And the ban on the use of mercury electrodes in chemical laboratories in several countries is unjustified and unwarranted. Every day many new organic and organometallic compounds as well as complexes and supramolecules are synthesized as promising pharmaceuticals, catalysts, agrochemicals, dyes, organic semiconductors, liquid crystals, molecules for photovoltaics, etc.etc. and their fundamental redox properties and reactivity in their oxidized or reduced state must be characterized and elucidated. Therefore polarography is currently very important in so called Molecular electrochemistry, discipline, where individual molecules in solution are investigated in detail. For full understanding, polarography can be combined in-situ with UVvis, IR or EPR spectrometry and the acquired data should be checked by quantum chemical calculations. For the further application the known thermodynamic, kinetic and structural data should be correlated with expected activities. For this presentation I selected one of the most recent investigations from our laboratory based mainly on polarography - the systematic research of nitro-substituted calixarenes, their electroreduction, stereoelectrochemistry and radical formation. In the second part some other interesting topics will be presented and discussed, like e.g. diphenyl isobenzofurans for singlet fission, Fischer carbenes or ferrocene derivatives. 12

13 1 SUPRAMOLECULAR PUZZLES: HOST-GUEST COMPLEXES IN METALLODRUG RESEARCH Radek MAREK 1,2* Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno CZ-62500, Czechia 2 CEITEC Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czechia *rmarek@chemi.muni.cz Supramolecular interactions govern many properties of systems ranging from the biological effects of drugs to the function of novel materials. These interactions are responsible for communication between individual molecules and formation of supramolecular host-guest assemblies. In this contribution, supramolecular metallocomplexes will be shown as prospective drugdelivery systems for administrating the novel anticancer metallodrugs based on platinum or ruthenium. 1 Our recent investigations of the host-guest systems containing Pt(IV) or Ru(II) anticancer cargo in cucurbit[n]uril carrier will be discussed. 2,3 Further, unprecedented paramagnetic NMR characterization of Ru(III) coordination compounds 4-6 and their hostguest complexes with macrocyclic cavitands (cyclodextrins or cucurbiturils) 7 will be demonstrated. Finally, perspectives of our research in the design and development of supramolecular cages and metallodrugs will be outlined. ACKNOWLEDGEMENT This work has received support from the Czech Science Foundation (Grant No S) and the Ministry of Education of the Czech Republic (Grant No. LQ1601). REFERENCES [1] Mjos K. D., Orvig C.: Chem. Rev., 114 (2014), [2] Chyba J. et al.: unpublished results. [3] Sojka M. et al.: Inorg. Chem., submitted. [4] Novotny J. et al.: J. Am. Chem. Soc., 138 (2016), [5] Novotny J. et al.: Inorg. Chem., 57 (2018), [6] Jeremias L. et al.: Inorg. Chem., 57 (2018), [7] Chyba J. et al.: Inorg. Chem., 57 (2018),

14 1 SIMPLE AND RAPID ELECTROCHEMICAL ASSAY FOR DETECTION OF HPV IN CLINICAL SAMPLES Martin BARTOŠÍK 1*, Ludmila JIRÁKOVÁ 1, Roman HRSTKA 1 RECAMO, Masaryk Memorial Cancer Institute, Zluty kopec 7, Brno, Czech Republic *martin.bartosik@mou.cz Persistent infection with human papilloma virus (HPV) can cause malignant tissue transformation and lead to various types of cancers, most often cervical cancer in women [1]. HPV16 and HPV18 are two most oncogenic high-risk HPV types which cause over 70% of HPV-positive cervical carcinomas. Current detection methods [2] involve complex protocols, need for skilled personnel and expensive reagents and instruments. Electrochemical methods of detection may circumvent these challenges by offering rapid, simple and inexpensive assays [3]. In our work, we focused on development of such assay by combining loop-mediated isothermal amplification (LAMP) reaction to amplify DNA from cancer cells, magnetic beads for improved selectivity and amperometric measurement performed on carbon electrode arrays for parallel measurements to speed up the protocol. We were able to determine and discriminate HPV16 and HPV18 types not only in cervical cancer cell lines, but more importantly in clinical samples isolated from cervical smears obtained during gynecological examinations [4]. Currently, we are adapting this method also for detection of oncoviral mrnas, E6 and E7 mrnas, which better reflect virus activity and may thus help to reveal disease progression before cellular changes become visible in classical cytological examination. ACKNOWLEDGEMENT The work has been supported by GACR S and MEYS-NPS I-LO1413. REFERENCES [1] Schiffman M., Castle P. E., Jeronimo J., Rodriguez A. C., Wacholder S.: Lancet 370 (2007), [2] Kurian E. M., Caporelli M. L., Baker S., Woda B., Cosar E. F, Hutchinson L.: Am. J. Clin. Pathol. 136 (2011), [3] Palecek E., Bartosik M.: Chem. Rev. 112 (2012), [4] Bartosik M., Jirakova L., Anton M., Vojtesek B., Hrstka R.: Anal. Chim. Acta 1042 (2018),

15 EFFECT OF THE LENGTH OF CYTOSINE OLIGONUCLEOTIDE BLOCKS ON THEIR ELECTROREDUCTION AT THE MERCURY ELECTRODE Miroslav FOJTA 1,2, Hana PIVONKOVA 1, Stanislav HASON 1, Zuzana BABKOVA 1, 1 Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, CZ Brno, Czech Republic 2 Central European Institute of Technology, Masaryk University, Kamenice 5, CZ Brno, Czech *fojta@ibp.cz Republic Behavior of nucleic acids (NA) at electrodes is influenced by their nucleobase composition, sequence and secondary structure. Mercury electrodes are still most frequently used tool for the studies of electrochemical reduction of natural nucleobases. Among them, cytosine and adenine are irreversibly electroreduced while reduction of guanine gives a product that can be electrooxidized back to guanine, giving a well-developed anodic signal. Moreover, signals of polyanionic NAs interacting with the negatively charged atomically smooth mercury electrode surface are strongly influenced by the NA structure, allowing sensitive detection of helix-coil transitions, DNA damage, interactions of DNA with small molecules as well as G- quadruplex formation. Our recent data indicate that interactions of NA chains with the mercury electrode surface are remarkably influenced by the presence of homonucleotide blocks. Homopyrimidine oligonucleotides exhibit 2D condensation at negatively charged electrode surface and stronger adsorption than homopurine ones. Cytosine blocks showed a tighter adsorption, compared to other homonucleotide blocks, as indicated by the dominance of C-E curves characteristic for homocytosine stretches obtained in competition experiments. Moreover, cytosine block exhibited an anomalous reduction behavior. Longer homocytosine stretches gave well developed reduction signals at unusually high ph values (above ph 8) at which random (but containing cytosine at levels corresponding to at least ¼ of all nucleobases) yielded no reduction peaks. Maximum ph values at which cytosine reduction was observed were dependent on the length of homocytosine blocks separated by other nucleotide sequences and followed similar trends as the propensity of the same nucleotides to formation cytosine tetraplexes - i-motifs (as assessed by CD spectra measurements). ACKNOWLEDGEMENT This work has been supported by the SYMBIT project reg. no. CZ /0.0/0.0/15_003/ financed from the ERDF. 15

16 CHARGE TRANSPORT IN EXTENDED BIPYRIDINIUM SINGLE MOLECULE JUNCTIONS Magdaléna HROMADOVÁ 1*, Viliam KOLIVOŠKA 1, Jakub ŠEBERA 1, Táňa SEBECHLEBSKÁ 1,2, Štěpánka NOVÁKOVÁ LACHMANOVÁ 1, Jindřich GASIOR 1, Pavel MORENO GARCIA 3, Gábor MÉSZÁROS 4, Michal VALÁŠEK 5 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, Prague, Czech Republic Department of Physical and Theoretical Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, Bratislava, Slovak Republic Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland Research Centre for Natural Sciences, HAS, Magyar Tudósok krt. 2, H-1117, Budapest, Hungary Karlsruhe Institute of Technology, Institute of Nanotechnology, P. O. Box 3640, Karlsruhe, Germany *hromadom@jh-inst.cas.cz Aim of this contribution is to compare charge transport properties of two photochemically addressable single molecule switches (see Figure below), which have the same length but differ in a torsion angle between individual aromatic rings of the conductance path. Single molecule conductance was obtained by scanning tunneling microscopy break junction technique that was complemented by theoretical analysis based on the density functional theory and non-equilibrium Green s function approach [1]. Figure: Chemical structures of extended bipyridinium cations 1 2+ and The conductance measurements were complemented by UV-VIS spectroscopy and electrochemical studies. Both molecules accept four electrons. Molecule 1 2+ is reduced in two two-electron steps, whereas molecule 2 2+ in three steps involving first transfer of two electrons, followed by two one electron waves [2,3]. We will show that the energy and shape of the LUMO is insensitive to the value of θ, but the difference in torsion angle θ leads to a sizable shift of the LUMO energy and single molecule conductance value in the metalmolecule-metal junction arrangement. Single molecule conductance of cation 2 2+ is 3.2 times higher than that for cation 1 2+ and an increase in the charge transport magnitude upon the photochemical cyclization of 1 2+ to 2 2+ is related to an enhanced electronic communication between pyridine and pyridinium moieties. The experimental conductance ratio is somewhat smaller than that obtained from the DFT/NEGF analysis, but DFT/NEGF is in a perfect agreement with the value calculated from torsion angles demonstrating that the investigated extended 4,4 -bipyridinium system follows the cos 2 θ law [3,4]. 16

17 ACKNOWLEDGEMENT This work has been supported by the Czech Science Foundation ( S). REFERENCES [1] Hromadová M., Kolivoška V.: Studying the Electrical Properties of Single Molecules by Break Junction Techniques. In: Encyclopedia of Interfacial Chemistry: Surface Science and Electrochemistry. (Ed. K. Wandelt), Elsevier, 5, (2018) [2] Nováková Lachmanová Š., Šebera J., et al.: Electrochimica Acta, 264 (2018) [3] Šebera J., Sebechlebská T., et al.: Electrochimica Acta, 301 (2019) [4] Venkataraman L., Klare J. E., et al.: Nature, 442 (2006)

18 PRINTED CIRCUIT BOARD ELECTRODES AS A PLATFORM FOR DISPOSABLE SENSING PARTS Karel LACINA Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czech Republic *lacinak@chemi.muni.cz Printed circuit boards (PCBs) are utilised in production of electronics. It combines mechanical support with conductive copper tracks for electronic elements in circuits. All employed methods and techniques are well-developed, robust, optimised and cheap. Sometimes, the contact pads are galvanically plated with gold to enhance their long-term stability compared to bare copper. These exposed gold pads can be used as sensing electrodes [1-3]. A routine, large scale monitoring, such as point-of-care (POC) systems, requires inexpensive and simple analytical platforms. If specifically designed, disposable and very cheap electrodes can be produced using PCB techniques (Figure 1). If the dimensions are optimized, estimated price for one piece can be $ (1.8 Kč). The crucial parameter for all electrochemical methods is the quality of a reference electrode. In fact, the reference electrode appeared to be the Achilles heel of electroanalytical disposable applications, where cheap and simple parts should be integrated. Use of external reference, additional plating of electrode surfaces with silver or combination with screenprinting was necessary. One of the possible solutions for this issue is to use biamperometry measuring with the electrode system consisting of two identical electrodes (e.g. two gold electrodes) [4]. An electroanalytical concept of biamperometry has been overlooked due to the widespread potentiostat-controlled experiments. In this case, no reference electrode is needed as the sensing electrodes are referenced to each other throughout the well-designed electrolyte. So, well-defined electrochemistry can be performed even with such simple and cheap setup. This system combination of PCB electrodes with biamperometry - has already been utilised in biosensing applications employing both enzymes and antibodies as biorecognition elements. The possible utilization of the introduced concept was proven on an exemplar determination of glucose by means of immobilized glucose oxidase. Affinity-based biosensing was performed with biamperometric electrode systems as well. Determination of the antibody (anti-human Serum Albumin) binding on the antigen-modified (HSA) surface was followed with measurement of electrochemical impedance. Interesting behavior of the signal was observed decrease of the impedance upon binding of the analyte in the environment of 5 mm ferro/ferricyanide. The behavior was explained as charge-dependent [3]. 18

