• National Academy of Science of Ukraine • • State agency on Science, Innovations and Informatization of Ukraine • • The State Fund for fundamental researches of Ukraine • • Institute of Bioorganic Chemistry and Petrochemistry NAS of Ukraine • • Ivan Franko National University of Lviv • • Taras Shevchenko National University of Kyiv • • Ukrainian Chemical Society • • Ukrainian Biochemical Society • • Ukrainian Biophysical Society •
PROGRAMME and ABSTRACTS of 3rd International Symposium:
«Intracellular Signaling and Bioactive Molecules Design» http://www.bpci.kiev.ua/isabmd/ 17-23 of September 2012 Lviv, Ukraine
International Advisory committee • • • • • • • • • • • • • • • • • • • • • • •
Kukhar V.P. academician, Prof. (Kyiv, Ukraine) Amini A. Prof. (New Brunswick, USA) Frebort I. Prof. (Olomouc, Czech Republic) Grimm B. Prof. (Berlin, Germany) Hardtke S.C. Prof. (Lausanne, Switzerland) Khripach V.A. Corresponding Member of NAS of Belarus, Prof. (Minsk, Belarus) Kumar P., Prof. (Singapore) Kuznetsov Vl.V. Prof. (Moscow, Russia) Li J., Prof. (Ann Arbor, USA) Litvinovskaya R.P., Prof. (Minsk, Belarus) Martinec J. Prof. (Prague, Czech Republic) Romanov G.A. Prof. (Moscow, Russia) Ruelland E. Prof. (Paris, France) Sakakibara H. Prof.(Yokohama, Japan) Savaldi-Goldstein S. Prof. (Haifa, Israel) Schmulling T. Prof. (Berlin, Germany) Strnad M. Prof. (Olomouc, Czech Republic) Valentova O. Prof. (Prague, Czech Republic) Van Der Straeten D. Prof. (Gent, Belgium) Voinikov V.K. Prof. (Irkutsk, Russia) Volotovski I.D. academician, Prof. (Minsk, Belarus) Wang X. Prof. (St. Louis, USA) Yamamoto Y.Y. Prof. (Gifu, Japan)
Programme committee Co-Chairs: • •
Kukhar V.P., Academician of NAS of Ukraine, Prof (Kyiv, Ukraine) Kotur B.Ya., Prof (Lviv, Ukraine)
Members of Program Committee: • • • • • • • • •
Andronati S.V., Academician of NAS of Ukraine, Prof. (Odessa, Ukraine) Blume Ya.B., Academician of NAS of Ukraine, Prof. (Kyiv, Ukraine) El'skaya A.V., Academician of NAS of Ukraine, Prof. (Kyiv, Ukraine) Kalychak Ya.M., Prof. (Lviv, Ukraine) Khamar I.S., Prof. (Lviv, Ukraine) Komisarenko S.V., Academician of NAS of Ukraine, Prof. (Kyiv, Ukraine) Kosterin S.O., Member of NAS Ukraine, Prof. (Kyiv, Ukraine) Kravets V.S., Prof. (Kyiv, Ukraine) Manko V.V., Prof. (Lviv, Ukraine)
2
• • • • • • • • •
Martynyuk V.S., Prof. (Kyiv, Ukraine) Miroshnichenko M.S., Prof. (Kyiv, Ukraine) Ostapchenko L.I., Prof. (Kyiv, Ukraine) Obushak M. D., Prof. (Lviv, Ukraine) Svizhenko V.O., Prof. (Kyiv, Ukraine) Sybirna N.O., Prof. (Lviv, Ukraine) Terek O.I., Prof. (Lviv, Ukraine) Vovk A.I., Member of NAS Ukraine, Prof.(Kyiv, Ukraine) Yarmoluk S., Prof. (Kyiv, Ukraine)
Organizing committee • • • • • • • • • • • • • • • • • • • • • • • •
Kravets V.S., Prof., (chair)(Kyiv, Ukraine) Baranov V.I., PhD (Lviv, Ukraine) Bila E.E., Assoc. Prof., PhD (Lviv, Ukraine) Boyko I.V., PhD student (Lviv, Ukraine) Chernomorchenko S.S. (Kyiv, Ukraine) Derevyanchuk M.V., PhD student (Kyiv, Ukraine) Djura N.M. PhD (Lviv, Ukraine) Fetsko Z.M., PhD student (Lviv, Ukraine) Iakovenko O.M., PhD (Kyiv, Ukraine) Kalachova T.A., PhD student (secretary) (Kyiv, Ukraine) Karpyn O.L., PhD (Lviv, Ukraine) Kobyletska M.S. PhD (Lviv, Ukraine) Kolesnikov Y.S., PhD (Kyiv, Ukraine) Kretynin S.V. (Kyiv, Ukraine) Mamchur O.V., PhD (Lviv, Ukraine) Patsula O.I. (Lviv, Ukraine) Pokhodylo N.T., PhD (Lviv, Ukraine) Pokotylo I.V., PhD student (Kyiv, Ukraine) Romanyuk N.D., PhD (Lviv, Ukraine) Tsvilunyuk O.M., PhD (Lviv, Ukraine) Turytsya V.V., PhD (Lviv, Ukraine) Vaschuk V.P., PhD student (Lviv, Ukraine) Velychko O.I. PhD (Lviv, Ukraine) Yavorska N.Y. (Lviv, Ukraine)
3
Acknowledgements The Organizing committee wants to express a sincere gratitude for the support of the Symposium by the following sponsors:
National Academy of Science of Ukraine The State Fund for fundamental researches of Ukraine State agency on Science, Innovations and Informatization of Ukraine Institute of Bioorganic Chemistry and Petrochemistry NAS of Ukraine Ivan Franko National University of Lviv Alsichrom Co Ltd. (www.alsichrom.com)
4
Map of Lviv city center
1. IFNUL Main building; Universytetska Str., 1 2. IFNUL Faculty of Biology; Hrushevskogo Str., 4
5
Programme Monday, September 17th 10.00–18.00 14.00–18.00
Arrival and Registration at hotel “Lviv” City walk
Tuesday, September 18th Ivan Franko National University of Lviv (IFNUL) main building; Universitetska Srt., 1 10.00–14.00 14.00–14.30
Registration at Ivan Franko National University of Lviv Opening Ceremony at Historic conference hall Welcome address: Valery P. Kukhar, Academician NAS of Ukraine, Head of the State Fund for Fundamental Researches of Ukraine Ivan O. Vakarchuk, Prof., President of Ivan Franko National University of Lviv Bogdan Y. Kotur, Prof. Volodymyr S. Kravets, Prof. Plenary lectures Moderators: Valery Kukhar, Bogdan Kotur
14.30–15.15
Hitoshi Sakakibara, Tokunaga H., Kiba T., Kojima M., Yokohama, Japan How the log-dependent cytokinin activation is important for plant growth and development?
15.15–16.00
Coffee break
16.00–16.45
Guo L., Li M., Xuemin Wang, Saint Louis, USA Lipid signaling: Connecting stress cues to metabolic and physiological response
16.45–17.30
Vladimir Khripach, Zhabinskii V.N., Litvinovskaya R.P., Minsk, Belarus Practical applications of brassinosteroids: a way that offers a challenge
18.00
Welcome party
6
Wednesday, September 19th IFNUL biology faculty building; Hrushevskogo Str., 4 Plenary lecture Moderators: Hitoshi Sakakibara, Prakash Kumar 9.30–10.15
Georgy Romanov, Moscow, Russia Studies of cytokinin receptors: from in vitro to in planta and vice versa
Lecture hall “B” Symposium Session 1B: Cytokinin signaling and application in biotechnology Moderators: Hitoshi Sakakibara, Georgy Romanov, Prakash Kumar 10.15–10.50
Verma V., Oruganti V., Anand G., Sivaraman J., Prakash P. Kumar, Singapore Interaction studies of cytokinin signaling proteins AHP1 and ARR4
10.50–11.20
Lukáš Spíchal, Olomouc, Czech Republic Chemical regulation of cytokinin status - potential applications in biotechnology and agriculture
11.20–11.50
Aniça Amini, Andreu F., Glévarec G., Rideau M. and Crèche J., New Brunswick, USA Role of cytokinins on the regulation of monoterpenoid indole alkaloids biosynthesis in Catharanthus roseus cell suspension cultures
11.50–12.20
Coffee break Moderators: Lukáš Spíchal, Aniça Amini,
12.20–12.40
Guzel Kudoyarova, Vysotskaya L.B., Akhiyarova G.R., Veselov S.Y, Ufa, Russia Cytokinin content in cells of different zones of wheat roots
12.40–13.00
Sergey Lomin, Wulfetange K., Yonekura-Sakakibara K., Sakakibara H., Heyl A., Schmülling T., Romanov G.A., Moscow, Russia Subcellular localization of arabidopsis and maize cytokinin receptors
13.00–13.15
Dmitry Krivosheev, Getman I.A., Romanov G.A., Moscow, Russia N6-(benzyloxymethyl)adenosine is an antagonist of cytokinin receptor CRE1/AHK4 of Arabidopsis Lunch Break
13.15–14.10
7
Symposium Session 2B: Role of hormones, protein kinases/phosphatases and cytoskeleton in signal transduction Moderators: Igor Volotovsky, Andriy Vovk, 14.10–14.40
Andriy Vovk, Kyiv, Ukraine Cell signaling and phosphatases as possible targets for synthetic compounds
14.40–15.10
Talanova V.V., Topchieva L.V., Natalia Repkina, Titov A.F., Petrozavodsk, Russia Hormone effect on the protease genes expression
15.10–15.30
Sigal Savaldi-Goldstein, Haifa, Israel Understanding coherent root growth through cell type-specific activity of brassinosteroids
15.30–16.20
Coffee break, Poster session Moderators: Pavel Karpov, Eliezer Lifschitz
16.20–16.35
Fedina E.O., Salnikov V.V., Karimova F.G. Kazan, Russia The role of 24-epibrassinolide in pea leaf senescence
16.35 – 16.55
Pavel Karpov, Sheremet Ya.A., Raevsky A.V., Yemets A.I., Blume Ya.B., Kyiv, Ukraine Casein kinases CKL6 and CK1D as important factors of plant microtubule regulation
16.55–17.20
Natalia Petrova, Karimova F.G., Kazan, Russia Changes in tyrosine phosphorylation of some regulatory proteins by oxidative stress
17.20–17.50
Victor Martynyuk, Tseyslyer Yu.V., Tsymbalyuk O.V., Shelyuk O.V., Nurishenko N.E., Kyiv, Ukraine Modification of functional state of muscle tissue upon influence of extremely low frequency electromagnetic field
18.00–19.00 20.00–22.00
Poster session Attending performance at Lviv Opera theatre
8
Lecture hall “C” Symposium Session 3C: Analysis of synthetic compound activity and application in living systems (parallel session 2B) Moderators: Oleg Kolodiazhnyi, Roman Lesyk 14.10–14.40
Oleg Kolodiazhnyi, Kyiv, Ukraine Methods of biocatalysis in molecular design and synthesis of new bioactive compounds
14.40–15.10
Roman Lesyk, Zimenkovsky B., Kaminskyy D., Kryshchyshyn A., Havryluk D., Subtel’na I., Atamanyuk D., Roman O., Zelisko N., Lviv, Ukraine Thiazolidinone and related heterocyclic systems in anticancer agents discovery
15.10–15.30
Stepan Pilyo, Kornienko A.N., Kozachenko A.P., Prokopenko V.M., Shablykin O.V., Brovarets V.S. Kyiv, Ukraine Synthesis and properties of new 4,5-difunctionally substituted 1,3-oxazoles
15.30–16.10
Coffee break Moderators: Roman Lesyk, Oleh Devinyak
16.10–16.40
Oleh Devinyak, Zimenkovsky B., Lesyk R., Uzhgorod, Ukraine Identification of the possible pharmacophore responsible for 4-thiazolidinones anticancer activity
16.40–17.10
Olena Mitlenko, Zhygadlo E.Y., Sharapa D.I., Gayday A.V., Levandovskiy I.A., Shubina T.E., Kyiv, Ukraine Preparation and thansformations of D3-trishomocubane tertiary derivatives.
