Abstracts
Gespeichert in:
| Datum: | 2013 |
|---|---|
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
2013
|
| Schriftenreihe: | Вiopolymers and Cell |
| Schlagworte: | |
| Online Zugang: | http://dspace.nbuv.gov.ua/handle/123456789/153700 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Abstracts / Вiopolymers and Cell. — 2013. — Т. 29, спецвипуск. — С. 1-30. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-153700 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1537002019-06-15T01:26:14Z Abstracts Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU 2013 Article Abstracts / Вiopolymers and Cell. — 2013. — Т. 29, спецвипуск. — С. 1-30. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/153700 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| spellingShingle |
Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU Abstracts Вiopolymers and Cell |
| format |
Article |
| title |
Abstracts |
| title_short |
Abstracts |
| title_full |
Abstracts |
| title_fullStr |
Abstracts |
| title_full_unstemmed |
Abstracts |
| title_sort |
abstracts |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2013 |
| topic_facet |
Materials of VII annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/153700 |
| citation_txt |
Abstracts / Вiopolymers and Cell. — 2013. — Т. 29, спецвипуск. — С. 1-30. — англ. |
| series |
Вiopolymers and Cell |
| first_indexed |
2025-07-14T05:11:25Z |
| last_indexed |
2025-07-14T05:11:25Z |
| _version_ |
1837597863535706112 |
| fulltext |
1
Local conformational changes and hydrophobic sites formation in
eukaryotic tyrosyl-tRNA synthetase studied by fluorescence probes and
computational modeling
L. O. Andriichuk., V. V. Mykuliak, A. I.Kornelyuk
andriychuck.lilia@yandex.ru
Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein
biosynthesis, which catalyzes the specific aminoacylation of homologous
tRNATyr. Local conformational changes of TyrRS contribute to the enzyme
functioning, but their nature and specific role are not studied yet. The first stage
of protein folding involves the formation of the molten globule state, but the
physiological significance of such conformation is not fully understood.
Usually, the molten globule state is characterized by higher exposure of
hydrophobic regions, which allows to monitor this state by fluorescent probes.
Аim of this work was to study the local conformational changes in the N-
terminal catalytic module of TyrRS (mini-TyrRS) and possible formation of
molten globule state by fluorescent probes binding.
Methods. Recombinant proteins were obtained by bacterial expression in
E.coli BL21(DE3)pLysE by standard methods. The binding of fluorescent
probes 1,8-ANS and bis-ANS by mini-TyrRS were performed in order to
monitor the formation of hydrophobic regions in the protein in the temperature
range of 25–42ºC. Computational modeling of the binding of 1,8-ANS and bis-
ANS on the surface of Hs TyrRS was performed with AutoDock Vina program.
Results. The interaction of 1,8-ANS and bis-ANS fluorescent probes with
TyrRS resulted in the significant increase of fluorescence intensity due to the
binding to hydrophobic sites on the protein surface. The unstable bis-ANS
binding with mini-TyrRS was detected at 25 ºC, but the temperature increase
resulted in a more stable dye binding. This indicates the formation of
nonspecific hydrophobic sites on the protein surface at 25ºC temperature.
Hence, increasing of the temperature to 42◦C induced conformational changes,
which lead to the formation of specific bis-ANS binding sites on TyrRS
surface. Computer simulation of molecular docking of 1,8-ANS and bis-ANS to
the HsTyrRS revealed several binding sites, one of which is common for the
both dyes. The common region was formed by Arg16, Lys265, Phe269,
Tpr290, Tyr292, and the Ser273, Ala291, Val293 for bis-ANS only. The energy
of interactions of 1.8-ANS and bis-ANS to this hydrophobic region of TyrRS
was about -6.1 and -5.7 kcal/mol, respectively.
Conclusion. Temperature-induced local conformational changes of TyrRS
were studied by 1,8-ANS and bis-ANS fluorescent probes binding. The
difference in the affinity and binding sites numbers on the mini-TyrRS surface
was observed in the 25-42ºC temperature range. Therefore, 1,8-ANS and bis-
ANS are sensitive fluorescent probes in order to detect the formation of
hydrophobic sites and possible molten globule state in proteins, especially at the
neurodegenerative diseases.