19 Figure: Photography of a set of 240 disposable biamperometric electrode systems prepared using Printed Circuit Board approach (left). Various architectures of electrodes can be designed example of biamperometric electrode system with different electrode areas (right). ACKNOWLEDGEMENT The work has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601) and by the Czech Science Foundation, grant nr Y. REFERENCES [1] La Belle J. T., Shah M, Reed J, Nandakumar V, Alford T. L., Wilson J. W., Nickerson C. A. and Joshi L.: Electroanalysis, 21 (2009), [2] Cui H, Xiong X, Gao B, Chen Z, Luo Y, He F, Deng S. and Chen L.: Electroanalysis, 28 (2016), [3] Lacina K, Sopoušek J, Čunderlová V, Hlaváček A, Václavek T and Lacinová V.: Electrochem. Commun., 93(2018), [4] Lacina K, Vanýsek P, Bednář P, Trnková L and Skládal P.: ChemElectroChem, 3 (2016),

20 SIMULTANEOUS ACTIVATION AND NANOMATERIAL MODIFICATION OF ELECTRODE SURFACES USING SPARK DISCHARGES Jan HRBÁČ 1 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic *jhrbac@mail.muni.cz The sensors capable to sensitively determine diverse analytes important in various research and industry applications often utilize electrochemical principles. Novel strategies to develop cheap and easy to use sensors, preferentially disposable and mass producible are intensely studied. Often, these sensors use inorganic nanoparticles (mostly metallic) or nanomaterials (graphene, nanotubes etc.) The fabrication of nanoparticle based sensors can be tedious, encompassing a number of steps, including nanoparticle synthesis, purification, embedding in a suitable matrix, application onto sensor surface, temperature stabilization etc. The specific problem of mass produced sensors is the surface poorly performing from the electrochemical point of view, exhibiting sluggish electron transfer between the electrode surface and analyte molecules or electrode surface and modifying nanomaterial, triggering the need to activate the electrodes prior to modification and use. As an alternative to mechanical polishing, often impractical with mass produced sensors, chemical activation (e.g. exposing to oxidiying agents), electrochemical activation (e.g. potential cycling) or low temperature plasma is used. A novel approach based on application of spark discharges offers simultaneous activation and nanomaterial modification of electrode surfaces. The lecture aims at presenting an overview of current work on spark discharge modified disposable electrodes [1-7]. REFERENCES [1] Riman D, Jirovsky D, Hrbac J, Prodromidis M.I.: Electrochem. Commun. 50 (2015), 20. [2] Bartosova Z, Riman D, Halouzka V, Vostalova J, Simanek V, Hrbac J, Jirovsky D.: Anal Chim Acta, 935 (2016), [3] Riman D, Avgeropoulos A, Hrbac J, Prodromidis M.I.: Electrochim. Acta, 165 (2015), 410. [4] Riman D, Spyrou K, Karantzalis A.E, Hrbac J, Prodromidis M.I.: Talanta, 165 (2017), 466. [5] Trachioti M.G., Hrbac J, Prodromidis M.I.: Sens. Actuators B, 260 (2018), [6] Trachioti M.G., Karantzalis A.E., Hrbac J, Prodromidis M.I.: Sensor. Actuat. B-Chem., 281 (2019), 273. [7] Trachioti M.G., Tzianni E.I., Riman D, Jurmanova J, Prodromidis M.I., Hrbac J.: Electrochim. Acta, 304 (2019),

21 1 2 3 STRUCTURAL STUDY OF ζ MONOMERIC MUTANT Tomáš BROM 1,2, Aneta KOZELEKOVÁ 1,2, Petr LOUŠA 1, Norbert GAŠPARIK 1,2, Central European Institute of Technology, Masaryk university, Kamenice 5, , Brno, Czech Republic National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic Center for Interdisciplinary Biosciences, Technology and Innovation Park P.J. Šafárik University, Jesenná 5, Košice, Slovakia *jozef.hritz@ceitec.muni.cz The dimeric character of proteins is often crucial for their functions and for their ability to bind phosphorylated binding partners [1] s form homo/heterodimers that are in dynamic equilibrium with monomers [2]. The dimeric form of proteins has been heavily studied, however, the structure and properties of the monomeric form remain insufficiently described. The monomerization of ζ is regulated in different ways, including phosphorylation of Ser58 located at the dimer interface [3]. The mutant of ζ with a double mutation was designed [4] that exhibits complete monomerization over a wide range of concentrations while retaining the characteristic α-helical structure based on the far-uv CD measurements. The monomeric character of the designed mutant was proved by analytical ultracentrifugation and small angle X-ray scattering. Differential scanning calorimetry showed that the stability of monomeric mutant is lower than ζ WT. Nuclear magnetic resonance provided partial insight into the structure. NMR HN-HSQC experiments revealed that the monomeric mutants possess very similar overall NMR fingerprints as ζ WT that could indicate similar fold of monomeric mutants. Taken together, these results offer additional insight into the monomeric form stability and structure of this important family of regulatory proteins. ACKNOWLEDGEMENT This work was supported by the research grant from the Czech Science Foundation, grant no. GA L. The results of this research have been acquired within the CEITEC 2020 (LQ1601) project. This work was supported by Ministry of Education, Youth and Sports within program INTER-ACTION (LTAUSA18168). REFERENCES [1] Aitken, A., Semin. Cancer Biol., 2006, 16, [2] X. Yang et al., Proc. Natl. Acad. Sci., 2006, 103, [3] Sluchanko, N. N., Gusev, N. B., Arch. Biochem. Biophys., 2008, 477, [4] Jandova, Z. and Hritz, J., Biochim. Biophys. Acta, 2018,1866(3),

22 ELECTROCHEMICAL ASSAY FOR microrna DETECTION USING SPECIFIC ANTIBODY AND HYBRIDIZATION CHAIN REACTION: APPLICATION TO CLINICAL SAMPLES Ludmila JIRÁKOVÁ 1*, Martin BARTOŠÍK 1 1 RECAMO, Masaryk Memorial Cancer Institute, Žlutý kopec 7, Brno, Czech Republic *408579@mail.muni.cz MicroRNAs (mirnas) are small non-coding RNA molecules with many regulatory functions, including cell differentiation, proliferation or apoptosis. Recent research demonstrated that mirnas can be associated with onset and progression of various types of cancer and are thus considered to be interesting biomarkers or therapeutic targets. Current detection methods involve long protocols, often with reverse transcription step or fluorescent labeling and require expensive and very sensitive fluorescent readout instruments. Aim of our project is to develop rapid, reliable and inexpensive electrochemical (EC) method for up-regulated mirnas detection. Our EC assay comprised mirna-specific hybridization probe and two biotinylated auxiliary probes (Figure). Target mirna separation from the sample was facilitated by protein G-magnetic beads, which were modified with special S9.6 antibody specifically binding RNA/DNA heteroduplexes [1]. EC signal, generated by enzymatic reaction catalyzed by horseradish peroxidase (HRP) conjugated to streptavidin [2], was measured by chronoamperometry. As a target mirna model, we used mir-21, which has already been shown to be up-regulated in a wide range of tumors and is thus well detectable in real samples. Following the optimization, we then detected other up-regulated mirnas on a wide panel of cancer cell lines as well as in total RNA samples isolated from precancerous cervical tissues. These results make the assay potentially useful in treatment response prediction or early cancer diagnostics. Figure: Detection system for mirna, including magnetic beads modified with S9.6 antibody and three different DNA probes to increase the sensitivity of the measurement in so-called hybridization chain reaction. 22

23 ACKNOWLEDGEMENT The work has been supported by GAČR S and MEYS-NPS I-LO1413. REFERENCES [1] Boguslawski S., Smith D. et al.: Journal of Immunological Methods, 89 (1986), 1, [2] Torrente-Rodríguez R., Ruiz-Valdepeñas Montiel V. et al: ACS Sensors, 1 (2016), 7,

24 1 2 AMPEROMETRIC IMMUNOSENSOR FOR DIAGNOSIS OF EUROPEAN FOULBROOD Zuzana MIKUŠOVÁ 1,2 *, Matěj PASTUCHA 1,2, Veronika POLÁCHOVÁ 1,2, Radka OBOŘILOVÁ 1, Petr SKLÁDAL 1,2, Zdeněk FARKA 2 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czech Republic * mikusovaz@mail.muni.cz Western honeybee is an important pollinator and therefore, an invaluable part of agriculture and biodiversity. Serious losses of honeybee colonies in recent years are attributed mostly to climate changes and various diseases. One of the most important microbial diseases of honeybees is European foulbrood (EFB), which is caused by pathogen Melissococcus plutonius [1]. From the economic and environmental point of view, the prevention of EFB spreading is crucial to prevent losses of honeybee colonies. Therefore, the development of an effective method for the detection of M. plutonius is necessary, ideally in the point-of-care (POC) format with sensitivity high enough to detect the pathogen before the clinical symptoms develop. Amperometry and electrochemical impedance spectroscopy (EIS) are highly sensitive and robust approaches compatible with POC testing [2]. Due to their low cost, portability and mass production capabilities, the electrochemical biosensors are typically based on screenprinted electrodes (SPEs). For proper functionality, the immunosensors require antibodies with high affinity and low cross-reactivity. Since there were no antibodies against M. plutonius available, we have prepared them in-house. Purified bacterial cell wall fraction was prepared for rabbit immunization. After 45 days, rabbit blood was collected and serum was prepared. Subsequently, the immunoglobulin G fraction was separated from the serum by liquid chromatography with protein G column. The final antibody in PBS was stored at 30 C for further use. Functionality of the prepared antibodies was verified using enzyme-linked immunosorbent assay (ELISA). The sandwich assay provided a limit of detection (LOD) of CFU ml 1. The ELISA was used to detect M. plutonius in real samples of bees, larvae and bottom hive debris, which are the matrices where this bacterium is typically present in the case of EFB infection [3]. For the electrochemical biosensing, the anti-melissococcus antibody was immobilized on the surface of SPE and allowed specific capture of bacteria. Non-specific binding was evaluated by incubating the sensor with Paenibacillus alvei instead of M. plutonius. The label-free EIS allowed to detect M. plutonius, however, the level of non-specific binding was very high, which was limiting for real samples analysis. Thus, better performance was obtained with amperometric sandwich assay, where the antibodies were conjugated with horseradish peroxidase (HRP). The Ab-HRP conjugate was binding to surface-captured immunocomplex and provided oxidation of 3,3,5,5 -tetramethylbenzidine (TMB) in presence of H 2 O 2. The electrochemical detection of current was based on the reduction of the enzymatically oxidized TMB on working electrode. For pure bacterial culture in buffer, the LOD was CFU ml 1. After optimization of amperometric immunosensor, real samples of bees 24

25 and larvae were tested with LODs CFU ml 1 and CFU ml 1, respectively. Time of analysis was only 2 hours compared to time-consuming laboratory assays such as ELISA [4]. The analysis of real bee and larvae samples confirmed the suitability of the developed immunosensor for in-field M. plutonius detection. Figure: (A) EIS response of antibody-modified SPE electrode to M. plutonius; (B) detection of M. plutonius based on amperometry in presence of H 2 O 2 after addition of TMB. The bacteria concentrations are expressed in CFU ml 1. ACKNOWLEDGEMENTS We thank Dr. Martin Faldyna and Dr. Lubomír Janda from the Veterinary Research Institute (Brno, Czech Republic) for collaboration on the development of polyclonal antibodies. This research has been financially supported by the Technology Agency of the Czech Republic (project TJ ) and Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). REFERENCES [1] Bailey L., Collins M. D.: J. Appl. Bacteriol., 53 (1982), [2] Mistry K.K., Layek K., Chell T.N., Chaudhuri C.R., Saha H.: Anal. Methods, 8 (2016), [3] Poláchová V., Pastucha M., Mikušová Z., Mickert M. J., Hlaváček A., Gorris H. H., Skládal P., Farka Z.: Nanoscale, 11 (2019), [4] Mikušová Z., Farka Z., Pastucha M., Poláchová V., Obořilová R., Skládal P.: Electroanalysis, 2019, submitted. 25