17.10–17.40
Alexandr Vasilenko, Turov K.V., Zyabrev V.S., Brovarets V.S., Kyiv, Ukraine Study of anticancer activity of sulfonyl derivatives of 1,3-thiasole
17.40–18.10
Natalia Finiuk, Zinchuk N., Romanyuk N.,Terek O., Mitina N., Zaichenko O., Stoika R., Lviv, Ukraine Toxicological studies of novel DMAEM-containing nanopolymers
18.10–19.00 20.00–22.00
Poster session Attending performance at Lviv Opera theatre
9
Thursday, September 20th IFNUL biology faculty building; Hrushevskogo Str., 4 Plenary lectures Moderators: Christian Hardtke, Victor Martynyuk 9.30–10.15
Christian S. Hardtke, Lausanne, Switzerland A vertical case of Systems Biology
10.15–11.00
Igor Volotovsky, Minsk, Belarus Calcium signaling in plant cell. Relation to other mediator systems
11.00–11.30
Eliezer Lifschitz, Haifa, Israel Florigen systemic signaling
11.30–12.00
Coffee break
Symposium Session 4B: Lipid signaling and stress responses Moderators: Xuemin Wang, Volodymyr Kravets 12.00–12.30
Jan Martinec, Kocourkova D., Pejchar P., Brouzdova J., Kravets V., Pokotylo I., Krckova Z., Valentova O., Prague, Czech Republic The role of non-specific phospholipase C in plant response to environmental stresses
12.30–13.00
Eric Ruelland, Delage E., Puyaubert J., Sasek M., Zachowski A., Paris, France Roles of phosphoinositides in the signalling of cold or salicylic acid in Arabidopsis thaliana
13.00–14.30
Lunch Break
14.30–15.00
Volodymyr Kravets, Kyiv, Ukraine Lipid signaling in plants
10
Symposium Session 5B: Salicylic acid and other stress hormones signaling Moderators: Eric Ruelland, Olga Valentová 15.00–15.20
Kalachova Tetiana, Kyiv, Ukraine Role of salicylic acid in RbohD mediated stomata movement
15.20–16.10
Janda M., Šašek V., Matoušková J., Burketová L., Olga Valentová, Prague, Czech Republic Salicylic acid signalling in Arabidopsis is mediated by phospholipase D
16.10–16.40
Alevtina Egorova, Yakovleva V.G., Tarchevsky I.A., Kazan, Russia Effect of methyl jasmonate on dicot rice and monocot pea root proteomes
16.40–18.00 18.00–19.00
Poster session Poster discussion session Moderators: Vladimir Khripach, Mykola Obushak, Georgy Romanov
19.30–22.00
Gala dinner
Friday, September 21st IFNUL biology faculty building; Hrushevskogo Str., 4 Lecture hall “B” Symposium Session 6B: Plant cell metabolism and growth regulation Moderators: Olga Terek, Alevtina Egorova, Elena Garmash 9.30–9.50
Elena Garmash, Dymova O.V., Malyshev R.V., Grabelnykh O.I., Borovik O.A., Khristin M.S., Plyusnina S.V., Kokovkina E.V., Voinikov V.K., Golovko T.K., Syktyvkar, Russia Light regulation of mitochondrial and chloroplast energy-dissipating systems activity during de-etiolation of wheat seedlings
9.50–10.20
Vladyslava Kovalska, Losytskyy M.Yu. and Yarmoluk S.M., Kyiv, Ukraine Fluorescent cyanine dyes for protein aggregation analysis
10.20–10.50
Coffee break
11
Lecture hall “A” Symposium Session 7A: Characteristics of cell signaling and hormonal regulation in animal systems Moderators: Lyudmila Drobot, Oleksandr Shkrabak 10.50–11.20
Samoylenko A., Kozlova N., Byts N., Ponomarenko O., Pasichnyk H., Bazalii A., Geraschenko D., Vorotnikov A., Kietzmann T., Lyudmila Drobot, Kyiv, Ukraine The adaptor protein Ruk/CIN85 acts as key signaling integrator of plasmogen activation system in breast adenocarconoma cells
11.20–11.45
Oleksandr Shkrabak, Veklich T., Bevza O., Cherenok S., Rodik R., Kalchenko V., Kosterin S., Kyiv, Ukraine Nа+,K+-ATP-ase of myometrium cell plasma membrane and calixarenes
11.45–12.15 12.15–13.45 14.00–18.00
Symposium closing ceremony Lunch Break Excursion
Saturday, September 22nd 10.00–16.00
Excursion
Sunday, September 23d Departure
12
Contents Page
14
Welcome Address (Valeryi P. Kukhar)
16
Oral presentations (in chronological order)
52
Poster presentations (in alphabetical order)
52
Session 1: Cytokinin signaling and application in biotechnology
57
Session 2: Role of hormones, protein kinases/phosphatases and cytoskeleton in signal transduction
74
Session 3: Analysis of synthetic compound activity and application in living systems
105
Session 4: Lipid signaling and stress responses
110
Session 5: Salicylic acid and other stress hormones signaling
119
Session 6: Plant cell metabolism and growth regulation
133
Session 7: Characteristics of cell signaling and hormonal regulation in animal systems
166
Author index
171
Sponsors 13
Welcome Address
14
CELL SIGNALING AT THE HUB OF MULTI-VECTOR STUDIES Kukhar V.P. Institute of Bioorganic Chemistry and Petrochemistry National Academy of Sciences of Ukraine, Murmanskaya 1, Kyiv, 02660, Ukraine, e-mail:
[email protected] On behalf of the Organizing Committee, it is a great pleasure for me to greet the participants of the 3rd International Symposium „Intracellular signaling and bioactive molecules design” which is held in Lviv, Ukraine. The subject of the symposium points at the importance that cell signaling studies gained nowadays and desire of committee to unite international scientific community in disclosure of its most recent problems. The object of the Symposium is to promote the progress of the study of cell signaling regulation – one of most topical points of world biochemistry, physiology, and molecular biology. Excitingly, solutions can be obtained coming from multiple directions. First of all, great progress have been made in research of functions and properties of natural signaling agents and hormones in living cells that allowed further in-depth analysis aimed at successful manipulation of metabolism reactions. From the other hand, synthesis and application of artificial growth regulators have proven to be beneficial and provided characteristics that possibly surpass that of natural analogues. Interdisciplinary studies directed at dissection of common signaling patterns in varied living species are also becoming more and more valued. Nevertheless, one of the most crucial problem of modern cell signaling research remain to be a discovery of specific primary effects evoked by biologically active compounds in cells leading to modulation of plant growth and development. Despite increasing elucidation of the molecular basis of growth regulators action in recent years, many fundamental questions remain to be solved. Thus receptors and other upstream signaling components remain to be identified and\or characterized for the majority of the phytohormones. Equally important are the revelation of hormonal networks and the integration of these networks within the morphogenetic programs. The previous symposiums organized by our research team - ISPAS in 2001 and PGSIHS in 2007 were based on scientific collaboration programs supported by INTAS fund. However, it is enjoyable that our current international symposium was to a great extent made possible due to the active role that The State Fund for fundamental researches of Ukraine plays in contemporary scientific life in Ukraine. Successfully, Ukrainian scientists are making more and more contribution to global cell signaling research initiatives. Studies are conducted on phytohormonal regulation of growth and development processes both on the cell and whole plant levels, adaptive role of growth regulators and phytohormones as well as agricultural use of synthetic and natural growth regulator substances. Good progress has been made regarding the disclosure of brassinosteriods functions in plants and characterization of novel types of plant phospholiapses. In addition significant advancements were achieved in synthesis of artificial cell signalling regulators with great applied potential. Though it is important that Ukrainian science will stay in tight bonds with renowned international research hubs that now provide cutting-edge ideas and technologies in cell signaling research. That is why it is crucial to bring experts from different fields of cell signaling studies together to join knowledge and share experience important for further progress. Dear participants, I sincerely hope that symposium will not only be scientifically rewarding but one that can bring new emotions, ideas, fruitful collaborations and far–going vistas.