2
Structural and energetic properties of the four biologically important
configurations of the А·Т and G·C DNA base pairs: comparative quantum-
chemical analysis
O. O. Brovarets’, D. M. Hovorun
“State Key Laboratory of Molecular and Cell Biology”
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
Institute of High Technologies, Taras Shevchenko National University of Kyiv
brovarets@list.ru
Aim. The main purpose of this work is to show and quantum-chemically
substantiate that a necessary and sufficient condition for the transition of the G·C
Watson-Crick (WC) base pair into the standard biologically important
configurations, characteristic for the А·Т base pair, namely – Hoogsteen (H),
reverse Hoogsteen (rH), WC and reverse WC (rWC), is its tautomerisation into
the G*·C* Löwdin’s (L) base pair with WC geometry formed by mutagenic
tautomers. Methods. Quantum-chemical modeling at the MP2/6-
311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory in the free state (ε = 1)
and in the continuum with a low dielectric constant (ε = 4) corresponding to
hydrophobic interfaces of protein–nucleic acid interactions. Results. It was
established for the first time, that H, rH, WC and rWC configurations of the A·T
and G·C DNA base pairs are isoelectronic and isomorphic structures with
similar dynamic properties. Based on these results, a non-ionisation mechanism
of the Hoogsteen "breathing" of the G·C DNA base pair, namely transformation
of the tautomerised G*·C* L base pair with WC geometry, stabilized by the
three O6H…N4, N3H…N1 and N2H…O2 H-bonds, into the electroneutral
G*·C* H base pair, stabilized by the three O6H…N4, N3H…N7 and C8H…O2
H-bonds, has been postulated. It is suggested that such scenario is activated only
in those cases, when DNA is not located in aqueous solution, but operates
together with proteins, and cytosine protonation at the N3 atom is precluded.
Moreover, it was shown for the first time, that the G*·C* L DNA base pair
can acquire as the A·T WC DNA base pair four biologically important
configurations, namely – WC, rWC, H and rH. This fact demonstrates rather
unexpected role of the tautomerisation of one of the WC DNA base pairs, in
particular, via double proton transfer: exactly the G·C→G*·C* tautomerisation
allows to overcome steric hindrances for the implementation of the above
mentioned configurations. Geometric, electron-topological and energetic
properties of the H-bonds that stabilise the studied base pairs, as well as the
energetic characteristics of the latters are presented. Conclusions. This study has
shown for the first time, that the G*·C* L DNA base pair can acquire as the A·T
WC DNA base pair four biologically important configurations, namely – WC,
rWC, H and rH. Based on these results, the tautomeric mechanism of the
Hoogsteen “breathing” [1], established experimentally [1,1], was suggested.
1. Frank-Kamenetskii M.D. // Artificial DNA: PNA & XNA. – 2011. – 2, N 1. – P. 1-3. Nikolova
E.N. et al. // Nature. – 2011. – 470, N 7335. – P. 498-502. Nikolova E.N. et al. // J. Am. Chem. Soc. –
2013. – 135, N 18. – P. 6766-6769.
3
Design, synthesis and evaluation of 2-phenylisothiazolidin-3-One-1,1-
Dioxides as a new class of human protein kinase CK2 inhibitors
M. O. Chekanov, A. R. Synyugin, O. V. Ostrynska and S. M. Yarmoluk
chekanov_maxx@mail.ru
Aim: The main goal of this work was identification of 2-
phenylisothiazolidin-3-one-1,1-dioxides as a new class of human protein kinase
CK2 inhibitors.
Methods: In order to reach that goal we used molecular modeling approach
in combination with combinatorial synthesis and high throughput screening.
Results: We have described the synthesis and in vitro evaluation of 64 new
2-phenylisothiazolidin-3-one-1,1-dioxide derivatives as a new class of CK2
inhibitors. We have identified this class of compounds using flexible docking of
the virtual library consisted of 1037 members [1]. The optimization based on
biological screening data and molecular modeling led to the identification of 3-
{[2-chloro-4-(1,1-dioxido-3-oxoisothiazolidin-2-yl)benzoyl]amino}benzoic
acid, a potent inhibitor of human protein kinase CK2 (ІС50 = 1.5 μМ).