26 1 STUDY OF CHARGE TRANSFER AND CHARGE TRANSPORT MECHANISM IN EXPANDED PYRIDINIUM MOLECULES Štěpánka NOVÁKOVÁ LACHMANOVÁ 1*, Jakub ŠEBERA 1, Jindřich GASIOR 1, Gábor MÉSZÁROS 2, Grégory DUPEYRE 3, Philippe P. LAINÉ 3, Magdaléna HROMADOVÁ 1 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, Prague 8, Czech Republic 2 Research Centre for Natural Sciences, HAS, Magyar Tudósok krt. 2, H-1117, Budapest, Hungary 3 Univ Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR CNRS 7086, 15 rue J-A de Baïf, 75013, Paris, France *stepanka.lachmanova@jh-inst.cas.cz Development of the daily used electronic devices is limited by the miniaturization of single electronic components. One of the possibilities, how to reach even smaller component size, is the exchange of traditional silicon-based technologies by the specialized molecules. Such molecules could serve as molecular wires, diodes or switches [1]. Expanded pyridinium derivatives rank among the promising candidates for the purpose of molecular electronics. Expanded pyridinium molecules are due to their electroactivity and relatively high single molecule conductance suitable model compounds for the study of correlation between the characteristics of electron transfer (electrochemical activity) and electron transport (single molecule conductance). Even though the linear dependence between the charge transfer heterogeneous rate constant and conductance was predicted by theoretical works, [2, 3] the conclusive experimental confirmation is missing [4, 5]. Electron transfer mechanism of four expanded pyridinium derivatives (for structure see figure below) were studied by various electrochemical techniques. Compounds 1 and 3 are reduced by two electrons in two separate one-electron steps. The charge transfer rate constants of both reduction steps were obtained and higher values were observed for the first electron transfer. On the other hand, only one two-electron signal was obtained for molecules 2 and 4. Only the charge transfer rate constant for the first electron transfer was measurable by the used methods. The contrast between the reduction mechanism is caused by the ability of the molecules to undergo the structural changes after the first electron reduction [6, 7]. The presence of pyridyl anchoring groups enabled the single molecule conductance measurements by the scanning tunneling microscopy break junction (STM BJ) technique. Statistical analysis of measured conductance curves showed the highest conductance value for the molecule 1 [6]. Figure: Chemical structure of compounds 1 to 4. 26

27 The obtained values of charge transfer rate constants for the first electron reduction were ploted against the values of single molecule conductance. The linear dependence was observed for the molecules 1, 2 and 4. The obtained correlation experimentally confirms the theoretical relationship of charge transfer and charge transport characteristics [2, 3]. The compound 3 deviated from this dependence. Considerably higher value of charge transfer rate constant was observed. The electrochemical behaviour of compound 3 is influenced by the locked conformation caused by the chemical structure of molecule 3. ACKNOWLEDGEMENT The work has been supported by the Czech Science Foundation ( S). REFERENCES [1] Metzger R. M.: Nanoscale, 10 (2018), [2] Nitzan A.: Isr. J. Chem. 42 (2002), [3] Berlin Y.-A.; Ratner M. A.: Radiat. Phys. Chem. 74 (2005), [4] Venkatramani R.; Wierzbinski E.; Waldeck D. H.; Beratan D. N.: Faraday Discuss. 174 (2014), 174, [5] Zhou X. S.; Liu L.; Fortgang P.; Lefevre A. S.; Serra-Muns A.; Raouafi N.; Amatore C.; Mao B. W.; Maisonhaute E.; Schöllhorn B.: J. Am. Chem. Soc. 133 (2011), [6] Nováková Lachmanová Š.; Šebera J.; Kolivoška V.; Gasior J.; Mészáros G.; Dupeyre G.; Lainé P. P.; Hromadová M.: Elchim. Acta 264 (2018), [7] Lachmanová Š.; Dupeyre G.; Tarábek J.; Ochsenbein P.; Perruchot C.; Ciofini I.; Hromadová M.; Pospíšil L.; Lainé P. P.: J. Am. Chem. Soc. 137 (2015),

28 1 BIOPHYSICAL ANALYSIS OF SILVER NANOPARTICLES AND USE THEIR ANTIMICROBIAL ACTIVITY IN 3D PRINTING Karel SEHNAL 1,3*, Martina STANKOVA 2, Michaela DOCEKALOVÁ 2,3, Zuzana TOTHOVA 2, Dagmar UHLIROVA 2, Branislav RUTTKAY-NEDECKÝ 1,3, Augustine Enakpodia OFOMAJA 4, Marta KEPINSKA 5, Rene KIZEK 1,2,3 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1946/1, Brno, Czech Republic 2 Department of Research and Development, Prevention Medicals, Tovární 342, 742 1, Czech Republic 3 Institute of Viticulture and Wine Production, Faculty of Horticulture, Valtická 337, Lednice, Czech Republic 4 Biosorption and Wastewater Treatment Research Laboratory, Department of Chemistry, Faculty of Applied and Computer Sciences, Vaal University of Technology, P. Bag X021, Vanderbijlpark, 1900, South Africa, 5 Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory Medicine, Wroclaw Medical University, Borowska 211, Wroclaw, Poland *karelsehnal15@gmail.com In the 21 st century there is a steady increase in the resistance of microorganisms to antibiotics. Poorly understood mechanisms have remained a major problem. Prevention of these infections is not easy [1]. According to the NIH (National Institutes of Health), 99,987 people die in the United States as a result of these infections which translates to an average of 271 deaths per day, accounting for more loss of human lives than from HIV AIDS, car accidents and breast cancer put together [2, 3]. Metal nanoparticles have shown significant antibacterial activity similar to the plant/animal materials used in traditional medicine. The study focuses on the preparation of silver nanoparticles (AgNPs) by green synthesis which utilizes plant extracts as reducing agents and their use in 3D printing. AgNPs obtained by this type of synthesis contain biomolecules bound to their surface. AgNPs and used plant extracts were studied in detail by physico-chemical methods (Table). A concentration of total protein in plant extract from S. officinalis prepared at different temperatures was determined by both biuret (BM) and pyrrogallol red (PM) methods. We also optimized a methodology for measuring of flavones using aluminium method. For determination of fenolic compounds Folin Ciocalteatu method was used. Antioxidant activity was measured using both ABTS (2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) and DPPH ((N,N-dimethyl)-1,4-diaminobenzene) method. Dried plant homogenized mixture was stirred in water at different temperatures (20, 40, 60, 80 C) for 60 minutes in a ratio of 1:20 followed by centrifugation (15 min, 4000 g). The extract was mixed with 0.1 M AgNO 3 (1:1), and the prepared nanoparticles were purified with methanol (1:1). After precipitation, methanol was removed and the AgNPs were dried. In addition, the purified AgNPs were dispersed in 18 MΩ of water and acetone (1:1, c = 3 mg/ml). AgNPs in this form were applied to the 3D fiber by means of a brush and a material was printed (Figure). 28

29 Silver nanoparticles have pronounced antibacterial properties. However, there is a concern about their persistence in the environment. An alternative is the surface treatment by biomolecules form plant extracts of such nanoparticles. We created unique material modified by AgNPs which has a potential antimicrobial activity. This material can be use in hospitals to reduce the number of infected patients by nosocomial infections. Figure: Optimalization of a methodology of a 3D filament modification. A) printed material without AgNPs, B) printed material with AgNPs (c = 3 mg/ml), C) printed material with AgNPs (c = 12 mg/ml), D) nonmodified filament E) modified filament by AgNPs (l filament = 40 cm; V AgNPs = 2 ml), F) modified filament by AgNPs (l filament = 120 cm; V AgNPs = 6 ml). Table: Characterization of plant extracts from S.officinalis by physico-chemical methods. Extraction temperature Extraction temperature Extraction temperature Extraction temperature Plant extract 20 C Color scheme Total protein (BM) Total protein (PM) Fenolic compounds Flavones Antioxidant activity ABTS Antioxidant activity DPPH Recovery (D) (g/l) (g/l) (g/l) (g/l) (GA g/l) (GA g/l) (%) S. officinalis (S) 0.27± ± ± ± ± ± ± C S. officinalis (S) 0.27± ± ± ± ± ± ± C S. officinalis (S) 0.30± ± ± ± ± ± ± C S. officinalis (S) 0.31± ± ± ± ± ± ± ACKNOWLEDGEMENT The work has been supported by H2020 CA COST Action CA15114, and INTER-COST LTC REFERENCES [1] Bailey, L. and M.D. Collins, Reclassification of Streptococcus pluton (White) in a new genus Melissococcus, as Melissococcus pluton nom. rev.; comb. nov. J. Appl. Bacteriol., (2): p [2] Heger, Z., et al., A Novel Insight into the Cardiotoxicity of Antineoplastic Drug Doxorubicin. Int. J. Mol. Sci., (11): p [3] Arora, A. and E.M. Scholar, Role of tyrosine kinase inhibitors in cancer therapy. J. Pharmacol. Exp. Ther., (3): p

30 [4] Reibenwein, J. and M. Krainer, Targeting signaling pathways in ovarian cancer. Expert Opin. Ther. Targets, (3): p [5] Meyer, M.R., New psychoactive substances: an overview on recent publications on their toxicodynamics and toxicokinetics. Archives of Toxicology, (10): p [6] Wintermeyer, A., et al., In vitro phase I metabolism of the synthetic cannabimimetic JWH-018. Analytical and Bioanalytical Chemistry, (5): p [7] Grigoryev, A., et al., Gas and liquid chromatography mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures. Journal of Chromatography B, (25): p [8] Fujita, K., Cytochrome P450 and anticancer drugs. Curr. Drug Metab., (1): p [9] He, D.D., et al., Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments. Elife,

31 POLYMER NANOSPHERE-ASSEMBLED SURFACE FOR BIOSENSING BASED ON ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY Jakub SOPOUŠEK 1*, Karel LACINA 1 1 Central European Institute of Technology, Masaryk University, Kamenice 753/ , Brno, Czech Republic *423048@mail.muni.cz Electrochemical impedance spectroscopy (EIS) emerges as the promising method for biosensing due to its label-free detection capabilities together with the high sensitivity. But despite all the praise, EIS has difficulties in differentiation of particular (bio)chemical processes as the output signal is affected by impedance of a bulk and by impedance at the solution/electrode interface. Moreover, bio-related surfaces are very complex, and it is thus highly challenging to design a reliable biosensing system where only an analyte generates measurable signal specifically. We develop an analyte-sensitive surface that provides dominant output signal changes upon interaction. Its modification layer consists mainly of spherical polystyrene nanoparticles, homopolymer poly-l-lysine, and protein human serum albumin (HSA). Immobilized HSA protein specifically interacts with the analyte, anti-hsa antibody present in a liquid sample. The biosensing system is designed in a way that it contains the assembly of spherical nanoparticles forming thus specifically large nanopores. As the nanoparticles are coated with the protein ligand (HSA), an interaction with specific antibodies results in the blockage of nanopores. This effect results in radical changes of impedance as the analytical response comes from differentiation between permeable and isolated state of the modification layer. Such surfaces can be applied for testing of blood sera to detect immunological response of a patient s pathologic state antigen will be immobilized onto the assembly of nanoparticles and the specific antibodies, present in patient s blood, will be detected. ACKNOWLEDGEMENT This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). 31

32 ELECTROCHEMICAL DETERMINATION OF INSULIN ON NiONPS MODIFIED CARBON ELECTRODES Ivana ŠIŠOLÁKOVÁ 1*, Jana HOVANCOVÁ 1, Renáta ORIŇAKOVÁ 1, Libuše TRNKOVÁ 2, Andrej ORIŇAK 1 1 Department of Physical Chemistry, Faculty of Science, Pavol Jozef Šafárik University, Moyzesova 11, Košice, Slovak Republic 2 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic *ivana.smoradova@student.upjs.sk Diabetes mellitus can be considered as the one of the most widespread disease in the world [1]. Thus a construction of highly sensitive, small, rapid, selective, accurate and cost-effective electrochemical sensor for glucose or insulin determination is called for [2]. This work deals with electrochemical determination of insulin on nanomodified screen printed carbon electrodes (SPCEs). Mentioned electrodes have significantly smaller size of working electrode (4 mm in diameter) compared to classic electrodes, achieving miniaturization of the system. SPCEs were modified by combination of multi walled carbon nanotubes (MWCNTs), chitosan and nickel oxide nanoparticles (NiONPs). NiONPs were used because of high electrical conductivity, low cost and excellent catalytic activity towards insulin oxidation because of the presence of NiO(OH) active species formed in alkaline solution. The correlation coefficient, limit of detection and sensitivity of prepared electrode was determined and compared to unmodified SPCE. NiONPs/chitosan-MWCNTs/SPCE displayed wide linear concentration range for insulin determination (0.25 µm 0.5 µm) with R 2 = 0.997, low limit of detection (94 nm) and excellent sensitivity of µa/µm. Having in mind the possible interaction of substances that can be contained in real samples, the influence of interferences (sucrose, glucose, ascorbic acid and lactic acid) on insulin determination at NiONPs/chitosan- MWCNTs/SPCE was studied. The cyclic voltammograms of above mentioned compounds did not show any measurable interference in insulin detection. Due to the small size and appropriate analytical characteristics, NiONPs/chitosan-MWCNTs/SPCE can be considered as the suitable candidate for electrochemical determination of insulin in real samples. Figure: Prepration of NiONPs/chitosan-MWCNTs/SPCE 32