15
Abstracts Oral presentations
16
HOW THE LOG-DEPENDENT CYTOKININ ACTIVATION IS IMPORTANT FOR PLANT GROWTH AND DEVELOPMENT? Sakakibara H., Tokunaga H., Kiba T., Kojima M. RIKEN Plant Science Center, Tsurumi, Yokohama 230-0045, Japan Cytokinins play a crucial role in the maintenance of stem cell activity in plants and in various aspects of growth and development. Spatiotemporal distribution of bioactive cytokinins is finely controlled by the metabolic enzymes. Cytokinins, such as N6-(∆2-isopentenyl)adenine (iP) and trans-zeatin (tZ), in higher plants are synthesized by adenosine phosphate-isopentenyltransferase (IPT), cytokinin trans-hydroxylase CYP735A, and cytokinin-specific phosphoribohydrolase LONELY GUY (LOG). Our recent studies in Arabidopsis and rice have demonstrated that IPTs, CYP735As, and LOGs are expressed in various parts during growth and development, and differentially regulate the synthesis of iP- and tZ-type cytokinins. Studies on the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants’ morphological and developmental phenotypes demonstrated a dominant role of the activation pathway mediated by LOGs for cytokinin production: the log1log2log3log4log5log7log8 septuple mutant in which all functional LOG genes were mutated by T-DNA insertion, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Further detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size, and that LOG3, LOG4 and LOG7 coordinately play a role for normal primary root growth in Arabidopsis. These results suggest that overlapping but differentiated functions of the members of the LOG gene family in meristem maintenance. We will outline recent progress in cytokinin biosynthesis and its regulation, especially focusing on the final step of cytokinin biosynthesis. Kurakawa, T. et al. (2007) Nature 445: 652-655. Kuroha, T. et al. (2009) Plant Cell 21: 3152-3169. Tokunaga，H. et al. (2012) Plant J. 69: 355-365.
17
LIPID SIGNALING: CONNECTING STRESS CUES TO METABOLIC AND PHYSIOLOGICAL RESPONSE Liang Guo, Maoyin Li, Xuemin Wang Biology, University of Missouri, St. Louis and Donald Danforth Plant Science Center Stresses, such as drought and nutrient deprivation, adversely impact plant growth and crop production. How are stress cues transduced into metabolic and growth alterations? Our studies have identified a number of lipid signaling cascades in plant response to environmental stresses. Many critical cellular events, such as intracellular signaling, vesicular trafficking, secretion, cytoskeletal rearrangements, are initiated by the assembly of protein complexes at specific sites in cellular membranes. Binding to lipid ligands is often required for the recruitment and assembly of these protein complexes. Membrane lipids are rich sources for generating intracellular messengers. Lipidomic analyses also reveal extensive modifications of membrane lipids under different stresses. The networking of lipid-based signaling pathways plays pivotal roles in connecting stress perception on membranes to metabolic and physiological responses.
18
PRACTICAL APPLICATIONS OF BRASSINOSTEROIDS: A WAY THAT OFFERS A CHALLENGE Khripach V.A., Zhabinskii V.N., Litvinovskaya R.P. Institute of Bioorganic Chemistry NAS of Belarus, 220141 Minsk, Kuprevich str., 5/2, Belarus Steroidal phytohormones brassinosteroids (BS) have a broad spectrum of regulatory activity in plants with respect to growth, development and adaptation to the environment. As a result of a long coevolution, BS developed into habitual food components of mammals. Till recently there were practically no attempts to investigate their specific effects in higher animals. Studies of BS effects in animal models have revealed a number of beneficial properties of these compounds which became prospective candidates for medicinal applications. One of the most interesting results is the effect of BS on the serum cholesterol levels in rats and humans that indicate their high efficacy as cholesterol-lowering agents for a wide range of doses. Together with the lowering of blood cholesterol, BS positively influenced some other blood factors, which are involved into the disease development, such as low-density and high-density lipoproteins and triglycerides. The very low BS content in natural sources makes chemical synthesis the only realistic approach for the preparation of these compounds for scientific and practical purposes. This aspect of BS studies has been the main subject of interest in our laboratory for many years and has led to synthesis of all most important BS. Our strategy was based on the use of new selective methods and reagents, some of them proved to be useful also for the preparation of sterols, ecdysteroids, and vitamin D metabolites. Low concentration of BS in plants creates many difficulties for their quantification that is absolutely necessary in their scientific studies and for efficient practical application. In the early period of BS studies the most fruitful analytical approaches were those based on GC-MS and HPLC of BS derivatives. The main drawback of all such apparatus approaches is the relatively long analysis time and the need of rather sophisticated equipment. A good alternative to the apparatus approaches proved to be immunochemical analysis. A number of immunochemical methods based on binding the proteins through spacers at C-6 or C-26 had been developed and used for determination of BS differing B-cycle and/or side chain functionality. In conclusion, our studies provided evidence that BS possess different activities in mammals similar to a certain extent to their stimulating effects in plants that creates good preconditions for multipurpose use of BS in medicine.
19
STUDIES OF CYTOKININ RECEPTORS: FROM IN VITRO TO IN PLANTA AND VICE VERSA Romanov G.A. K.A. Timiryazev Institute of Plant Physiology RAS, 127276 Moscow, Botanicheskaya, 35, Russia Cytokinins are ones of the most known and important plant hormones. Discovered more than half a century ago, cytokinins consistently attract the researcher attention by a variety of effects on plant growth and development, participation in adaptation to external conditions, the possibility of applications in biotechnology, agriculture, medicine and even cosmetics. However, the molecular mechanism of action of cytokinins has long remained unknown, and began to become clear only in the 21st century, after the discovery of receptors for these phytohormones. As it turned out, the plants have adapted to cytokinin signaling a two-component signal transduction system, imported from prokaryotic organisms. The lecture will cover recent advances in research of the molecular basis for perception and transduction of cytokinin signal. Emphasis will be placed on cytokinin receptors, their domain and spatial structures, subcellular localization, signaling activity, effect of mutations, ligand-binding properties, and phylogeny. This work was supported by Program of Presidium of the Russian Academy of Sciences (RAS) “Molecular and Cell Biology” and grants nn° 11-04-00614 and 11-04-90491 of the Russian Foundation for Basic Research. Recommended literature: Lomin SN, Romanov GA (2008) The analysis of hormone-receptor interaction. Theoretical and practical aspects. Russ J Plant Physiol 55:244–258 Romanov GA (2009) How do cytokinins affect the cell? Russ J Plant Physiol 56:268–290 Kravets VS, Kolesnikov YS, Kretynin SV, Getman IA, Romanov GA (2010) Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system. Physiol Plant 138:249–255 Lomin SN, Yonekura-Sakakibara K, Romanov GA, Sakakibara H (2011) Ligand-binding properties and subcellular localization of maize cytokinin receptors. J Exp Bot 62:5149–5159 Stolz A, Riefler M, Lomin S.N, Ashazi K, Romanov GA, Schmülling T (2011) The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors. The Plant J 67:157–168 Wulfetange K, Lomin SN, Romanov GA, Stolz A, Heyl A, Schmülling T (2011) The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol 156:18081818 Heyl A, Riefler M, Romanov GA, Schmülling T (2012) Properties, functions and evolution of cytokinin receptors. Eur J Cell Biol 91:246–256 Lomin SN, Krivosheev DM, Steklov MYu, Osolodkin DI, Romanov GA (2012) Receptor properties and features of cytokinin signaling. Acta Naturae in press
20
INTERACTION STUDIES OF CYTOKININ SIGNALING PROTEINS AHP1 AND ARR4 Vivek Verma1, Vidya Oruganti1, Ganesh S. Anand1, Jayaraman Sivaraman1, Prakash P. Kumar1,2 1
Department of Biological Sciences, National University of Singapore (NUS), Singapore 117543 Temasek Life Sciences Laboratory, NUS, 1 Research Link, Singapore 117604 Corresponding author email:
[email protected]
2
Cytokinins, a major class of plant hormones, regulate various developmental processes including cell division and differentiation. The cytokinin signaling pathway is similar to the bacterial two component system. In Arabidopsis, the signal is perceived by receptors, transferred to Arabidopsis Histidine Phosphotransfer Proteins (AHPs) in the cytosol, which then relay it to the Arabidopsis Response Regulators (ARRs) in the nucleus. AHPs interact with type-A ARRs. However, how this interaction takes place and the ensuing changes in the ARRs after receiving the signal are not well understood. To address this gap, we generated a homology model of AHP1-ARR4 (∆16-175) using the crystal structure of a homologous protein complex from yeast as the template. Based on the model, several amino acid residues from ARR4 that are likely to be involved in the phosphate transfer and the protein interaction interface were identified. To test this experimentally under in vitro conditions, recombinant AHP1 and ARR4 were generated for Hydrogen-Deuterium Exchange (HDX) mass spectrometry. Additionally, selected amino acids were mutated, and the mutant proteins were tested for their interaction with AHP1 in yeast-two hybrid system. Interestingly, some of the single amino acid mutations are capable of aborting the interaction, indicating their critical role for the interaction. Independent verification of the interactions was also done in Arabidopis mesophyll protoplasts using Bimolecular Fluorescence Complementation (BiFC) assay. Furthermore, to study the impact of mutations on functional aspects of ARR4 in planta, hextuple type-A ARR knock-out (arr3 arr4 arr5 arr6 arr8 arr9) mutant was complemented with ARR4 (wild-type and selected single amino acid mutants). The various transgenic lines were examined for their cytokinin sensitivity, which further reinforced the physiological impact of the mutated amino acids. Our data show that the AHP1-ARR4 interaction mechanism is highly conserved between yeast and plants.