Its binding selectivity was checked with the usage of two serine/threonine
(ASK1, Aurora А1) and one tyrosine protein kinase (FGFR1). It was found that
3-{[2-chloro-4-(1,1-dioxido-3-oxoisothiazolidin-2-yl)benzoyl]amino}benzoic
acid in vitro exhibited selectivity towards inhibition of СК2.
Using the biochemical tests and molecular docking we have investigated the
relationship “chemical structure-biological activity” (SAR) of 2-
phenylisothiazolidin-3-one-1,1-dioxides. As a result, the binding mode of the
synthesized compounds with CK2 ATP-acceptor site has been proposed.
Conclusions: Hereby, we have identified 2-phenylisothiazolidin-3-one-1,1-
dioxides as a new class of human protein kinase CK2 inhibitors using molecular
modeling, combinatorial synthesis and high throughput screening techniques.
Keywords: 2-phenylisothiazolidin-3-one-1,1-dioxides, CK2 inhibitors,
Molecular Docking.
1. Chekanov MO, Synyugin АR, Lukashov SS. Yarmoluk SM. The synthesis of 2-
phenylisothiazolidine-3-one dioxide derivatives as inhibitors of protein kinase CK2. Ukr
Bioorg Acta 2009;7,1:62-64.
4
Comparative conformational analysis of 2'-deoxyguanosine and 2'-
deoxyadenosine with their oxidized analogs: a quantum chemical study
K. Gavrilyuk, O. Zhurakivsky, D. M.Hovorun
gavrilyuk.katarina@gmail.com
Aim. To compare conformational ability of dG and dA and their eight
position oxidized nucleosides (8-Oxo-7,8-dihydro-2'-deoxyguanosine, 8oxodG
and 2'-deoxy-8-oxo-adenosine, 8oxodA), thus, to answer a biologically
important question “Could oxidized bases in comparison with non-modified
easier become DNA-like syn-conformations?”
Methods. The methods of the quantum-chemical density functional method
on the MP2/6-311 + + G (d, p) / / DFP B3LYP/6-31(d, p) theory level in
isolated state (ε=1) were used in this work.
Results. The complete conformational family of 8oxodG containing 95
conformers with relative Gibbs energy lying in the range of 0-8, 56 kcal / mol
and the family of 8oxodA consisting 87 structures with 8.74 kcal / mol under
standard conditions were shown for the first time. Comprehensive structural
features of conformers and characteristics of intramolecular H-bones stabilizing
the conformers are presented. 8oxodG and 8oxodA data were compared with
similar data for dG and dA for the first time.
Conclusions. Oxidation of dG and dA at the C-8 position triggering their
lightweight ability to become syn conformated in comparison with canonical
nucleosides was shown for the first time. These results are useful to explain the
structural mechanism of mutagenic action at eight position oxidation of purine
bases of DNA.
5
In silico design of protein kinase FGFR1 inhibitors as potential anti cancer
drugs
A. A. Gryshchenko, V. G. Bdzhola, А. О. Balanda, S. S. Lukashov,
T. P. Ruban, L. L. Lukash, S. M. Yarmoluk
Institute of Molecular Biology and Genetics NAS of Ukraine
a.a.grischenko@imbg.org.ua
Aim. Fibroblast Grow Factor Receptor 1 (FGFR1) tyrosine kinase plays an
important role in cell proliferation and angiogenesis control. Enhanced protein
kinase activity due to activating mutations or overexpression has been
implicated in several angiogenic pathologies and different cancers. Therefore,
discovery of inhibitors of FGFR1 kinase has substantial potential therapeutic
value. The aim of the present study is search for FGFR1 inhibitors with
antiproliferative activity, which can be used for anticancer drug development.
Methods. Receptor-based virtual screening of 100000 chemical compounds
collection was used to search for novel FGFR1 inhibitors. Docking of the
compound collection was performed with program Autodock. Consensus
scoring, checking of hydrogen bonding with a kinase hinge region and visual
inspection were used for docking results filtering. In vitro inhibition tests were
carried out with γ-32P-АТP assay. The antiproliferative activity was measured
by MTT assay.
Results. From virtual screening several novel FGFR1 inhibitors were found.