33 ACKNOWLEDGEMENT The work has been supported by the projects VEGA 1/0074/17 of the Slovak Scientific Grant Agency, APVV of the Slovak Research and Development Agency, VVGS-PF and VVGS-PF of Pavol Jozef Šafárik University in Košice. REFERENCES [1] Kanaka-Gantenbein C., Mastorakos G., Chrousos G.P., Ann. N. Y. Acad. Sci., 997 (2003) [2] Prasad B.B., Madhuri R., Tiwari M.P., Sharma P.S., Electrochim. Acta. 55 (2010)

34 1 2 AMINOFERROCENE: DETERMINATION OF THE ACIDITY OF UNSTABLE COMPOUNDS Jakub VĚŽNÍK 1,2*, Libuše TRNKOVÁ 1, Karel LACINA 2 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czech Republic *jveznik@mail.muni.cz Ferrocenes are quite popular in electrochemistry, due to the simplicity of redox reactions of iron core. While ferrocene is nonpolar and thus of limited use, its cyclopentadienyl ring opens way for many interesting modifications. One of which is aminoferrocene (FcNH 2 ), the -NH 2 group increases solubility in polar solvents, introduces chemical reactivity and most importantly ph sensitivity to the ferrocene complex. Due to the proximity of NH 2 group and Fe 2+ ion in FcNH 2, the redox potential of FcNH 2 is influenced by protonation and vice versa the Fe 3+ ion changes acidity of the complex upon oxidation. Electrochemical observation of this interplay can be used to deduce the acidity of the Fe 3+ complex. Such approach is useful for complexes that are unstable in their oxidized form. pk a constants determined using electrochemical methods are obtained through kinetic processes, therefore great care needs to be taken when interpreting these results. While the limiting redox potential for FcNH 2 is easily obtained from electrochemical results (Figure), the limiting potential for FcNH 3 + strongly varies depending on the type of electrode used. This is anomalous from other ferrocene derivates, namely ferroceneboronic acid [1] and ferrocenecarboxylic acid [2], where both limiting redox potentials are easily obtainable. In our contribution, we tried to uncover the correct pk a value of aminoferrocene through different electrochemical methods both in buffered and unbuffered solutions. Spectrophotometry was used along to validate our findings. 34

35 Figure: Redox potential of 1 mm aminoferrocene (FcNH 2 ) in dependence on ph. Standard three electrode setup was used with different working electrodes: Gold, platinum, glassy carbon and boron doped diamond (BDD). The intercepts of the slope and the limiting values of redox potential equates to pk a of FcNH 2 (pk a = 5.8). Determination of pka of FcNH + 3 was affected by the type of working electrode (glassy carbon and boron doped diamond) and was not obtainable for gold and platinum electrodes. ACKNOWLEDGEMENT This research has been financially supported by the project CEITEC 2020 (LQ1601), MUNI/A/1359/2018 and by the Czech Science Foundation, grant nr Y. REFERENCES [1] Moore A.N.J., Wayner D.D.M.: Can. J. Chem., 77 (1999) [2] De Santis G, Fabbrizzi L, Licchelli M, Pallavicini P: Inorganica Chim. Acta, 225 (1994)

36 EFFECT OF NEWLY DESIGNED STABLE CFTR-MESSENGER RNA ON TRANSFECTION OF HUMAN CYSTIC FIBROSIS AIRWAY EPITHELIUM Lucie BOŘEK-DOHALSKÁ 1 *, Tomáš KOBLAS 2, Kateřina PECKOVÁ 1, Jan KRÁL 1, Marie 1 STIBOROVÁ 1, Pavel DŘEVÍNEK 3, Petr HODEK 1 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 2, Czech Republic 2 Institute for Clinical and Experimental Medicine, Vídeňská 1958/9, Prague 4, Czech Republic 3 Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and University Hospital Motol, V Úvalu 84, Prague 5, Czech Republic * borekdol@natur.cuni.cz Cystic fibrosis (CF) is the most frequent lethal autosomal recessive disease within the Caucasian population. This disorder results from mutations in the gene for cystic fibrosis transmembrane conductance regulator (CFTR). The F508del mutation of CFTR allele gives raise a misfolded protein causing functional abnormalities in chloride ion secretion in the apical membrane of epithelial cells. The major clinical manifestation of the CF disorder occurs in respiratory tract. Thick and sticky mucus blocks the airways. Consequently, the ability for clearance of microorganisms is impaired. Thus, airways of CF patients are susceptible to chronic microbial infections predominantly with Pseudomonas aeruginosa and Burkholderia cepacia complex which significantly contribute to morbidity and mortality in CF patients. The gene therapy seems to be a promising way for CF patients. Attempts for the gene replacement in clinical applications are focused on mrna approaches as the mrna transfection is effective without need of reaching the nucleus [1]. In addition, using this approach the risk of mutagenesis does not need to be considered. The aim of our study was to synthesize a stable CFTR mrna containing 3'-0-Mem7G(5')ppp(5')G cap (ARCA cap), 25% pseudouridine triphosphate, 25% 5-methylcytidine triphosphate and a 200poly(A) tail. This ARCA cap should allow better in vitro translation of mrna than that with a standard cap analog. In order to study the delivery and expression of an introduced gene, human airway cells with the CF genetic disorder, CuFi, and airway cells derived from a healthy subject, NuLi, were employed. The modified mrna construct containing a marker gene coding for GFP in a combination with Lipofectamine MessengerMAX was initially used for the cell transfection. The production of GFP was detected by immunofluorescence assay and Western blotting. Then, the cells were transfected with CFTR mrna construc and the CFTR expression at the protein level was assayed using immunostaining techniques. The CFTR function, as a chloride channel, was studied using a fluorescent probe MQAE sensitive to halides [2]. Finally, the effect of CFTR mrna transfection on the adhesion of P. aeruginosa (ST 966) on CF cells was assessed in a fluorescent adherence assay [3]. To prove our concept of human NuLi and CuFi cell transfection the mrna construct containing a marker gene coding for GFP was tested. The efficient GFP expression (based on 36

37 GFP fluorescence) was detected 48 hrs after the cell line treatment. Under optimized conditions CuFi cells were transfected with CFTR mrna construct. Immunofluorescence staining revealed that the levels of total CFTR protein are similar to that detected in nontransfected NuLi cells. Next, the iodide efflux mediated by CFTR protein was measured in CuFi cells treated with CFTR mrna. After 24 hrs from the transfection the iodide efflux was clearly stimulated by forskolin and reduced by the specific inhibitor CFTR inh -172 to the level similar in NuLi cells. Finally, the adhesion of P. aeruginosa to CuFi cells after transfection with CFTR mrna was significantly lowered compared to untreated CuFi cells. In conclusion the present data suggest that the transfection of non-polarized CF human epithelial cells with novel CFTR-mRNA increased expression of the CFTR protein up to the level of healthy cells and their functional restoration occurred, too. Moreover, transfected cells are more resistant to adhesion of P. aeruginosa. ACKNOWLEDGEMENT The financial support from the grant UNCE /2012 is highly acknowledged. REFERENCES [1] Bangel-Ruland N, Tomczak K, Fernández Fernández E, Leier G, Leciejewski B, Rudolph C., Rosenecker J, Weber W.M.: J. Gene Med, 15 (2013), [2] Mahlangu D.A., Dix J.A.:Anal. Biochem., 325 (2004), [3] Nosková L., Kubíčková B., Vašková L., Bláhová B., Wimmerová M., Stiborová M., Hodek P.: Sensors (Basel), 15 (2015),

38 ITP ANALYSIS OF SWEET BEVERAGES AND BEERS Sandra BUGDOLOVÁ 1*, Michaela BAHELKOVÁ 1, Přemysl LUBAL 1, Marta FARKOVÁ 1 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic *451511@mail.muni.cz Isotachophoresis (ITP) is a modern analytical method which provides information about qualitative and quantitative composition of the sample [1]. The sample is injected between leading (LE) and terminating (TE) electrolyte which ions have higher (LE) and lower (TE) mobility than all ions present in the sample. When the voltage is applied on inert Pt electrodes placed in solution, the ions are separated into individual zones according to their decreasing electrophoretic mobility[2,3]. A B 1-cyclamate sodium, 2-saccharin 1-sulphate, 2-malate, 3-succinate, 4-citrate, 5-phosphate Figure: ITP records of selected drinks: Sprite light beverage (A), beer Staropramen Smíchov (B) The ITP method was applied for analysis of samples of sweet beverages, alcoholic and nonalcoholic beers when the content of artificial sweeteners (e.g. acesulfame, cyclamate, saccharin) and some anions (e.g. sulphate, malate, succinate, citrate, phosphate) was determined (see Fig. 1). The analyses were carried out on electrophoretic analyzer EA 102 (Villa Labeco, Slovakia) with conductivity detector. ACKNOWLEDGEMENT The work has been supported by Masaryk University (MUNI/A/1359/2018). REFERENCES [1] Křivánková L, Herrmannová M, Bartoš M, Vytřas K.: J. Sep. Sci., 29 (2006), [2] Boček P, et al.: Analytická kapilární izotachoforéza. Academia, Praha [3] Wilson I. D. (Ed.): Encyclopedia of Separation Science ITP chapter. Academic Press, San Diego

39 1 APOFERRITIN NANOCAGE AS A PROMISING DOXORUBICIN NANOCARRIER AND ITS EFFECTS ON NEUROBLASTOMA CELL LINES Tereza ČERNÁ 1,2*, Radek INDRA 1, Katarína VAVROVÁ 1, Jan HRABĚTA 2, Tomáš ECKSCHLAGER 2, Zbyněk HEGER 3, Vojtěch ADAM 3, Marie STIBOROVÁ 1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, , Prague, Czech Republic 2 Department of Pediatric Hematology and Oncology, 2 nd Medical Faculty, Charles University and University Hospital Motol, V Úvalu 84, , Prague, Czech Republic 3 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemědělská 1, Brno, Czech Republic *terezcern@gmail.com Abstract Although doxorubicin (DOX) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier. Apoferritin with encapsulated DOX (APODOX) was prepared and tested. APODOX was compared with free DOX in field of cytotoxicity, apoptosis, histone phosphorylation and localization in neuroblastoma UKF-NB-4 cells. 1. INTRODUCTION Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common solid extracranial tumor in children and a major cause of neoplastic death in infancy [1]. Prognosis of high risk neuroblastoma is poor because of gradually developing tumor cell chemoresistance [2]. Conventional cancer treatment based on cytostatic therapy is highly toxic not only for cancer cells, but also for normal ones [1-3]. Currently nanocarriers are promising agents to improve drug therapeutic index, divert ABC-transporter mediated drug efflux mechanism and selectively target tumor cells [4]. Apoferritin is a protein composed of 24 polypeptide subunits, structurally arranged to create an internal cavity, which is naturally used for storage of iron ions; but artificially it can be employed for carrying of any molecule of interest [4-6]. To enhance the targeting ability of apoferritin to cancer cells, it is possible to modify its surface with antibodies. To evaluate potential application of this technology for cancer therapy, the aim of this study was to compare the cytotoxic effects of anthracycline antibiotic doxorubicin encapsulated into apoferritin (APODOX) [5,6] and free doxorubicin (DOX) on neuroblastoma cells and chemoresistant subline derived from this cell line in vitro. Moreover, the surface of apoferritin was modified with targeting antibodies against CD133 and GD2, antigens expressed on neuroblastoma cells. 2. MATERIALS AND METHODS Apoferritin with encapsulated DOXO (APODOX) was prepared [5,6] and its effect on tumour cells was analysed. The nanocarrier was compared with free doxorubicin in field of cytotoxicity, apoptosis, histone phosphorylation and localization in cells. Using the Western blot analysis, expression of TfR 1 and SCARA5 receptors in tested neuroblastoma cells (UKF-NB-4) was also analysed [5]. 39