21
CHEMICAL REGULATION OF CYTOKININ STATUS - POTENTIAL APPLICATIONS IN BIOTECHNOLOGY AND AGRICULTURE Lukáš Spíchal Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic Cytokinins are important plant hormones regulating many aspects of plant growth and development. Recent studies on transgenic plants with altered cytokinin metabolism or signalling revealed interesting consequences of cytokinin deficiency or disruption of cytokinin perception. Modulation of cytokinin levels by exogenous application has also shown possible applications in agriculture. Recently we have described new groups of potent inhibitors of cytokinin degradation and perception derived from cytokinins, and synthetic cytokinins with enhanced cytokinin activity. In presented report rational design and targeted development of these new generations of cytokinin derivatives will be described. The effects of resulting compounds on plant growth, development and stress tolerance will be presented and potential application in biotechnology and agricultural will be demonstrated.
Supported by Grants No. ED0007/01/01 Centre of Region Hana for Biotechnological and Agricultural Research; MSM 6198959216; and 501/10/1450 from Czech Science Foundation.
22
ROLE OF CYTOKININS ON THE REGULATION OF MONOTERPENOID INDOLE ALKALOIDS BIOSYNTHESIS IN CATHARANTHUS ROSEUS CELL SUSPENSION CULTURES 1
Aniça Amini, Françoise Andreu, Gaëlle Glévarec, Marc Rideau and Joël Crèche EA 2106 Biomolécules et Biotechnologies Végétales, Faculté de Pharmacie, Université François Rabelais de Tours, 31 Avenue Monge, F-37200 Tours, France. 1 Present address: Biotechnology Center for Agriculture and the Environment, Rutgers University, 59 Dudley Road, New Brunswick, NJ, 08901, USA. Catharanthus roseus (Madagascar periwinkle) is a medicinal plant accumulating monoterpenoid indole alkaloids (MIAs), some of which exhibit strong anti-cancer activity. In Catharanthus roseus cell cultures, phytohormones modulate MIAs accumulation, and some of these phytohormones can interact with each other. We have shown synergistic effects between CK and methyljasmonate both on MIAs accumulation and on transcript level of genes encoding enzymes of the MEP and seccoiridoïd pathways (Crdxr and Crg10h). CK and gibberellin (GA) exerted antagonistic effects on alkaloid accumulation in a dose-dependant manner and at low concentrations. These effects were also observed on Crdxr and Crg10h gene transcript levels with low doses of GA. We have also shown that endogenous gibberellins inhibit MIAs accumulation. Thus, in presence of GA biosynthesis inhibitor, paclobutrazol, the accumulation of MIAs is dramatically increased. We also have established that phospholipase D (PLD) regulates the expression of CrRR3, encoding a type A response regulator (a protein involved in CK signalling), via its product, phosphatidic acid. On the other hand, PLD had no effect on CrRR1, suggesting that response regulators may be differentially regulated by CK in periwinkle cells. Finally, studies on the relationship between CK signal transduction and MIAs biosynthesis pathway have been developed using a dexamethasone (DEX) inducible RNAi system targeting elements of the early steps of CK signaling. In transgenic cell lines, in presence of DEX, the up-regulation of Crdxr and Crg10h genes by CK was completely abolished. Theses results clearly show a relationship between the canonical CK signaling and the MIAs biosynthetic pathway. Keywords: Cytokinin, Catharanthus roseus, Monoterpenoid indole alkaloids, Gene regulation, Hormonal interaction.
23
CYTOKININ CONTENT IN CELLS OF DIFFERENT ZONES OF WHEAT ROOTS Kudoyarova G.R., Vysotskaya L.B., Akhiyarova G.R., Veselov S.Y* Institute of Biology, Ufa Research Centre, RAS and Bashkirian State University*, Russia Distribution of cytokinins was detected in cross-sections of wheat roots using the immunostaining procedure with primary antibodies against zeatin riboside and colloidal gold-labelled secondary antibodies coupled with silver enhancement. Cytokinins were quantified by enzyme immunoassay following thin layer chromatography (Kudoyarova et al., 2011), reliability of the method being confirmed by HPLS-MS assay of cytokinins in whole wheat roots. Zeatin derivatives were the most abundant cytokinins in wheat plants. Antibodies had high specificity to free zeatin bases and their conjugates. However applied paraformaldehyde–glutaraldehyde tissue fixation enabled detection of zeatin alone (active cytokinin form). Several controls were processed to confirm specificity of the method. Thus no immuno-reaction was observed in root sections processed with pre-immune serum, addition of synthetic zeatin riboside with specific antibodies drastically reduced immunostaining, while plant treatment with exogenous zeatin prior to fixation greatly increased it. Both cytokinin quantification and immunolocalization revealed greatest hormone accumulation in the root tips, which is in accordance with literature data (Aloni et al., 2005; Rijavek et al., 2011). In this zone cytokinin specific immunostaining was uniform, suggesting even contents of cytokinins in the cells. The intensity of cytokinin staining decreased in elongation zone paralleled by a decline in cytokinin content quantified by enzyme immunoassay, which was likely to be due to high activity of cytokinin oxidase detected by us in root tips. Cells of elongation zone were non-uniformly stained, cytokinin signal being weaker in the cortex than in the central cylinder. Unexpectedly cytokinin signal was more intensely visualized at the beginning of differentiation zone marked by appearance of root hairs, which could not be attributed to changes in cytokinin oxidase activity or release of zeatin from its conjugated forms. The second peak of cytokinins may be due increased phloem unloading in this zone. However it remains to explain how this increase may be brought about. Whatever the mechanism of cytokinin accumulation in this zone may be, the effect was highly reproducible and we tried to reveal its physiological significance. Confocal microscopy detected appearance of autofluorescence of lignified vessels in the section just above the zone with increased cytokinin signal. Since cytokinins have been shown to induce the process of lignification, local cytokinin maximum may be related to differentiation of xylem.
24
SUBCELLULAR LOCALIZATION OF ARABIDOPSIS AND MAIZE CYTOKININ RECEPTORS 1
Lomin S.N., 2Wulfetange K., 3Yonekura-Sakakibara K., 3Sakakibara H., 2Heyl A., 2Schmülling T., 1 Romanov G.A. 1 Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia; 2Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences, Freie Universität Berlin, Berlin, D–14195, Germany;3RIKEN Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan Cytokinin receptors are transmembrane sensory hybrid histidine kinases. They are encoded by a small family of genes. Three genes in Arabidopsis and at least seven genes in maize encoding cytokinin receptors were identified. We have studied the localization of cytokinin receptors in Arabidopsis and maize cells by means of a variety of approaches. Analysis of cytokinin high-affinity binding to membrane fractions separated by aqueous polymer two-phase partitioning showed that in Arabidopsis seedlings and maize roots the maximal binding was associated with endomembranes. Similar results were obtained when Arabidopsis double mutants expressing only one cytokinin receptor were used. In another approach, cytokinin receptor genes were fused with a fluorescent protein GFP. These receptor-GFP constructs were transiently expressed in leaves of Nicotiana benthamiana (Arabidopsis genes) or maize mesophyll leaf protoplasts (maize genes). The subcellular fluorescence pattern was visualized using confocal microscopy. In all cases including maize protoplasts (receptors ZmHK 1-2) or tobacco leaves (receptors AHK 2-4) the fluorescence pattern corresponded to the endoplasmic reticulum (ER). In the third approach we have separated microsomes containing native or Myc-tagged cytokinin receptors by sucrose gradient centrifugation followed by immunoblotting. In case of maize we have used specific antibodies against native ZmHK1. In case of Arabidopsis transgenic plants were produced expressing the receptors AHK2 or AHK3 fused with marker peptide 4xMyc at the C-terminus. The immunoblotting of cell membrane fractions with corresponding antibodies showed the best colocalization of cytokinin receptors with the ER marker BiP. Also the Mg2+-dependent density shift in receptor positions typical of ER membrane proteins was demonstrated. On the basis of all these results we can conclude that cytokinin receptors are localized mainly in endomembranes (most likely in the ER) and not in the plasma membrane, as was previously believed. This work is supported by Program of Presidium of the Russian Academy of Sciences (RAS) “Molecular and Cell Biology” and grant n° 11-04-00614 of the Russian Foundation for Basic Research. References Wulfetange K, Lomin SN, Romanov GA, Stolz A, Heyl A, Schmülling T (2011) The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum. Plant Physiol 156:18081818 Lomin SN, Yonekura-Sakakibara K, Romanov GA, Sakakibara H (2011) Ligand-binding properties and subcellular localization of maize cytokinin receptors. J Exp Bot 62:5149–5159 Lomin SN, Krivosheev DM, Steklov MYu, Osolodkin DI, Romanov GA (2012) Receptor properties and features of cytokinin signaling. Acta Naturae in press
25
N6-(BENZYLOXYMETHYL)ADENOSINE IS AN ANTAGONIST OF CYTOKININ RECEPTOR CRE1/AHK4 OF ARABIDOPSIS Krivosheev D.M., Getman I.A., Romanov G.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Cytokinins are classic phytohormones and represent a large group of natural and synthetic compounds, derivatives of adenine or phenylurea. Antihormones, which can suppress the effect of hormone at the level of its perception, are a useful tool in studies of the molecular mechanism of hormone action. These antihormones are called receptor antagonists; they inhibit the effect of respective hormones in the most specific manner. Receptor antagonists are widely used in studying the mechanism of action of animal hormones. In the studies of hormonal regulation in plants, attempts to obtain and use antihormones were also made. Only very recently, the first compounds that have shown real anticytokinin activity (i.e., the ability to inhibit cytokinin signaling at the level of their receptors) have been found. These anticytokinins competitively inhibited the binding of cytokinin to Arabidopsis receptors and did not cause hormonal effects in bioassays by themselves. These compounds were derivatives of the well known cytokinin 6-benzylaminopurine (BAP): N6-(2hydroxy-3-methylbenzylamino)purine (PI-55) and N6-(2,5-dihydroxybenzylamino)purine (LGR991). We have revealed the anticytokinin activity of an another adenine derivative, N6(benzyloxymethyl)adenosine (BOMA). This compound fundamentally differs from the two anticytokinins described earlier by the presence of the oxygen residue in the chain of atoms connecting the aromatic ring with the N6 of adenine as well as by the absence of modifications of the benzene residue. At the beginning, we have tested a number of synthetic adenosine derivatives for cytokinin activity in bioassays in planta (Kolyachkina et al., 2011). For the majority of the compounds, we have obtained a clear correlation between the ability to bind to the cytokinin receptor and the ability to cause a response in the bioassay. However, in the case of compound BOMA, this correlation was missing: this substance was capable of binding to receptors, especially to the CRE1/AHK4 receptor (KD for CRE1/AHK4 and AHK3 was about 230 and 3500 nM, respectively), but the reaction in the bioassay was extremely weak (for the AHK3 receptor) or absent (for the CRE1/AHK4 receptor). This finding suggested that the compound exhibited anticytokinin activity, especially for the CRE1/AHK4 receptor, whose affinity for BOMA was quite high but the typical cytokinin signaling was absent. To verify this hypothesis, we tested the ability of BOMA to suppress the physiological effects of the typical cytokinin BAP. Adenine derivative PI-55, which had previously been shown to be a competitive antagonist of cytokinin receptor CRE1/AHK4 (Spichal et al., 2009), was taken as a control. Bioassays with seedlings of Arabidopsis mutant PARR5:GUS expressing the only CRE1/AHK4 receptor showed that BOMA significantly inhibited the effect of BAP when seedlings were treated simultaneously with these compounds. The effects of BOMA and anticytokinin PI-55 almost coincided corroborating the anticytokinin properties of BOMA. Thus, as a result of this study, the group of the newly found anticytokinins was supplemented with a new antagonist of cytokinin receptor CRE1/AHK4: BOMA, which differs markedly in structure from the anticytokinins found earlier. This work was supported by the Program of the Presidium of the RAS “Molecular and Cellular Biology” and the Russian Foundation for Basic Research (project nos. 11-04-00614, 11-04-90491).
26
CELL SIGNALING AND PHOSPHATASES AS POSSIBLE TARGETS FOR SYNTHETIC COMPOUNDS Vovk A.I. Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, 02660, Kyiv-94, Murmanska str.1, Ukraine. E-mail:
[email protected] Various phosphatases are involved in cellular activities such as cell growth, differentiation, migration and other processes to catalyze dephosphorylation of different proteins and low molecular weight phosphates. Some of the known roles of the phosphatases in the regulation of the processes in plant and animal cells may be attributed to mechanisms of cell signaling. Together with protein kinases, protein phosphatases and some acid and alkaline phosphatases are important to control the phosphorylation/dephosphorylation balance. Protein tyrosine phosphatases regulate a number of biochemical processes governed by dephosphorylation of phosphotyrosine residues in proteins. Intracellular protein tyrosine phosphatase 1B (PTP1B) is known to be implicated in dephosphorylation of activated insulin receptor and leptin receptor-associated kinase JAK2 and is considered as a negative regulator of insulin and leptin signaling. Therefore PTP1B is one of the most promising therapeutic targets for potential treatment of type 2 diabetes and obesity. There is growing interest in developing potent and selective inhibitors of this enzyme. Recently the new derivatives of carboxylic, phosphonic, sulfonic acids, heterocyclic and other compounds have been described as PTP1B inhibitors. This report summarizes literature data on mechanisms of functioning and inhibition of protein tyrosine phosphatases by bioactive synthetic compounds. It includes also the analysis of the studies carried out in our laboratory,1-4 of both experimental and theoretical nature, which dealt with the inhibition of phosphatases by phosphonic acid derivatives bearing thiourea, isoindolin-1-one, calixarene and other fragments as molecular scaffolds. Some of the compounds were found to be effective inhibitors of alkaline phosphatase from different sources, human prostatic acid phosphatase, Yersinia PTP and human PTP1B, and other protein tyrosine phosphatases in vitro with apparent inhibition constants in the micromolar range. Some of the inhibitors of PTP1B showed high selectivity over TC-PTP, LAR-PTP, PTP-β. The possible inhibition mechanisms have been explained using computational docking of inhibitor into active centre of the enzyme. Special approaches were proposed for binding models of the inhibitors with PTP1B. (1) A.I. Vovk, I. M. Mischenko, V.Yu. Tanchuk, G.A. Kachkovskii, S.Yu. Sheiko, O.I. (2) Kolodyazhnyi, V.P. Kukhar. Bioorg. Med. Chem. Lett. 2008, 18, 4620–4623. (2) A.I. Vovk, L.A. Kononets, V.Yu.Tanchuk, A.B. Drapailo, V.I. Kalchenko, V.P. Kukhar. J. Incl. Phenom. Macrocycl. Chem. 2010, 66, 271-277. (3) A.I. Vovk, L.A. Kononets, V.Yu. Tanchuk, S.O. Cherenok, A.B. Drapailo, V.I. Kalchenko, V.P. Kukhar. Bioorg. Med. Chem. Lett. 2010, 20, 483-487. (4) V.Yu. Tanchuk, V.O. Tanin, A.I. Vovk. Chem. Biol. Drug Design, 2012 doi:10.1111/j.1747-0285.2012.01370.x
27
HORMONE EFFECT ON PROTEASE GENES EXPRESSION Talanova V.V., Topchieva L.V., Repkina N.S., Titov A.F. Institute of Biology of Karelian Research Centre RAS, 185910 Petrozavodsk, Pushkinskaya, 11, Russia It is known, that hormones such as abscisic acid (ABA) and cytokinins play an important role in plant defense and adaptation mechanisms under unfavorable environmental conditions. As well as one of the nonspecific plants response to various stressors is the enhancement of the rate of biodegradation and especially proteolysis which catalyzed by proteases enzymes. In this process proteases, degrading proteins and thus prevent the accumulation of damaged proteins and peptides in the cells. Taking into account the above the goal of our work was to analyze the effects of exogenous ABA and cytokinin on the proteases gene expression in wheat seedlings hardened by low temperatures. The experiments were performed on seedlings of winter wheat (Triticum aestivum L.) cv. Moskovskaya 39 grown for 7 days. Seedlings were subjected to hardening low-temperature treatment (5ºC) during 7 days. Some seedlings were placed on 0.1 mM ABA (ICN, USA) or 0.01mM synthetic cytokinin 6-benzylaminopurine (ICN, USA) one day before hardening. The genes expression level of Сys (encoding cystein protease) and inCys (encoding proteases inhibitor cystatin) in leaves was analyzed by Real-Time PCR. The results obtained showed that, at normal temperature (220C) ABA treatment did not affect on the Cys gene expression in wheat seedlings. Under low temperature (50C) with exogenously applied ABA an abrupt growth of the Cys transcript content took place with the maximum reached after 5 hours after beginning of influence and saved on higher level until the end of experiment. The similar dynamics was found under exposure low temperature with cytokinins. A 24-hours-long ABA treatment of seedlings prior to cold exposure resulted in the growth of the inCys gene transcripts. Whereas during chilling were shown the increase of inCys gene only at the final stage of it. The changes in inCys gene expression under low temperatures with cytokinin were not found. Based on this results we can conclude that ABA and cytokinin were increased the proteases gene expression under low temperature thus took place in protect mechanisms of plants to different stress-factors. This study was supported by the Russian Foundation for Basic Research (project no. 10-04-00650a)
28
UNDERSTANDING COHERENT ROOT GROWTH THROUGH CELL TYPE-SPECIFIC ACTIVITY OF BRASSINOSTEROIDS Sigal Savaldi-Goldstein Technion-Israel Institute of Technology, Israel Multiple hormonal pathways, acting in a subset of cells, can promote or restrict organ growth. Our goal is to understand how information from distinct cell-types is integrated to achieve coordinated growth. Here, we focus on brassinosteroids (BRs) and ask how their spatial activity affects the root meristem and the expansion of cells in the elongation zone. BRs are required to maintain normal cell cycle activity and cell expansion of meristematic cells (Hacham et al, 2011). These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. Because the extent of mitotic activity and differentiation is tightly linked to auxin gradient we further asked how the BR pathway integrates with current proposed models for PIN regulation. We found that BRs impinge on auxin distribution through distinct regulatory modes, including transcriptional and post-transcriptional regulation of specific PIN auxin efflux carriers (Hacham et al, 2012). Importantly, the activity of the BR receptor BRI1 in the epidermis is sufficient to regulate meristem size and controls the expression of the QC and the steleenriched MADS-BOX gene AGL42. Thus, AGL42 is a molecular readout for cell-cell communication identified in our work. Our current approaches addressing how local BR activity maintains coherent root growth will be discussed.