40 3. RESULTS AND DISCUSSION Cell viability was assessed using Alamar Blue assay, the real time impedance based platform (xcelligence) and apoptosis rates were measured by flow cytometer using Annexin V and DAPI staining. DNA double-strand breaks (phosphorylation of H2A.X) and caspase-3 activation were detected by flow cytometry. Subcellular location of cytostatic loaded apoferritin and free cytostatic was analyzed by confocal microscopy. Here, we have shown that apoferritin can carry and deliver a high dose of doxorubicin into UKF-NB-4 neuroblastoma cancer cells. The effect of APODOX on sensitive and resistant neuroblastoma cells is similar to that of free DOX, but percentage of sensitive neuroblastoma cells with DNA double-strand breaks after APODOX treatment was higher than after free cytostatic. In addition, apoferritin loaded DOX are cytotoxic in hypoxic conditions (1% O 2 ). Further, using fluorescence microscopy, we have shown that apoferritin can deliver drugs inside cancer cells and the drug exerts their effect thereof. Entry of APODOX and free DOX into sensitive and resistant cells was similar. We suppose that apoferritin is targeted to the several cancer cells (i.e. neuroblastoma) through TfR 1 and/or SCARA5 which are overexpressed in a neuroblastoma cell line. To enhance the specificity of APODOX, we tested modification of its surface with different antibodies (anti-cd133 and anti GD2) targeted to neuroblastoma cells. The specificity of targeted nanocarrier is dependent on a type and concentrations of antibodies. The results found in this study seem to be promising, because encapsulation does not affect toxicity of cytostatic and improves drug stability. Moreover, entry of APODOX is significantly lower into non-malignant cells than into cancer cells. 4. ACKNOWLEDGEMENT This work was supported by GACR ( S) and Charles University (GAUK ) 5. REFERENCES [1] Brodeur G.M.: Nat Rev Cancer, 3 (2003), [2] Maris J.M., et al.: Lancet, 369 (2007), [3] Cerna T., et al.: Int J Mol Sci. 19 (2018), article E164 [4] Dostalova S., et al.: ACS Appl. Mater. Interfaces 8 (2017), [5] Indra R., et al., Toxicology 419 (2019), [6] Dostalova S., et al..: Scientific Reports, 8 (2018),

41 ANALYSIS OF METABOLISM AND DNA ADDUCT FORMATION BY ARISTOLOCHIC ACIDS I AND II IN RATS IN VIVO Alena DEDIKOVA 1, Frantisek BARTA 1, Petr HODEK 1, Jaroslav MRÁZ 2, Šárka DUŠKOVÁ 2, Eva FREI 1, Heinz H. SCHMEISER 3, Volker M. ARLT 4, Marie STIBOROVA 1* Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2 Centre of Occupational Health, The National Institute of Public Health, Srobarova 48, , Prague 10 Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, Germany Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK *stiborov@natur.cuni.cz Abstract A plant extract aristolochic acid (AA) is a mixture of AAI and AAII that causes Aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial malignancies. The impact of exposure of rats to AAI or AAII alone and their combination on metabolism of AA and their genotoxicity was investigated. The higher formation of the nitroreduction metabolites of AAII than AAI, aristolactam II (AlacII) than aristolactam I (AlacI), respectively, was found in rats exposed to AAs. Likewise, the higher levels of AAII- than AAI-derived DNA adducts were produced. The results demonstrate that induction of NAD(P)H:quinone oxidoreductase (NQO1) reducing AAs to species forming AA-DNA adducts, which prevails the induction of cytochromes P450 (CYP) oxidatively detoxifying AA, potentiates AA-derived genotoxicity. 1. INTRODUCTION Exposure to AA, a natural mixture of plant alkaloids aristolochic acid I (AAI) and II (AAII), causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), which are both diseases associated with a high risk of urothelial malignancy (UUC) [1]. Nitroreduction of AAs is required to exert their carcinogenic properties (i.e. UUC development) [1,2]. One of the most efficient enzymes reductively activating AA to species forming AA- DNA adducts [predominantly 7-(deoxyadenosin-N 6 -yl)-aristolactam I (da-aai) which causes specific AT TA transversions in the TP53 tumor suppressor gene in tumors from AAN and BEN patients] is NAD(P)H:quinone oxidoreductase 1 (NQO1) [1,2]. AAI is reductively activated, too, but also oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by cytochromes P450 (CYP) 1A1 and 1A2 [2,3]. Besides CYP1A/2, rat CYPs of the 2C subfamily also oxidize AAI [2,3]. A balance between activation and detoxification reactions of individual AA components, AAI and AAII, can influence the AA-induced AAN or BEN/UUC development. Therefore, the aim of this study was to investigate the impact of exposure of rats to AAI or AAII alone and to combination of both compounds on expression and activities of enzymes dictating AA metabolism, on formation of individual metabolites of AAI and AAII and on AA-derived DNA adducts in rats in vivo. 41

42 2. MATERIAL AND METHODS Rats were treated with AAI or AAII or AAI together with AAII (a ratio of 1:1). Expression of enzymes was determined by Western blotting and their activities with marker substrates [2,3]. AA metabolites were determined in urine of rats by HPLC and mass spectrometry. DNA adducts were analyzed by 32 P-postlabeling [2,3]. 3. RESULTS AND DISCUSSION Strong induction of NOQ1 activity in liver, up to more than 10-fold, was caused by exposure of rats to AAs, mainly by exposure to the combination of both compounds. However, the combined treatment leads to higher NQO1 activity only in the liver, but not in kidney. Compared to control (untreated) rats, oxidation of Sudan I, 7-ethoxyresorufin-O-deethylase and 7-methoxyresorfin-O-demethylase, marker activities of CYP1A1, 1A1/2 and 1A2, respectively, were increased in livers of exposed animals, while they were almost not influenced in kidney. The diclofenac 4'-hydroxylation reaction, a marker for CYP2C6, was also elevated by treatment of rats with AAs, while 16 -hydroxylation of testosterone, a marker for CYP2C11, decreased by this treatment. Elevated activities of CYP1A1/2 and 2C6 correlated with an increase in oxidation of AAI to its detoxification metabolite, AAIa. This O- demethylated metabolite of AAI, AAIa, was found to be the major metabolite of AAI found in rat urine in vivo, while aristolactam II (AlacII) is the major metabolite of AAII. AlacIa is formed both from AAI and AAII, being the final metabolite of both compounds. Up to three AA-derived DNA adducts were formed in liver, kidney and lung of rats; da-aai, 7- (deoxyguanosin-n 2 -yl)-aristolactam I (dg-aai), 7-(deoxyadenosin-N 6 -yl)-aristolactam II (da-aaii) and 7-(deoxyguanosin-N 2 -yl)-aristolactam II (dg-aaii). The highest levels of AA-DNA adducts were formed in rat kidney, followed by those in liver and lung. Compared to rats treated with AAI or AAII alone, the sum of total levels of AA-DNA adducts were higher in all tested organs of rats exposed to a mixture of AAs. The results demonstrate the additive effects of exposure of rats to AAI combined with AAII on AA-genotoxicity. 4. ACKNOWLEDGEMENT The work was supported by GACR ( S). 5. REFERENCES [1] Gökmen M.R., et al.: Annals of Internal Medicine, 158 (2013), [2] Stiborova M., et al.: Archives of Toxicology, 90 (2016), [3] Stiborova M., et al.: International Journal of Molecular Sciences 8 (2017), pii: E

43 1 DIRECT ELECTRODEPOSITION OF SILVER AMALGAM PARTICLES ON SCREEN PRINTED SILVER ELECTRODES USING DOUBLE PULSE CHRONOAMPEROMETRY Pavlína HAVRANOVÁ 1*, Lukáš FOJT 1, Aleš DAŇHEL 1 Institute of Biophysics of the Czech Academy of Sciences, v.v.i., Královopolská 135, Brno, Czech Republic *vidlakova@ibp.cz Searching for alternatives to mercury electrodes exhibiting an analogous electrochemical behavior represents an important contribution to the research in electroanalytical chemistry. Different solid composite electrodes can serve as substitutes for liquid mercury. Silver amalgam was found to be one of the most suitable alternative electrode material with its electrochemical behavior very close to mercury electrodes [1,2]. The silver solid amalgam electrodes (AgSAE) are usually prepared by mixing silver powder with liquid mercury [2]. According to its composition based on ratio of silver and mercury, resulting material can be liquid (<10% Ag), paste (10 15% Ag), solid (15 80% Ag), or overamalgamed metal (>80% Ag), what increase its application variability. However, silver amalgam can be prepared by its direct electrodeposition on various conductive materials from the solution of both Ag + and Hg 2+ ions [3]. This work is focused on the preparation of silver amalgam particles (AgAP) by its direct electrodeposition on disposable screen printed silver electrodes (SPAgE) using double pulse chronoamperometry from the solution containing soluble Ag + and Hg 2+ salts. Different parameters of the chronoamperometry (pulse and grow potentials and times) and various Ag/Hg ratios in the solution were optimized during electrodepositions of silver amalgam particles. This work confirmed the SPAgE as suitable platform for direct electrodeposition of silver amalgam particles from small volume of the solution decreasing its consumption. Simultaneous electro-reduction of silver and mercury ions could be applied for preparation of nanostructured and sufficiently stable silver amalgam particles with controlled distribution and surface coverage. Its electroanalytical application was proved by detection of model organic nitrocompound, 4-nitrophenol. ACKNOWLEDGEMENT This work was supported by The Czech Science Foundation (grant Y) and a part of the work was carried out with the support of CEITEC Nano Research Infrastructure (MEYS CR, ). REFERENCES [1] Yosypchuk B, Novotny L.: Crit. Rev. Anal. Chem., 32 (2002), 2, [2] Yosypchuk B., Barek J.: Crit. Rev. Anal. Chem.,39 (2009), 3, [3] Danhel A., Ligmajer F., at al.: J. Electroanal. Chem., 821 (2018),

44 METABOLISM OF TYROSINE KINASE INHIBITOR CABOZANTINIB BY LIVER MICROSOMES Radek INDRA 1*, Tomas JURECKA 1, Katarina VAVROVA 1, Petr POMPACH 1, Zbynek HEGER 2,3, Vojtech ADAM 2,3, Marie STIBOROVA 1 1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 6, Prague 2, Czech Republic 2 Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic 3 Central European Institute of Technology, Brno University of Technology, Purkynova 123, Brno, Czech Republic * indra@natur.cuni.cz Abstract The metabolism of cabozantinib was studied by microsomes isolated from human livers and livers of several animal models (rat, rabbit and mice). The metabolites were separated and identified by LC/MS. Microsomes of all tested animal models generate six metabolites. On the contrary only three metabolites were generated by human microsomes. The predominant metabolite with all tested liver microsomes is cabozantinib N-oxide. 1. INTRODUCTION Cabozantinib is oral drug that was approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treatment of medullary thyroid cancer in 2012 and 2014, respectively. In 2016, both agencies approved the drug also for treatment of kidney cancer [1, 2]. It is an inhibitor of tyrosine kinases affecting vascular endothelial growth factor receptor-2 (VEGFR-2), hepatocyte growth factor receptor (HGFR) and rearranged during transfection (RET) [3]. Although cabozantinib is used for treatment, only limited information is known about its metabolism and efficiency of its potential metabolites. 17 individual metabolites were identified after single oral dose of cabozantinib to healthy volunteers in plasma, urine and faeces, but individual enzymes responsible for their formation are still mystery [4]. 2. MATERIALS AND METHODS Cabozantinib (50 μm) were incubated with liver microsomes from human livers and livers of several animal models (rat, rabbit and mice at 37 C in open tubes. The microsomal protein concentration was 0.5 mg/ml. After 20 minutes of incubation the reaction was stopped by ethyl acetate and extraction with this solvent was done. Organic phases of individual samples were evaporated, dissolved in methanol (50 μl) and analysed on HPLC. Individual metabolites were separated using C18 column by acetonitrile/acetate buffer mobile phase and identified by mass spectrometry. 3. RESULTS AND DISCUSSION Because only limited information is known about efficiencies of cabozantinib metabolites and available data indicate their lower efficiency, the knowledge of cabozantinib metabolic 44

45 pathway is crucial for improvement of treatment and prognoses. Microsomes isolated from mice, rabbits and rats generate six metabolites. The predominant metabolite was identified as cabozantinib N-oxide. Other metabolites such as desmethyl cabozantinib, monohydroxycabozantinib and cabozantinib amide cleavage product were also identified to be formed. Pre-treatment of rats with inducers of cytochromes P450 (CYP) enzymes influences the amount of metabolites formed. Inducers of CYPs of a family 1 and CYP2E1 decrease the amount of cabozantinib metabolites, in contrast to an inducer of CYP2B that slightly increases the amount of formed metabolites. Pregnenolone carbonitrile (PCN, an inducer of CYP3A) causes a significant increase in cabozantinib metabolism. These results demonstrate the importance of CYP3A in cabozantinib metabolism. In contrast to animal samples, human liver microsomes were less efficient in cabozantinib metabolism. Human microsomes generate only three metabolites. Cabozantinib N-oxide was the predominant metabolite and the two other metabolites were identified as monohydroxy cabozantinibs. On the base of activity of individual cytochrome P450 enzymes in human liver microsomes from single donors, the main isoform responsible for cabozantinib transformation seems to be CYP3A4. Nevertheless, the identification of individual enzymes and the mechanisms of their action in the cabozantinib biotransformation need further investigation. Therefore, they are the subjects of additional studies of our laboratory 4. ACKNOWLEDGEMENT This work was supported by GACR ( S). 5. REFERENCES [1] Food and Drug Administration (FDA) [2] European Medicines Agency (EMA) [3] Song E-K., et al.: International journal of cancer,136 (2015), 8, [4] Lacy S., e al.: Drug Metabolism and Disposition, 43 (2015), 8,