29
CASEIN KINASES CKL6, CK1D AND CKL2 AS IMPORTANT FACTORS OF PLANT MICROTUBULE REGULATION Karpov P.A., Sheremet Ya.A., Raevsky A.V., Yemets A.I., Blume Ya.B. Institute of Food Biotechnology and Genomics , Natl. Academy of Ssci. of Ukraine, 04123, Оsipovskogo, 2а, Кyiv-123, Ukraine,
[email protected] Casein kinases 1 are multifunctional serine/threonine phosphotransferases expressed ubiquitously in all eukaryotic organisms. It was shown recently that mammalian CK1s regulate microtubule dynamics and their substrates are such proteins as tubulin and tau. [PMID:18945931]. Also, it is known, that mammalian CK1δ binds and phosphorylates animal EB1 (MAP1) [PMID:22123863]. It was demonstrated that Arabidopsis thaliana casein kinases 1 (CK1) are associated with cortical microtubules (MT) in vivo and phosphorylates tubulin in vitro [PMC2593671]. Nevertheless role of CK1s in regulation of plant MTs remains an open issue to date. Blastp-scanning and subsequent bioinformatic analysis revealed presence of 18 CK1-like kinases in A. thaliana. According to the BLAST protocol, the maximum "Score" (456) belongs to A. thaliana KC1D (UniProt: P42158, identity = 78 %, similarity = 92 %). We reconstructed spatial structure of all CK1s catalytic domains from Rattus norvegicus (α, β, γ1-3, δ, and ε) and their homologs from A. thaliana. Structural superimposition of catalytic domains confirms high similarity of CK1 (α, β, γ1-3, δ, and ε) from R. norvegicus and 13 (from 18 in total) A. thaliana homologs: KC1D, CKI1, CKL2, CKL3, CKL4, CKL5, CKL6, CKL7, CKL8, CKL9b, CKL10, CKL12, CKL13. Structural aspects of CK1s interaction with inhibitor D4476 demonstrate that this inhibitor interacts with all isoforms of animal and plant CK1 in the similar ATP-competitive manner. Results of ligand docking, molecular dynamics and chemogenomic analysis of D4476 complex with rat CK1δ and 13 plant isoforms revealed similar ligand binding with rat CK1δ and CKL12, CKL5, CK1D, CKL2, CKL4 and CKL3 from A. thaliana. It was demonstrated that CK1-specific inhibitor D4476 had strong effects on growth and morphology of A. thaliana primary roots. The effects of CK1 inhibition by D4476 on microtubule organization (spatial reorientation of cortical MTs) was also proved with confocal scanning microscopy of GFPMT labeled Arabidopsis plants. Basing on C-terminal homology we identified motifs of these protein kinases involved presumably in MT-binding of plant СK1D (with MAP1) and CKL6 (with tubulin). Summarizing these results and literature data [PMID: 18945931, PMC2593671, 22123863] we can assume significant role of CK1s in the regulation of plant microtubules through direct tubulin (isoform CKL6) and EB1 (isoform СK1D) phosphorylation.
30
CHANGES IN TYROSINE PHOSPHORYLATION OF SOME REGULATORY PROTEINS BY OXIDATIVE STRESS Petrova N.V., Karimova F.G. Kazan Institute of Biochemistry and Biophysics RAS, 420111 Kazan, Lobachevski, 2/31, Russia Recently reactive oxygen species (ROS) are increasingly discussed from the perspective of their involvement in cellular signaling, besides of their toxic effects. In this case effects of ROS are determined by concentration. Hydrogen peroxide is toxic only at high concentrations that exceed the capacity of antioxidant systems of cells. Signaling splashes in hydrogen peroxide concentration are not beyond the physiologically acceptable concentrations (Bystrova, Budanova, 2006) and redox homeostasis is maintained by antioxidant systems. The specificity of action of hydrogen peroxide required for a second messenger is determined by protein sulfhydryl groups with a low ionization constant, which can be oxidized at physiological pH in the cell. One of such proteins is tyrosine phosphatase, which catalyses reaction of protein dephosphorylation on tyrosine and work in antagonism with the tyrosine protein kinases. Tyrosine phosphorylation is an important mechanism in cellular signaling. Redox regulation of plant tyrosine phosphorylation is little studied. We have shown redox-dependent changes in tyrosine phosphorylation level of the range of plant regulatory proteins in early responses to stress. Proteins were identified by MALDI-TOF mass spectrometry after separation by 2D-electrophoresis. The level of tyrosine phosphorylation was detected by immunoblotting with monoclonal anti-phosphotyrosine antibodies (PY20). Changes in the tyrosine phosphorylation level under treatment with redox agents (H2O2, dithiothreitol) were observed for the following regulatory proteins: ABA-responsive protein ABR17, transcriptional regulatory protein, 14-3-3-like protein, guanine nucleotide binding protein subunit beta-like protein. Changes in the tyrosine phosphorylation level of regulatory proteins determine the adaptation strategy of organisms to various stress factors.
31
MODIFICATION OF FUNCTIONAL STATE OF MUSCLE TISSUE UPON INFLUENCE OF EXTREMELY LOW FREQUENCY ELECTROMAGNETIC FIELD Martynyuk V.S., Tseyslyer Yu.V., Tsymbalyuk O.V., Shelyuk O.V., Nurishenko N.E. Taras Shevchenko National University of Kyiv, 64, Volodymyrs'ka St., Kiev, 01061, Ukraine The aim of our study was to investigate the effect of weak electromagnetic fields of extremely low frequency (EMF ELF) on some functional parameters of smooth muscle and ATP-ase activity of skeletal muscle actomyosin (AM). In particular, the effects of ELF EMF on spontaneous and K +induced and also acetylcholine-induced contraction of smooth muscle strips of rat caecum were studied. At the same time the influence of the EMF ELF on the ATPase activity of AM in vitro that isolated from rabbit skeletal muscle was estimated. Contractions of circular smooth muscle strips were induced by application of acetylcholine (ACh, 10 μM) in Krebs’ solution, as well as high-potassium solution (K+, 80 mM). The AM of skeletal muscle from rabbit was isolated by the Perry’s method with modifications. The exposure of AM solutions (2 mg/ml) in EMF ELF was 1, 2 and 3 hours at 37oC. EMF (meander wave) with frequency 8 Hz and 25 μT was created by using the special generator G6-28 and Helmholtz coils. The study of the spontaneous activity of smooth muscle strips showed that the effect of EMF ELF exposure caused a statistically significant (p500) have not been considered as drug candidates, as they do not meet the Lipinski’s rule. However, such molecules with extensive surface and diverse geometry are more suitable as compared to low-molecular compounds for targeting both allosteric sites and macromolecular interactions. The cage compounds with encapsulated metal ion (clathrochelates) allow to build the structure from a single center in eight different directions using a variety of substituents for the design of inhibitors whose shapes closely match the macromolecular surfaces of complex topology, including protein– protein and protein–DNA interfaces Here we report the first example of efficient transcription inhibition by clathrochelates in an in vitro system based on T7 RNA polymerase, a simple and convenient model in the search for transcription inhibitors and in the study of biological activity of organic and metalloorganic compounds. The testing of a series of new mono- and dimeric iron(II) clathrochelates 1-2 revealed the efficient structure- and concentration-dependent inhibition of transcription by these compounds. Most monomeric complexes 1 inhibited RNA polymerase in micromolar concentrations (IC50 2.5-9.5 µM), and compounds containing mercaptobenzoic acid residues (1e and 1f) were active in nanomolar region (IC50 0.5 µM). F B
F B O N N O
O O N N Fe2+ N
N OO
O N
R
N O
R
1d R = NH(CH2)4OH
O O N N Fe2+ N
N OO
O N R
R
B F
B F
1a R = CH3
F B
2a R = H
O N Fe2+
N N OO B F
O N N O
2d R = N
1b R = C6H5 1e R = S-(meta)C6H4COOH
2b R = NH2
2e R = S-(meta)C6H4COOH
1c R = NH2
2c R = NH(CH2)2OCH3
2f R = S-(para)C6H4COOH
1f R = S-(para)C6H4COOH
Bis-clathrochelates 2 were even more potent inhibitors active in micro- and nanomolar concentration range. Again, complexes containing bulky m- and p-mercaptobenzoic acid fragments appeared to be the most efficient inhibitors. IC50 values for the most active compounds 2d, 2e and 2f were 0.59, 0.43 and 0.31 µM, respectively. So the activity of clathrochelates is close to that of the most potent metalocomplex transcription inhibitors known to date (IC50 0.1-0.5 µM). This result is rather surprising since the tested complexes do not contain intercalative groups typical for active inhibitors. None of the studied complexes inhibited topoisomerase I in the in vitro DNA relaxation test system in 20-50 µM concentration suggesting that they hardly are efficient DNA binders. So the most probable mechanism of transcription inhibition could involve the interaction of complexes with polymerase or enzymatic complex, rather than with DNA template.
92
MOLECULAR DOCKING AND DESIGN OF NOVEL THIENOPYRIMIDINES AND TRIAZOLES Shyyka O.Ya., Pokhodylo N.T., Matiychuk V.S., Obushak M.D. Ivan Franko National University of Lviv, Kyryla i Mefodiya 6, Lviv 79005, Ukraine Substituted thienopyrimidines and triazoles are well-known for their diverse biological activity. Novel ring systems of thieno[3,2-e][1,2,3]triazolo[1,5-a]pyrimidines, thieno[2,3-e] [1,2,3]triazolo[1,5-a]pyrimidines and 5-amino-1-aryl-1H-1,2,3-triazole-4-carboxamides were obtained by base-catalyzed cycloaddition domino reactions of new heterocyclic 2-azidothiophenes, 3-azidothiophenes and other organic azides with activated methylene nitriles1,2. New polycyclic system of thieno[3,2-e]pyrazolo[1,5-a]pyrimidines was prepared via one-pot reaction of hydrazonoyl halides with activated acetonitriles1. Furthermore, the solvent-free reaction of thienotetrazoles with based N-nucleophiles under mild conditions afforded hitherto rare 2-R3,R4-amino-5-R1-6-R2thieno[2,3-d]pyrimidine-4(3Н)-ones and 2-R3,R4-amino-5-R1-6-R2-thieno[3,2-d]pyrimidine-4(3Н)ones with high yields3. Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Under this aim, in silico methods have proved to be a useful tool in drug discovery. Molecular docking is used to identify correct poses of ligands in the binding pocket of drug target proteins and to predict binding affinity. OpenEye Scientific Software package has been successfully used to perform molecular docking and identify the most active inhibitors of EGFR tyrosine kinase among synthesised thienopyrimidines. As a result, some promising anticancer drugs such as thienopyrimidines were identified and selected for further investigations in National Cancer Institute (USA).
References: 1. N.T. Pokhodylo, O.Ya. Shyyka, R.D. Savka, M.D. Obushak, Phosphorus, Sulfur, and Silicon and the Related Elements, 185, 10, 2092-2100 (2010). 2. N.T. Pokhodylo, V.S. Matiychuk, and M.D. Obushak, Tetrahedron, 65, 2678–2683 (2009). 3. N.T. Pokhodylo, V.S. Matiychuk, and M.D. Obushak, Tetrahedron, 64, 1430–1434 (2008).
93
2’-5’-OLIGOADENYLATE ALTERS SOME PROTEINS CONFORMATION Skorobogatov O. Yu.1, Kozlov O. V.1, Zhukov I.2,3, Tkachuk Z. Yu.1 1 Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine 150, Zabolotnogo Str., Kyiv - 143, Ukraine, 03680 2 Center of Excellence EN-FIST. Dunajska 156, 1000 Ljubljana, Slovenia 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences. Pawinskiego 5a, 02-106 Warsaw, Poland Dephosphorylated 2’-5’-oligoadenylates (2’-5’A) are low molecular weight cell activity mediators. They are known to have a wide range of biological activities, particularly inhibiting the reverse transcriptase, acting as protein kinase inhibitors, and regulating the topoisomerase I activity. The mechanisms, underlying these effects, are still being under investigation by scientists. It was suggested, that such influence can either be a result of protein conformation changes due to it’s interaction with 2’-5’A. The aim of current study was to estimate whether 2’-5’A affects the target proteins conformation or not. Fourier-transformed infrared spectroscopy (FTIR). FTIR spectra of calmodulin (CaM) and S100A1 Ca2+-binding proteins were recorded and then examined. After FTIR spectra were obtained, their further analysis let us postulate the presence of conformational changes in protein structure. In particular, amide I and II bands, which mainly correspond to C=O and N–H groups vibrations respectively, were of key interest for us, since these two are known to be the most sensitive sensors of protein structure changes. For both cases of CaM and S100A1 interacting with 2’-5’A, amide I and II bands shifts were detected: 3 cm-1 and 5 cm-1 blue shifts for pure CaM and CaM–2’-5’A complex, and 1 cm-1 and 4 cm-1 red shifts for pure S100A1 and S100A1–2’-5’A complex respectively. These data made it possible for us to postulate the occurrence of conformational changes in CaM and S100A1 structures due to complex formation with 2’-5’A. Since FTIR studies did not let us straightly identify the concrete amino acid residues interacting with 2’-5’A molecule, our efforts currently are being pointed toward applying methods, capable of giving more detailed information about complexes being studied.
94
SYNTHESIS OF POTENT TYROSINE KINASE INHIBITORS Smolii O.B., Kucher O.V., Muzychka L.V. Institute of Bioorganic Chemistry and Petroleum Chemistry NAS of Ukraine, 02094 Kyiv, Murmanska,1, Ukraine. Derivatives of рyrrolo[2,3-d]pyrimidine are known to be selective inhibitors of JAK3-kinase [1] and Akt-kinase [2]. Pyrido[2,3-d]pyrimidines can exhibit inhibitory activity towards Src and Abl tyrosine kinases [3]. New derivatives of condensed heterocyclic systems (II, III, IV) have been prepared from 4,6-dichloropyrimidine-5-carboxaldehyde (I). Compounds II, III, IV can be considered as scaffolds for design of potent tyrosine kinase inhibitors. O
Cl
H
N Cl
N I
Cl
NR1R2 OH
N N R
N II
O
Cl NH2
N N O
N
O
OH O
N
O
N
N H IV
III
[1] M.E. Flanagan et al. // J. Med. Chem., 2010, 53, 8468. [2] T. McHardy et al. // J. Med. Chem., 2010, 53, 2239. [3] C. Antczak et al. // Biorg. Med. Chem. Lett., 2009, 19, 6872.
95
O
SYNTHESIS AND SEPARATION OF BISSUBSTITUTED DERIVATIVES OF PENTACYCLO[6.3.02,6.03,10.05,9]UNDECANE 1
Solovyan A.A., Gayday A.V., Levandovskiy I.A., 2Shubina T.E. 1 National Technical University of Ukraine «KPI», 03056 Kyiv, Pobedu av.,37, Ukraine; 2 CCC, University Erlangen-Nuremberg, 91052, Germany, Erlangen, Nagelsbach str. 25. The leading area of modern organic chemistry is a synthesis of compounds with rigidly fixed substituents for examining their activity on various receptor sites. We have developed several methods to synthesize derivatives of D3-trishomocubane. The first step of synthesis of D3-trishomocubane halogen derivatives included obtaining haloalcohols and haloкetones. Haloalcohols 2-4 were synthesized from the Cs- trishomocubane-8,11-diol 1 and hydrohalic acids. Also the D3-trishomocubane-4,7-diol 5 and ether 6 were received as by-products. The chloroalcohol 2 also could be obtained by two other ways (Scheme 1). HX
H OH OH
H
105
0C
+
+
OH X
OH
O
HO 2 X= Cl 3 X=Br 4 X=I
1
5
HCl
6
AlCl3 OH
OH
O
Cl
Cl
6
2
2
Scheme 1. Synthesis of D3-trishomocubane halogen derivatives. Oxidation of halogen alcohols 2-4 by Jones reagent mixture of haloкetones 7-9 was obtained (Scheme 2). CrO3 OH
O
H2 SO4 X
X
7 X= Cl 8 X=Br 9 X=I
2 X= Cl 3 X=Br 4 X=I
Scheme 2. Synthesis of D3-trishomocubane halogen derivatives. Separation of 4-brom-D3-trishomocubane-7-ones 6 was performed by recrystallization from C6H14/EtOAc (7:3). Thus, endo-isomer was obtained as sole product and its structure was confirmed by X-ray analysis and COSY/NOESY spectra. The obtaining of 4-brom-D3-trishomocubane-7carboxylic acid included several steps such as synthesis of epoxides 10 and aldehydes 11. In the case of bromacids mixture formation we found the conditions for their separation using the recrystallization (Scheme 3). NaH Br H
O 8
BF 3.Et2 O O
(CH3 )3 SOI Br H
10
CrO3 O H 2SO4
Br H
H 11
Scheme 3. Synthesis of 7-bromo-D3-trishomocuban-4-carboxylic acid.
96
O
Br H
OH 12
COMPARATIVE ANALYSIS OF BINDING SITE CONFORMATIONS OF PTP1B Tanchuk V.Yu., Tanin V.O., Vovk A.I. Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02660, Kyiv-94, Ukraine, e-mail:
[email protected] Protein tyrosine phosphatase 1B (PTP1B) is one of the most promising therapeutic targets for potential treatment of type 2 diabetes and obesity. There is a great variety of crystal structures of this enzyme available in the RSCB Protein Data Bank. We have found 91 PDB files which include 102 chains of PTP1B. We think that in cases like this, where a substantial amount of crystallographic data exists, all enzyme active centre conformations should be clustered by their similarity and cluster centroids used as an ensemble of structures for further docking. The available conformations of PTP1B have been analyzed and clustered by a specially designed software tool ACTPDBCMP (Active Part of PDB Comparison), which finds a specified group of residues in PDB files, performs pairwise comparison of the obtained fragments, builds a distance matrix, finally performs hierarchical cluster analysis and determines the mobility of each residue.1 The program was used to analyze conformations of two fragments: i) 32 residues of catalytic and vicinal binding sites of PTP1B and WPD loop; ii) the same fragment and a 110-120 loop. It was found that the conformations can be divided into 5 clusters. The centroids of the clusters (1NL9, 1PH0, 2CNF, 1Q6M, 2CM8) form a representative ensemble of conformations that can be used for design of compounds applicable as potent and selective inhibitors of PTP1B. The difference between clusters is sometimes striking. It means that all types of binding (represented by clusters) should be tested. It has been also found that the loop 110-120 exists in 4 main conformations, one of which is prevailing. The other 3 conformations appear only in case of closed WPD loop, especially when the enzyme is represented by a dimeric structure. This research was supported by the Science and Technology Center in Ukraine (Project No. 5215). 1 V.Yu. Tanchuk, V.O. Tanin, A.I. Vovk. Chem. Biol. Drug Design, 2012 doi:10.1111/j.17470285.2012.01370.x
97
MODIFIED VERSION OF AUTODOCK TO DEAL WITH LARGE BINDING POCKETS AND ACCELERATE BINDING PROCESS Tanchuk V.Yu., Poyarkov A.A., Poyarkova S.A., Chmutov A.M., Prokopenko V.V. Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02660, Kyiv-94, Ukraine, e-mail:
[email protected] Investigation of a novel class of thrombin inhibitors resulted in a modified version of AutoDock. Docking studies were employed to position the inhibitors into the active site of thrombin to determine the probable binding conformation. Investigation of a large cavity of the active site of thrombin demonstrated the weaknesses of AutoDock 4.2 docking algorithm. Improvements include the ability to apply one or several spatial constraints on docking poses. In this case one or several atoms are positioned in some predefined areas. It might be very helpful if it is known which groups play important role in binding of inhibitors. These improvements also include the ability to make a sequential search where the best conformation is passed to the next run. iii) The ability to use charges calculated by MOPAC 2009 PM6 method which is known to improve binding poses. As a result it has been shown that the interaction of lengthy hydrophobic chains of the inhibitors with thrombin’s loop 60 plays an important role, which makes possible to explain high selectivity to thrombin of liporetro-D-peptides – a novel class of thrombin inhibitors published earlier. The new method of constrained docking that simplifies calculations and improves accuracy can be applied for other target enzymes. This research was supported by the Science and Technology Center in Ukraine (Project No. 5215).