46 1 2 COMPARISON OF EFFICIENCIES OF PEROXIDASES TO OXIDIZE THE ANTICANCER DRUG ELLIPTICINE AND THEIR INFLUENCING BY VANDETANIB, LENVATINIB AND CABOZANTINIB Matúš KOLÁRIK 1, Radek INDRA 1, Vojtěch ADAM 2, Zbyněk HEGER 2, Marie STIBOROVÁ 1* Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic *stiborov@natur.cuni.cz Abstract The plant alkaloid ellipticine and inhibitors of tyrosine kinases (TKIs) vandetanib, lenvatinib and cabozantinib are efficient drugs suitable for treatment of several cancers, including tumors of thyroid glands. Here, ellipticine oxidation by several peroxidases was investigated in detail and the effects of the tested TKIs on this oxidation were evaluated. Horseradish peroxidase, followed by lactoperoxidase, myeloperoxidase and cyclooxygenase I are capable of oxidizing ellipticine. Moreover, oxidation of ellipticine by thyreoperoxidase, the enzyme playing role in thyroid glands, was found for the first time. The TKIs had essentially no effect on ellipticine oxidation; only cabozantinib stimulates the reaction catalyzed by horseradish peroxidase, while the ellipticine oxidation by lactoperoxidase is inhibited by lenvatinib. 1. INTRODUCTION The drugs utilized for cancer chemotherapy have usually a narrow therapeutic index, and often the produced responses are only palliative as well as unpredictable [2]. The targeted therapy is directed against cancer-specific targets and signaling pathways and thus provides more limited nonspecific mechanisms [3]. The most promising drugs to target the cancer cells are inhibitors of receptor tyrosine kinases (TKIs) [4] and DNA-damaging drugs targeted to cancer cells due to their metabolism [5-7]. Ellipticine and its derivatives are the DNA-targeted anticancer agents effective against certain tumors of the thyroid gland (i.e. anaplastic thyroid carcinoma), ovarian carcinoma, breast cancer and osteolytic breast cancer metastasis [5-7]. TKIs vandetanib, lenvatinib and cabozantinib are inhibitors targeting VEGFR subtypes 1 and 2, EGFR and the RET-tyrosine kinase, thus considered as multiple TKIs. These TKIs have already been approved for treating patients suffering from thyroid cancer and renal cell carcinoma [8-10]. In cancer chemotherapy, serious clinical consequences may occur from small alterations in drug metabolism affecting drug pharmacokinetics. Ellipticine anticancer efficiencies are dependent on its metabolism leading both to the activation metabolites causing DNA damage (covalent DNA adducts) and their detoxification to products that are excreted. Ellipticine is oxidized by cytochromes P450 (CYP) and peroxidases. The CYP enzymes generate up to five metabolites, 9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7- hydroxyellipticine and N 2 -oxide of ellipticine. 12-Hydroxy-, 13-hydroxyellipticine and 46

47 ellipticine N 2 -oxide are metabolites that form reactive species binding to DNA. Peroxidases mediate formation of ellipticine dimer that is considered as a detoxification metabolite and ellipticine N 2 -oxide activating this drug to species generating ellipticine-dna adducts [5-7]. In contrast to ellipticine, the knowledge on metabolism of TKIs is scarce. 2. MATERIAL AND METHODS HPLC was utilized for separation of ellipticine metabolites formed by peroxidases. 3. RESULTS AND DISCUSSION Oxidation of ellipticine by horseradish peroxidase, lactoperoxidase, myeloperoxidase and cyclooxygenase I leads to formation of dimer and ellipticine N 2 -oxide [5-7]. However, the efficacy of thyreoperoxidase, the enzyme playing a role in proper function of thyroid glands, to catalyze the reactions has not been investigated, yet. Therefore, the aim of this study was to investigate this feature. Here, we found for the first time that thyreoperoxidase oxidizes ellipticine. Its efficiency to oxidize ellipticine is comparable to that of lactoperoxidase, myeloperoxidase, and cyclooxygenase I in the presence of hematin. A number of studies testing the effectiveness of individual anticancer drugs alone or in combination with other cytostatics demonstrated that such combination can have additive and/or even synergistic effects on treatment regimen. The tested TKIs vandetanib, lenvatinib and cabozantinib had low, if any, effects on oxidation of ellipticine by peroxidases; only cabozantinib slightly stimulates oxidation of ellipticine by horseradish peroxidase, while lenvatinib inhibits the reaction catalyzed by lactoperoxidase. The study might provide a rationale for the clinical evaluation of the combination of DNA-damaging anticancer drugs and TKIs. 4. ACKNOWLEDGEMENT The work was supported by GACR (grant S). 5. REFERENCES [1] Heger Z., et al.: International Journal of Molelcular Sciences, 14 (2013), [2] Arora A., et al.: Journal of Pharmacology and Experimental Therapeutics, 315 (2005), [3] Reibenwein J., et al.: Expert Opinon on Therapeutic Targets, 12 (2008), [4] Hartmann, J.T., et al., Current Drug Metabolism, 10 (2009), [5] Stiborova M., et al.: International Journal of Cancer, 120 (2007), [6] Stiborová M., et al.: Biochimica et Biophysica Acta, 1814 (2011), [7] Stiborova M. and Frei E.: Current Medicinal Chemistry, 21 (2014), [8] Greenhill C.: Nature Reviews Endocrinology, 13 (2017), 688 [9] Roviello G., et al.: Expert Opinion on Investigational Drugs, 27 (2018), [10] Abdelaziz A., Vaishampayan U.: Expert Review in Anticancer Therapy, 17 (2017),

48 DEVELOPMENT OF INSTRUMENTATION FOR COULOMETRIC TITRATIONS Matěj KUČERA 1*, Jiří VOLÁNEK 1, Pavel KRÁSENSKÝ 1, Přemysl LUBAL 1, Zdeněk FARKA 2, Marta FARKOVÁ 1 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic 2 CEITEC MU, Masaryk University, Kamenice 5, Brno, Czech Republic *437254@mail.muni.cz This contribution is focused on the development and testing of coulometric analyzer, which was employed for in situ generation of analytical agent. The goal of the project was construction of a new coulometer, which ensures the exact and precise automatic coulometric titration. Coulometry is one of the basic electroanalytical methods. Two main approaches are direct and indirect coulometry, based on monitoring of the current required to convert the substance of interest into a precisely defined product (direct), and/or the amount of current required to produce an equivalent amount of reagent which takes place in the reaction with selected analyte (indirect). The main advantage of indirect coulometry is the possibility of in situ generation of reactive unstable ions that may serve as titration agents [1,2]. The newly constructed device is based on an integrated circuit with a connected screen and electrode connection inputs. The measurement set-up consists of a beaker in which the coulometric titration takes place, and a pair of generator Pt electrodes connected to a controllable DC source for coulometric analyzer. Reference and Pt indicator electrodes are utilized for potentiometric detection of equivalence point. The titrations in this work are based on oxidization of analyte by iodine, which was coulometrically generated from alkaline potassium iodide. Other additives in solution were oxalic acid, which ensures the stability of analyzed ascorbic acid, and starch, which serves as an indicator forming blue-colored inclusive compounds with iodine. Firstly, the sample solution of ascorbic acid was titrated and its oxidation over time was recorded. Oxidation of ascorbic acid occurs due to oxygen in the air, even in solution, where metal ions have the same effect. All titrations of ascorbic acid for different concentrations were evaluated by observing the blue color of starch-iodine inclusion complex. However, the color of titration solution is very weak at the equivalence point, and thus the end of titration is subjective [3]. Secondly, the purity of sodium thiosulphate was checked using two ways of equivalence point estimation (i) again using visual detection with starch, and (ii) by potentiometric indication using the second-derivative curve [4] (Figure), which allowed to achieve significantly improved results under the optimized experimental conditions. 48

49 de2 (V) E (V) 0.3 A Q (mc) 0.8 B Q (mc) Figure: The potentiometric titration of thiosulphate by iodine generated by coulometer. (A) Normal record of titration curve; (B) the second derivation of the titration curve. ACKNOWLEDGEMENT This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic CEITEC 2020 (LQ1601) and by Masaryk University (MUNI/A/1359/2018). REFERENCES [1] Padilla Mercado J. B., Coombs E. M., De Jesus J. P., Bretz S. L., Danielson N. D.: J. Chem. Educ., 95 (2018), 5, 777. [2] Beilby A. L., Landowski C. A.: J. Chem. Educ., 47 (1970), 3, 238. [3] Bertotti M., Vaz J. M., Telles R.: J. Chem. Educ., 72 (1995), 5, 445. [4] Tanaka T., Hayashi H., Komiya Y., Nabekawa H., Hayashi H. Bunseki Kagaku 56 (2007), 5,

50 1 ON ELECTROCHEMISTRY OF 1-PENTYL-3-(1-NAPHTOYL)INDOLE AND 1-PENTYL-3-(2-METHOXYPHENYLACETYL)INDOLE Michaela OBLUKOVÁ 1,2, Romana SOKOLOVÁ 1, Radomír ČABALA 2, Ilaria DEGANO 3 J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 3, Prague 8, Czech Republic, michaela.oblukova@jh-inst.cas.cz 2 Charles University, 1st Faculty of Medicine, Kateřinská 1660/32, Prague 2 3 Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, Pisa, Italy The electrochemistry properties of 1-pentyl-3-(1-naphtoyl)indole (hereinafter referred to as JWH-018 ) and 1-pentyl-3-(2-methoxyphenylacetyl)indole (hereinafter referred to as JWH- 250 ) were studied. These compounds belong to heterogenous group of synthetic cannabinoids, which have been appeared in the drug market recently. These substances are abused for their psychoactive effect and serve as alternative to classical drugs (heroin, cocaine, ecstasy) due to their legal status [5]. In human organism, JWH-018 and JWH-250 are rapidly and completely metabolized and thus parent compounds are often undetectable. To detection of these compounds is necessary to know their metabolism pathways [6, 7]. In view of the fact that the electron transfer reactions play an important role in metabolic pathways, this study is focused on electrochemical investigation of oxidation and reduction of JWH-018 and JWH-250. This report is based on cyclic voltammetry, UV/Vis and IR spectroelectrochemistry in nonaqueous media combined with HPLC-ESI-MS/MS detection of reactions products. JWH-018 JWH-250 Figure: Chemical structure of JWH-018 and JWH-250 ACKNOWLEDGEMENT The work has been supported by the Czech Science Foundation (GAČR S). REFERENCES [1] Meyer MR.: Archives of Toxicology, 90 (2016), 10, [2] Wintermeyer A et al.: Analytical and Bioanalytical Chemistry, 398 (2010), 5, [3] Grigoryev A et al.: Journal of Chromatography B., 879 (2011), 25,

51 1 2 OXIDATION OF A TYROSINE KINASE INHIBITOR VANDETANIB BY RAT ENZYMATIC SYSTEMS IN VITRO Marie STIBOROVÁ 1*, Radek INDRA 1, Petr POMPACH 1, Zbyněk HEGER 2, Vojtěch ADAM 2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic *stiborov@natur.cuni.cz Abstract Oxidation of vandetanib, an inhibitor of tyrosine kinases, by rat enzymatic systems in vitro was investigated. The metabolites of vandetanib were identified to be N-desmethylvandetanib and vandetanib N-oxide. Rat CYP2C11 is the most efficient enzyme oxidizing vandetanib to N-desmethylvandetanib, whereas rat FMO1 and 3 are responsible for generation of vandetanib N-oxide in rat liver microsomes. 1. INTRODUCTION The drugs utilized for cancer chemotherapy have usually a narrow therapeutic index, and often the produced responses are only palliative as well as unpredictable [2]. One of the most promising drugs are inhibitors of receptor tyrosine kinases (TKIs) [4]. Vandetanib is a TKI indicated for the treatment of symptomatic or progressive medullary thyroid cancer in patients with unresectable locally advanced or metastatic disease. In cancer chemotherapy, serious clinical consequences may occur from small alterations in drug metabolism affecting drug pharmacokinetics [8]. Although metabolism of vandetanib has been partially studied, its oxidation by enzymatic systems in detail is still missing [5]. Therefore, the target of this study was to investigate the metabolism of vandetanib; enzymatic systems of rats, which were considered to mimic the fate of vandetanib in humans [5], were used as a model for such a study. 2. MATERIAL AND METHODS HPLC was utilized for separation of vandetanib metabolites and mass spectroscopy for their structural characterization. Expression of rat hepatic cytochromes P450 (CYP) and flavincontaining monooxygenases (FMO) was analyzed by Western blotting. 3. RESULTS AND DISCUSSION Rat liver microsomes oxidized vandetanib to N-desmethylvandetanib and vandetanib N-oxide. Their formation was dependent on NADPH, which serves as cofactor for both POR-mediated CYP catalysis and FMO-mediated oxidative reactions in liver microsomes. These results indicate that vandetanib oxidation in hepatic microsomes is mediated by CYPs and/or FMOs. In rat liver, CYPs of the 2C subfamily were identified to be mainly expressed [6] and confirmed by Western blotting in our study. Further, FMO1 and 3 were found by Western blotting to be expressed in rat liver microsomes. 51