98
QSAR MODELS FOR PREDICTION OF PTP1B INHIBITION BY STRUCTURALLY DIVERSE COMPOUNDS Tanchuk V.Yu., Tanin V.O., Vovk A.I. Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02660, Kyiv-94, Ukraine, e-mail:
[email protected] Intracellular protein tyrosine phosphatase 1B (PTP1B) is known to be implicated in insulin receptor dephosphorylation and considered a negative regulator of insulin signal transduction. Therefore PTP1B is one of the most promising therapeutic targets for potential treatment of type 2 diabetes and obesity. There is growing interest in developing potent and selective inhibitors of this enzyme. The goal of this investigation was to create a vast database of chemically diverse PTP1B inhibitors and build a publicly available QSAR model. We have collected 2237 compounds from more than 20 sources. A publicly available model1 is called “PTP1B inhibition (pI), 22336”. This is a simple regression model Y = 0.36137399 - 0.0027220813·ALogPS_logP - 0.0019107998·ALogPS_logS + 0.93312871·PTP1B inhibition(pI)(PTP1B inhibition(pI), 22334), where ALogPS_logP and ALogPS_logS are LogP and LogS values calculated by ALogPS, PTP1B inhibition(pI)(PTP1B inhibition(pI), 22334) is a prediction by another more complex model (PTP1B inhibition(pI)(PTP1B inhibition(pI), 22334). The later model (22334) has been built using 1902 descriptors calculated by Adriana, EState, ALogPS and Dragon packages. This is an ANN model trained by Supersab algorithm with 1000 iterations, 3 neurons, and 5-fold cross-validation. Parameters of this model are R2=0.70, q2=0.70, RMSE=0.69, MAE=0.50. The regression model built upon it (22336) is slightly better with R2=0.80, q2=0.80, RMSE=0.62, MAE=0.45. 1 The resulting dataset has been uploaded into online chemical database with modeling environment (OCHEM) located at http://ochem.eu with a name PTP1B.
99
PROTEIN TYROSINE PHOSPHATASE 1B INHIBITORS BASED ON A CALIXARENE SCAFFOLD 1
Trush V.V., 2Cherenok S.O., 1Tanchuk V.Yu., 2Kalchenko V.I., 1Kukhar V.P., 1Vovk A.I. 1 Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02660, Kyiv-94, Ukraine. E-mail:
[email protected]; 2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Murmanska, 5, 02660, Kyiv-94, Ukraine PTP1B is a member of superfamily of the protein tyrosine phosphatases, which are known to modulate phosphotyrosine-dependent signaling pathways. As a negative regulator, PTP1B may involve in insulin and leptin signal transduction. Because of the role of this enzyme in regulating type 2 diabetes and obesity, this enzyme is considered as a promising target for natural and synthetic inhibitors. As bioisosteric analogues of phosphotyrosine group, phosphonic acid derivatives may be potent inhibitors of PTP1B. The biological properties of synthetic phosphonic acids as well as the therapeutic potential of bisphosphonates generate interest in studying new bioactive compounds. We have already shown that preorganizing the phosphonic acid fragments on calix[4]arene platform provides a promising approach for the design of efficient inhibitors of Yersinia protein tyrosine phosphatase.1 In this study, we report for the first time the properties of calix[4]arene methylenebisphosphonic and hydroxymethylenebisphosphonic acids as inhibitors of PTP1B in vitro. The results demonstrated that methylenebisphosphonic acids based on a calixarene scaffold are competitive inhibitors of PTP1B with inhibition constants in the low micromolar range. The results indicate that some of them, especially hydroxymethylenebisphosphonic acids, are potent time-dependent inactivators of PTP1B. It was shown also that hydroxymethylenebisphosphonic acids were about 6- to 11-fold more potent inhibitors of PTP1B over TC-PTP. Methylenebisphosphonic acid derivatives showed about 4- to 5fold selectivity over TC-PTP. Binding models of the inhibitors with PTP1B were predicted by using AutoDock. These results point to the necessity for further investigation of the inhibitors of PTP1B based on a calixarene scaffold. The work was supported by the State Program Nanotechnologies and Nanomaterials through grants 5.18.2.8 (IBOPC) and 5.16.1.2/10 (IOC). 1 A.I. Vovk, L.A. Kononets, V.Yu. Tanchuk, S.O. Cherenok, A.B. Drapailo, V.I. Kalchenko, V.P. Kukhar.Bioorg. Med. Chem. Lett. 2010, 20, 483–487.
100
INHIBITION OF PROTEIN TYROSINE PHOSPHATASE 1B BY POLYCARBOXYLIC DERIVATIVES OF [60]FULLERENE 1
Trush V.V., 1Tanchuk V.Yu., 2Khakina E.A., 2Troshin P.A., 1Vovk A.I. 1 Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Murmanska, 1, 02660 Kyiv-94, Ukraine, E-mail:
[email protected]; 2 IPCP RAS, Semenov Prospect 1, Chernogolovka, 141432, Russia Protein tyrosine phosphatases catalyze dephosphorylation of phosphotyrosine residues in proteins and are involved in signal transduction pathways during cell growth, differentiation, movement, and other cellular processes. PTP1B negatively regulates insulin and leptin signalling by the dephosphorylation of the activated insulin receptor or leptin receptor-associated kinase, JAK2. Therefore, PTP1B is being considered as one of the promising target for new drug development to treat diabetes and obesity. With increasing understanding of the physiologic role of PTP1B in insulin and leptin signaling, there is growing interest in studying new inhibitors of this enzyme. Many unique fullerene-based compounds have been described to display an anticancer, antimicrobial and other biological activity which makes them a good candidate for the applications in therapy, drug delivery and diagnostics of human diseases. Recently, highly selective reactions of C60Cl6 with thiols have been used to synthesize bioactive water-soluble [60]fullerene derivatives bearing five carboxyethylthio or carboxydecylthio groups.1 According to this, we have investigated properties of the synthesized fullerene derivatives as inhibitors of PTP1B. We hypothesized that the preorganization of five fragments of carboxylic acid on the surface of the fullerene molecule would result in improved inhibitory activity, through their better coordination with positively charged amino acid residues near the active site of the enzyme. Under assay conditions in vitro with p-nitrophenylphosphate or phosphorylated polypeptide TRDIpYETDYYRK (pTyr1146) used as substrate, both fullerene compounds at high nanomolar concentrations inhibited the enzyme activity in a dose-dependent manner. Moreover, they showed high selectivity with respect to PTP1B thus discriminating it among other investigated PTPs, such as ТС-РТР, PTP-β and LAR-PTP. These results suggest that water-soluble fullerene derivatives might be useful in a new strategy for the design of potent and highly selective inhibitors of PTP1B. 1 E. A. Khakina, A. A. Yurkova, A. A. Peregudov, S. I. Troyanov, V.V. Trush, A. I. Vovk, A. V. Mumyatov, V. M. Martynenko, J. Balzarini, P.A. Troshin. Chem. Commun., 2012, doi:10.1039/c2cc32517a.
101
2-5-OLIGOADENYLATE AND ITS DERIVATES INTERACTION WITH SOME PROTEINS Tkachuk V.V.1, Tkachuk L.V.1, Dubey L.V.1 1 Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine 150, Zabolotnogo Str., Kyiv - 143, Ukraine, 03680 The process of protein-ligand binding is known to be easily determined by fluorescence spectra changes of either protein or ligand. Oligoadenylates do not possess their own fluorescence at room temperature, thus the binding was studied by protein fluorescence changes, caused by aromatic amino acid residues – tryptophan and tyrosine. Fluorescence spectroscopy essay was used to investigate human serum albumin (HSA), human interferon, human immunoglobulin G, calmodulin and insulin interactions with natural trimer 2’-5’-A3 and its derivates, particularly 3’-substituted 8aminoadenosine (2’-5’A3-NH2), inosine (2’-5’A3-ino), cordycepine (2’-5’A3-cord), epoxyadenosine (2’-5’-A3-epo), and thiophosphate (2’-5’-A3-thio). Experimental data showed that 2’-5’-A3, 2’-5’-A3-NH2, 2’-5’-A3-epo, 2’-5’-A3-ino, 2’-5’-A3-cord and 2’-5’-A3-thio have 55%, 67%, 45%, 40%, 38% and 37% quenching effect on HSA’s fluorescence respectively at 280 nm excitation wavelength. At 296 nm substantial quenching was reported only in case of interaction with 2’-5’-A3-NH2. Calmodulin’s fluorescence was drastically quenched after binding of 2’-5’-A3 (69%) and 2’-5’-A3-epo (55%) at 280 nm; and interferon fluorescence quenching value equaled 50% after interaction with 2’-5’-A3-NH2. In the case of insulin interaction with 2’-5’-A3-NH2 the qunching value equaled 87%, with 2’-5’-A3 – 42%, 2’-5’-A3-cord – 60%, with 2’-5’-A3-ino – 55%, and with 2’-5’-A3-epo – 46% at 280 nm. In case of immunoglobulin interacting with different 2’-5’-oligoadenylates, weak fluorescence quenching (57%) was observed at 280 and 296 nm respectively. It was shown, that the value of fluorescence quenching depended on 2’-5’-A3 sample structure and it decreased as 2’-5’-oligoadenylate concentration raised. Based on experimental data obtained, considering different fluorescence quenching efficiency of particular 2’-5’-oligoadenylates, the probable role of tyrosine and tryptophan in such interactions was suggested.
102
3,4-DYHIDROISOCOUMARIN-3-CARBOXAMIDES: SYNTHESIS AND BIOACTIVITY Turytsya V.V., Matiychuk V.S., Obushak M.D. Department of Organic Chemistry, Ivan Franko National University of Lviv, Kyryla i Mefodiya 6, Lviv 79005, Ukraine Biological activity of the 200 3,4-dihydroisocoumarin-3-carboxamides was investigated in silico using online-service PASS. Results revealed a prediction for 15 compounds with the highest probability of antimicrobial, antifungal, antiprotozoal, antiischemic and cardioprotective biological activity. These compounds showed high probability to be active index (Pa) in one or more types of biological activity (range 0.78–0.92) and low Pa in toxicity as well as low probability to be inactive index (Pi