52 In order to identify individual CYPs and/or FMOs oxidizing vandetanib and estimate their contribution to the oxidation process, three approaches were utilized: (i) use specific inducers of individual CYPs in a rat model; (ii) use selective CYP and FMO inhibitors in rat microsomes; and (iii) use recombinant rat CYPs. Under the experimental conditions used, rat microsomes oxidized vandetanib to N- desmethylvandetanib most efficiently when those were isolated from livers of rats pretreated with PCN (rich in CYP3A), followed by those isolated from livers of PB-pretreated rats (rich in CYP2B and 2C) and those isolated from livers of control (uninduced) rats in which CYP2C enzymes are highly expressed. Microsomes from BaP-pretreated rats and ethanol-pretreated rats were also capable of oxidizing vandetanib, but to lesser extent. These findings suggest that CYPs of the 3A subfamily, followed by those of the 2B/2C subfamilies, might play a role in vandetanib oxidation to N-desmethylvandetanib. On the contrary, vandetanib N-oxide was mainly formed by microsomes of control (uninduced) rats. The generation of N- desmethylvandetanib was attenuated by inhibitors of CYP3A and 2C subfamilies in rat microsomes, while the FMO inhibitor methimazol decreased the formation of vandetanib N- oxide in this rat subcellular system (microsomes). These results indicate that CYP3A and/or 2C are mainly responsible for the formation of N-desmethylvandetanib and FMO1 and 3 mainly for the generation of vandetanib N-oxide. Indeed, rat recombinant CYP2C11>>3A1>3A2 were most efficient to oxidize vandetanib. Based on the results showing the velocities of vandetanib oxidation to N-desmethylvandetanib in experimental systems containing recombinant CYP enzymes and the relative amounts of CYP enzymes expressed in rat livers, the contributions of individual CYPs to this reaction in rat livers were evaluated. The highest contribution to vandetanib oxidation to N-desmethylvandetanib in rat livers was attributed to CYP2C11 (~79%), followed by CYP3A (~20.3%). 4. ACKNOWLEDGEMENT The work was supported by GACR (grant S). 5. REFERENCES [1] Heger Z., et al.: International Journal of Molecular Sciences, 14 (2013), [2] Reibenwein J., et al.: Expert Opinon on Therapeutic Targets, 12 (2008), [3] Hartmann, J.T., et al., Current Drug Metabolism, 10 (2009), [4] Fujita K.: Current Drug Metabolism, 7 (2006), [5] Martin P., et al., Clinical Therapeutics, 34 (2012), [6] Nedelcheva V. and Gut I.: Xenobiotica, 2 (1994),

53 1 APPLICATION OF LOW-FIELD 1 H NMR SPECTROSCOPY IN ANALYTICAL CHEMISTRY Libuše SYCHROVÁ 1, Přemysl LUBAL 1*, Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic *lubal@chemi.muni.cz NMR spectroscopy is mostly applied for structural analysis of organic compounds, but less for quantitative analysis, however, it is absolute analytical method [1]. A benchtop NMR spectrometer Spinsolve Ultra 60 (Magritek, Germany) is low-field equipment suitable for fast measurements of 1 H NMR spectra of aqueous solutions of compounds because it efficiently enables to suppress analytical signal of water [2]. This work is focused on testing of this instrumentation for both qualitative and quantitative analysis of selected analytes. The quantitative analysis of primary alcohols (methanol, ethanol - see Figure) and polyalcohols (propylene-glycol, 1,3-propanediol, glycerol, ethylene- glycol) in binary and ternary mixtures with water was carried out The analytical procedure was verified for determination of alcohols in wine, plum brandy and cooling liquid used in automobiles. CH 3 OH CH 2 CH 3 OH OH Figure: NMR spectrum of selected analytes: ethanol (upper), methanol (middle), water (lower) 53

54 ACKNOWLEDGEMENT The work has been supported by Masaryk University (MUNI/A/1359/2018). REFERENCES [1] ROUESSAC, Francis a Annick ROUESSAC. Chemical analysis: modern instrumentation methods and techniques. Chichester: John Wiley [2] Downloaded April 28 th,

55 1 2 3 STUDY ON ENCAPSULATION OF LENVATINIB AND ELLIPTICINE INTO NANOTRANSPORTERS; EXPERIMENTAL AND THEORETICAL APPROACHES Paulína TAKÁCSOVÁ 1, Radek INDRA 1, Zbyněk HEGER 2, Vojtěch ADAM 2, Ivan BARVÍK 3, Marie STIBOROVÁ 1* Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, Prague 2, Czech Republic *stiborov@natur.cuni.cz Abstract Although lenvatinib, a tyrosine kinase inhibitor, is an efficient anticancer drug, it also produces several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier. The procedure for construction of an apoferritin nanoparticle with encapsulated lenvatinib, similar to that for construction of apoferritin-ellipticine particles, did not lead to encapsulation of lenvatinib into apoferritin. Theoretical approaches investigating the encapsulation processes for lenvatinib and ellipticine into apoferritin nanoparticles explained this phenomenon. 1. INTRODUCTION The receptor tyrosine kinases (TKs) are the enzymes that selectively phosphorylate the hydroxyl moieties of tyrosine residues on signal transduction molecules with a phosphate moiety from adenosine triphosphate [4]. Inhibitors of these enzymes are one of the most promising anticancer drugs used for treatment of several cancers in a last decade [4]. Lenvatinib, belonging to this group of inhibitors, is used for treatment of certain tumours of thyroid glands [3] and metastatic renal cell carcinoma [4]. Lenvatinib is an oral multitargeted TK inhibitor signaling networks implicated in tumor angiogenesis. Ellipticine is another anticancer drug that exhibits high efficiencies in antineoplastic action [5,6]. DNA damage is responsible for ellipticine s biological effects. There are, however, several phenomena that can cause a limited usage of lenvatinib and ellipticine and/or their limited anticancer efficiencies [3,6]. Thus, we are aimed to develop efficient and reliable methods for targeted delivery of these anticancer drugs and to prepare the drugs in forms that exhibit lower side effects and lead to an increase in their anticancer effects. One of the aims is to develop nanocarriers containing this drug. Apoferritins, which are responsible for the storage and transfer of iron [9] can provide the much needed properties of nanocarriers. Indeed, a nanotransporter with encapsulated ellipticine was prepared and found to be capable of inhibiting the growth of cancer cells, while is less efficient to healthy cells [9]. The aims of this study were to prepare nanocarriers based on apoferritin as well as on lipids bearing lenvatinib. Besides the experimental procedures, theoretical approaches investigating the 55

56 encapsulation processes for ellipticine and lenvatinib into apoferritin nanoparticles were utilized. 2. MATERIAL AND METHODS The procedure for preparation of apoferritin nanoparticles with encapsulated lenvatinib were essentially the same as for preparation of nanotransporters bearing ellipticine [9]. The solvents used to dissolve of both drugs were only the difference. Lenvatinib dissolved in ethylene glycol, ethanol and DMA with ratio of 1:2 with water or ethanol were used. 3. RESULTS AND DISCUSSION The theoretical model of ellipticine and lenvatinib interactions with an apoferritin cavity, as well as the model of their encapsulation obtained by computer modeling indicated that in contrast to ellipticine, lenvatinib seems not to be suitable for preparation of apoferritin nanoparticles. The differences in values of pka of ellipticine (pka = 7.05) and lenvatinib (pka = 5.4) and the experimental conditions used for preparation of nanoparticles of these drugs were suitable only for preparation of ellipticine-loaded apoferritin nanoparticles. Ellipticine exists during the procedure of apoferritin nanoparticles predominantly in a protonated state. Therefore, it easily interacts with the core of apoferritin rich in acidic amino acid residues, being properly distributed in the nanoparticle. On the contrary, since lenvatinib occurs in its neutral form during preparation of nanoparticles, it does not properly interact with a cavity of apoferritin nanoparticle. The neutral molecules of lenvatinib are precipitated in the particle. The unsuccessful experimental preparation of lenvatinib-loaded apoferritin nanoparticles confirmed that lenvatinib is not suitable for its preparation. Since the experimental preparation of apoferritin nanoparticles was not successful, we used liposomes as lenvatinib nanocarriers in further experiments. Even though the construction of liposome nanoparticle with encapsulated lenvatinib was successful, the amount of prepared nanoparticles was very low and, therefore, not relevant for cancer therapy. The results of this study indicate that the theoretical model elaborated to follow the encapsulation procedure can serve for screening of potentially suitable drugs before the experimental apoferritin nanoparticle preparation. 4. ACKNOWLEDGEMENT The work was supported by GACR (grant S). 5. REFERENCES [1] Reibenwein J., et al.: Expert Opinon on Therapeutic Targets, 12 (2008), [2] Hartmann, J.T., et al., Current Drug Metabolism, 10 (2009), [3] Greenhill C.: Nature Reviews Endocrinology, 13, (2017), 688 [4] Roviello G., et al.: Expert Opinion on Investigational Drugs 27 (2018), [5] Stiborová M., et al.: Biochimica et Biophysica Acta, 1814 (2011), [6] Stiborová M. and Frei E.: Current Medicinal Chemistry, 21 (2014), [7] Dostalova S., Cerna T., Hynek D., et al.: ACS Applied Materials and Interfaces, 8 (2016), [8] Liang MM, Fan KL, Zhou M, et al.: Proceedings of the National Academy of Sciences of the United States of America, 111 (2014), [9] Indra R., et al., Toxicology 419 (2019),

57 1 METALLOTHIONEIN AND SELENITE IN BRDIČKA REACTION Libuše TRNKOVÁ 1*, Jan SLAVÍK 2, Jaromír HUBÁLEK 2,3 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic 2 Central European Institute of Technology, Brno University of Technology, Purkyňova 123, Brno, Czech Republic 3 Faculty of Electrical Engineering and Communication, Brno University of Technology, Technická 10, Brno, Czech Republic *libuse@chemi.muni.cz Metallothionein II (MT II) is a cysteine-rich, low-molecular weight protein with a high affinity to heavy metals; it is a single-polypeptide chain with the molecular weight 6-7 kda, consisting of amino acid residues structured into two domains; α-domain with four and β-domain with three binding sites for bivalent ions (Figure 1a). The higher MT concentration in tissues reflects a higher amount of toxic heavy metals, oxidative stress, and DNA damage. The MT II has recently been considered as one of the potential cancer markers (Figure 1b). Figure 1: a) Metallothionein and b) its functions in tissues of mammals and non-mammals Selenium (Se) pertains to the essential part of the human diet and a low Se level in the diet is generally associated with various diseases (e.g., Kashin Beck disease). Selenium is incorporated into selenoproteins through selenocysteine having the active selenol group ( SeH). Selenium in the form of amino acids is suitable for any food supplementation and it can be easily absorbed compared to inorganic Se, as is sodium selenite (a potential chemotherapeutic agent). The aim of this research was the study of interaction of MT II and selenite by means of the catalytic reaction of hydrogen evolution at a mercury electrode, known under the name of the Brdička reaction. The Brdička reaction has been widely used for protein analysis, even though its mechanism was not completely elucidated. In addition, the mechanism of Brdička reaction may vary from the case to the case. But what matters is that this reaction is usually performed in a solution of ammonia buffer and Co(NH 3 ) 6 Cl 3 in differential pulse voltammetric mode at mercury or amalgam electrodes. We investigated the electrode processes of MT II (rabbit liver metallothionein) at a mercury electrode in the presence of sodium selenite (Na 2 SeO 3 ) by using differential pulse voltammetry. The interaction between MT II and Na 2 SeO 3 was analyzed via the hydrogen evolution catalytic peaks Cat2. 57

58 It was found that with the increasing selenite concentration, cobalt in MT is replaced by selenium. When concentration of Na 2 SeO 3 increases above the MT binding capacity, only selenite ions are responsible for Cat2 signals. Figure 2: The schematic dependence of the Cat2 peak height on the Na 2 SeO 3 concentration of with respect to all participating components. At lower selenite concentrations both components (Co-MT and Se-MT) contribute to the processes resulting in the peak Cat2. The concentration of selenite (25 µg/cm 3 ) indicates the binding capacity of MT for metal ions in this experiment. We have answered the questions: (i) how selenite participates in Brdička reaction, (ii) which competitive behavior of selenium against cobalt should be expected and (iii) what is the sequence of reaction processes in the modified Brdička reaction. Our new interpretation leading to complete description of the Brdička s mechanism is presented. Our results can be helpful in biochemical and clinical studies involving selenium compounds as potential chemotherapeutics. ACKNOWLEDGEMENT The work has been supported by the project CEITEC Nano Research Infrastructure (ID LM , MEYS CR, ) and SIX Research Center in the Czech Republic (the grant LO1401 INWITE is gratefully acknowledged). REFERENCES [1] Brdička R.: Collect Czech Chem Commun, 5 (1933) 112. [2] Heyrovsky J., Kůta J.: Principle of polarography. Publishing House of the CAS, Prague [3] Palmiter R.D: Proc Natl Acad Sci USA, 91 (1994) [4] Raspor B., Paic M., Erk M.: Talanta, 55 (2001)109 and J. Electroanal. Chem., 503 (2001) 159. [5] Adam V., Baloun J., Fabrik I., Trnkova L., Kizek R.: Sensors, 8 (2008) [6] Trnkova L., Kizek R., Vacek J.: Bioelectrochemistry, 56 (2002) 57. [7] Ganther H.E.: Biochemistry 7 (1968) 2898 and Biochemistry, 10 (1971) [8] Misra S., Boylan M., Selvam A., Spallholz J.E., Björnstedt M.: Nutrients, 7 (2015)

59 1 OXIDATION POTENTIALS OF GUANINE SPECIES Iveta TŘÍSKOVÁ 1, Alan LIŠKA 2, Jiří LUDVÍK 2, Libuše TRNKOVÁ 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic 2 J. Heyrovsky Institute of Physical Chemistry of the CAS, Dolejškova 3/2155, CZ Prague, Czech Republic *175126@mail.muni.cz Guanine (G) belongs to the most easily oxidizable nucleobases which is well known from various experimental and theoretical studies. Our contribution is directed towards studies, in which we compare theoretical oxidation potentials with their experimentally measured values for guanine (G), guanosine (Guo), deoxyguanosine (dguo), guanosine -5 - monophosphate (GMP) and 2 - deoxyguanosine -5 - monophosphate (dgmp). Figure 1: Guanine derivatives G guanine: 2-amino-1H-purin-6(9H)-one, Guo guanosine: 2-amino- 1,9-dihydro-9-β-D-ribofuranosyl-6H-purin-6-one, GMP guanosine-5 -monophosphate. For determination of experimental oxidation potentials of all G species linear sweep voltammetry was applied using polymer pencil graphite electrodes (ppeges). It was found that the oxidation process for all studied derivatives is irreversible and strongly ph dependent. Anodic peak potentials increase in the order G << dgmp < GMP < dguo < Guo and correlate well with the calculated thermodynamic redox potentials. For the determination of theoretical oxidation potentials the structures of G and its derivatives were optimized and the identities of minima were verified by vibration frequency calculations. Redox equilibria were modeled in terms of corresponding thermochemical cycles. The changes in free energy were calculated at DFT level using two different functionals: (a) general purpose B3LYP functional, and (b) a more specific ωb97x-d functional, both based on a 6-31+G(d) set. Conformity of theoretical and experimental data for radicals (cationic or neutral, respectively) indicates that the deprotonation process of G differs from its analogues whereas the oxidation process of all species takes place on the imidazole ring. 59

60 Figure 2: The linear sweep voltammograms of G (black), Guo (green full line), GMP (red), dguo (green dashed line) and dgmp (red dashed line) on the ppege. The scan rate 400 mv/s; the concentration of G and its derivatives 50 µmol L -1 ; phosphate acetate buffer (ph 5). ACKNOWLEDGEMENT Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum provided under the programme "Projects of Large Research, Development, and Innovations Infrastructures" (CESNET LM ), is greatly appreciated. The authors A.L. and J.L. are grateful to the institutional support RVO and the authors I.T. and L.T. thank Bc. M. Bosakova for her technical assistance in voltammetric experiments. REFERENCES [1] Steenken S.: Chem. Rev., 89 (1989) 503. [2] Li Q., Batchelor-McAuley C., Compton R.G.: J. Phys. Chem. B, 114 (2010) [3] Brett A.M.O., Matysik F.M.: Bioelectrochem. Bioenerg., 42 (1997) 111. [4] Ferapontova E.E., Electrochim. Acta, 49 (2004) [5] Navratil R., Kotzianova A., Halouzka V., Opletal T., Triskova I., Trnkova L., Hrbac J.: J. Electroanal. Chem., 783 (2016) 152. [6] Shields G.C., Seybold P.G.: Computational approaches for the prediction of pka values, CRC Press, [7] Becke A.D.: J. Chem. Phys., 98 (1993) [8] Chai J.D., Head-Gordon M.: Phys.Chem. Chem. Phys., 10 (2008) [9] Frisch M., Trucks G., Schlegel H., Scuseria G., Robb M., Cheeseman J., Scalmani G., Barone V., Petersson G., Nakatsuji H., Gaussian 16, revision A. 03, Gaussian Inc., Wallingford CT (2016). [10] Tomasi J., Mennucci B., Cammi R.: Chem. Rev., 105 (2005)

61 θ (mdeg) APPLICATION OF CD SPECTROSCOPY IN ANALYTICAL CHEMISTRY Anna VACULÍKOVÁ 1*, Marta FARKOVÁ 1, Přemysl LUBAL 1 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic *461086@mail.muni.cz Many biological compounds exhibit optical activity, which is due to asymmetric carbon on which four different substituents are attached. This configuration leads to two structurally different forms of one substance, when both forms have the ability to rotate the plane of polarized light, one turns to the left side (L-) and the second one to the right one (D-). This phenomena can be monitored by circular dichroism (CD) spectroscopy when the ellipticity is measured in the region of absorption band and this parameter can be used for structural analysis as well as for quantitative determination. However, if L- and D- forms are present in solution in ratio 1:1, i.e. racemic optically inactive mixture, they does not exhibit any rotatation. In our case, the L- and D- forms of tryptophan in aqueous solution were measured The spectroscopic measurements were done at Jasco J-810 spectropolarimeter (Jasco, Japan) in the wavelength range of nm. CD-spectra of pure L- and D-tryptophan and their racemic mixture are presented (see Fig. 1). It was demonstrated, that CD spectroscopy can be employed for quantitative analysis and determination of purity of chiral aminoacid (Tryptophan) λ (nm) D-tryptofan L-tryptofan Racemic mixture Figure: CD-spectra of aqueous solution of L- and D-tryptophan and mixure 1:1 (c = 0,05 mm, ph = 4) ACKNOWLEDGEMENT The work has been supported by Masaryk University (MUNI/A/1359/2018). 61

62 1 2 3 METABOLISM OF THE TYROSINE KINASE INHIBITOR LENVATINIB BY HUMAN HEPATIC MICROSOMES AND CYTOCHROMES P450 Katarina VAVROVÁ 1, Radek INDRA 1, Petr POMPACH 1, Zbyněk HEGER 2, Vojtěch ADAM 2, Tomáš ECKSCHLAGER 3, Kateřina KOPEČKOVÁ 3, Marie STIBOROVÁ 1* Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, Czech Republic Department of Chemistry and Biochemistry, Laboratory of Metallomics and Nanotechnology, Mendel University in Brno, Zemedelska 1, Brno, Czech Republic Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic *stiborov@natur.cuni.cz Abstract. Using human hepatic microsomes containing cytochrome P450 (CYP) enzymes and human recombinant CYPs, the oxidation of the tyrosine kinase inhibitor lenvatinib was investigated. The metabolites of lenvatinib were found to be O-desmethyllenvatinib, N-depropylated lenvatinib and the lenvatinib N-oxide. Of all tested human recombinant CYP enzymes, the CYP1A1, 1A2, 2A6, 2B6, 2C19, 3A4 and 3A5, oxidize lenvatinib to O-desmethyllenvatinib. CYP2B6, 3A4 and 3A5 are also responsible for oxidation of lenvatinib to N-descyclopropyllenvatinib, while the only CYP3A4 generates N-oxide of lenvatinib. 1. INTRODUCTION The receptor tyrosine kinases (TKs) are the enzymes that selectively phosphorylate the hydroxyl moieties of tyrosine residues on signal transduction molecules with a phosphate moiety from adenosine triphosphate [4]. Inhibitors of these enzymes are one of the most promising anticancer drugs used for treatment of several cancers in a last decade [4]. Lenvatinib is drug that belongs to this group of inhibitors that is used for treatment of certain tumors of the thyroid gland [3] and metastatic renal cell carcinoma [4]. Lenvatinib is an oral, multitargeted tyrosine kinase inhibitor (TKI) of vascular endothelial growth factor receptors (VEGFR1-VEGFR3), fibroblast growth factor receptors (FGFR1-FGFR4), platelet-derived growth factor receptor (PDGFR)α, rearranged during transfection (RET), and v-kit (KIT) signaling networks implicated in tumor angiogenesis. Overall, in cancer chemotherapy, serious clinical consequences may occur from small alterations in drug metabolism affecting drug pharmacokinetics. Nevertheless, there is only little insight in the metabolism of TKIs at this point, which is surprising since they are used on a daily basis in hundred thousand of patients. The target of this study was to investigate the the in vitro metabolism of lenvatinib in detail; human hepatic microsomes and human recombinant cytochromes P450 (CYPs) expressed in Supersomes TM were utilized for such a study. 2. MATERIAL AND METHODS HPLC was utilized for separation of lenvatinib metabolites formed by enzymatic systems and mass spectrometry for their structural characterization. 62

63 3. RESULTS AND DISCUSSION Based on preliminary studies using human hepatic microsomes, lenvatinib was suggested to be oxidized by cytochromes P450 (CYPs), mainly by CYP3A4, to its O-demethylated metabolite, a desmethylated form of lenvatinib. However, no direct prove of this suggestion was demonstrated. Therefore, the aim of this study was to investigate the metabolism of lenvatinib by human microsomal enzymes in vitro in detail. Two major lenvatinib metabolites, O-desmethyllenvatinib and N-descyclopropyllenvatinib were formed by incubation of lenvatinib with human hepatic microsomes. The generation of both these metabolites was attenuated by inhibitors of the CYP1A and 3A subfamilies in these subcellular systems. These results suggest that CYP3A and 1A are mainly responsible for the oxidation of lenvatinib in human livers. Of all tested human recombinant CYP enzymes, the CYP1A1, 1A2, 2A6, 2B6, 2C19, 3A4 and 3A5, oxidize lenvatinib to O-desmethyllenvatinib. CYP2B6, 3A4 and 3A5 are also responsible for oxidation of lenvatinib to N-descyclopropyllenvatinib. Another metabolite lenvatinib N-oxide, which is a minor oxidation product of lenvatinib, was generated only by CYP3A4. The presence of cytochrome b 5, which serves as electron donor to CYP enzymes, plays an essential role in lenvatinib oxidation, catalyzed by the CYP3A4-catalyzed formation of all metabolites. The results found in this study approved the knowledge showed by the preliminary studies, suggesting that lenvatinib is oxidized to O- desmethyllenvatinib, N-descyclopropyllenvatinib and lenvatinib N-oxide. Further, they specified the efficiencies of individual CYPs in lenvatinib oxidation reactions, and demonstrated an essential role of cytochrome b 5 in oxidation of lenvatinib by CYP3A4. The study of the enzyme kinetics of lenvatinib oxidation by the tested enzymatic systems is under way in our laboratory. 4. ACKNOWLEDGEMENT The work was supported by GACR (grant S). 5. REFERENCES [1] Reibenwein J., et al.: Expert Opinon on Therapeutic Targets, 12 (2008), [2] Hartmann, J.T., et al., Current Drug Metabolism, 10 (2009), [3] Greenhill C.: Nature Reviews Endocrinology, 13 (2017), 688 [4] Roviello G., et al.: Expert Opinion on Investigational Drugs 27 (2018),

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81 XIX. Workshop of Biophysical Chemists and Electrochemists Book of abstracts Editor: Libuše Trnková Technical adjustment: Iveta Třísková Published by Masaryk University, Brno st edition ISBN

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