This work presents the results of testing 4 inhibitors classes. The most active
compound among 4-phenyl-1H,4H-pyrano[2,3-c]pyrazoles was 4-[4-(1,3-
benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]-6-propylbenzene-1,3-diol (1)
with IC50 8.5 µM. The most active compound among thieno[2,3-b]pyridines
was 3-amino-6-cyclopropyl-4-(thiophen-2-yl)thieno[2,3-b]pyridine-2-carbox-
amide (2) with IC50 1.8 µM. The most active compound among 4-phenyl-
1H,4H-pyrano[2,3-c]pyrazoles was 6-amino-4-(2,5-dimethoxyphenyl)-3-me-
thyl-1H,4H-pyrano[2,3-c]pyrazole-5-carbonitrile (3) with IC50 2.5 µM. The
most active compound among (2Z)-3-(3-phenyl-1H-pyrazol-4-yl)prop-2-
enenitriles (2E)-2-(1,3-benzothiazol-2-yl)-3-(3-phenyl-1H-pyrazol-4-yl)prop-2-
enenitrile (4) with IC50 1.8 µM. Compounds 4-[4-(1,3-benzothiazol-2-yl)-5-
(trifluoromethyl)-1H-pyrazol-3-yl]benzene-1,3-diol (5), 3-amino-6-(4-chloro-
phenyl)-4-(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (6), 3 and 4
were tested on lung cancer cell line A549 for antiproliferative activity. All
tested compounds inhibited proliferation, the most active compound was 6.
Conclusions. In this study four new classes inhibitors of protein kinase
FGFR1 with antiproliferative activity were found. The identified classes can be
used for further development of FGFR1 inhibitors and anticancer drugs.
6
Role of PH domain of BCR/ABL fusion protein in cytoskeleton remodeling.
D. S. Gurianov, I. V. Kravchuk, O. V. Maliuta, M. V. Dybkov, G. D. Telegeev
dmitriy.gurianov@gmail.com
Background: Reciprocal translocation between short arms of 9 and 22
chromosomes leads to generation of fusion gene BCR-ABL. To date, three
forms of such fusion genes were identified and named according to molecular
weight of their products: p185, p210, and p230. Difference in molecular
weights is due to the absence or presence of certain domains of BCR protein,
such as Pleckstrin-homology (PH) and Dbl-homology (DH) domains. The
different forms of BCR-ABL protein are associated with different
myeloproliferative disorders: p185 – with acute lymphoblastic leukemia, p210 –
with chronic myelogenous leukemia, and p230 – with true thrombocytopenia. A
shorter form of BCR-ABL leads to a more aggressive phenotype of disorder,
while p210 and p230 lead to more mild and slow development. Therefore, it is
important to determine the role of above mentioned domains in signaling and
regulatory cellular pathways.
According to the earlier research of Miroshnichenko D. et. al. 23 protein-
protein interactions were identified between DH-PH part of BCR and various
cellular proteins of K562 cell line. Among them, several may be grouped by
functional similarity. For instance, cortactin (CTTN), cytokeratin type 10
(KRT10), tubulin beta 1 chain (TUBB1), and Collagen IV alpha-1 polypeptide
(COL4A1) are involved in cytoskeletal remodeling, membrane signaling, and
cell adhesion. As BCR-ABL p210 is associated with abnormal cytoskeletal
behavior, altered adhesion, and localization on cell cortex, a role of PH domain
in these processes should be determined and previous data on protein-protein
interactions verified by more sensitive methods. Aim. To determine cellular
localization and protein-protein interactions of PH domain of BCR protein with
COL4A1, CTTN, KRT10, and TUBB1 proteins. Methods. pCMV-SPORT6-
based constructs with full coding sequences of COL4A1, KRT10, and TUBB1
were acquired from Open Biosystems. Construct with full CTTN sequence was
kindly provided by Pontus Aspenstrom (Karolinska Institute, Sweden). CTTN
and TUBB1 were amplified by PCR with specific primers and ligated to
pBluescriptSKII(+) on EcoRI-BamHI and EcoRV sites, respectively. Further,
CTTN was subligated to pGEX4T2 on EcoRI-NotI, to pCMV-myc on EcoRI-
BglII, and pECFP-C3 on EcoRI-BamHI. TUBB1 was subligated to pGEX4T3
on SalI-NotI, pcDNA4HisMaxC on EcoRV-NotI, and pECFP-C3 on HindIII-
EcoRI. Coding sequence of COL4A1 was cut from pCMVSPORT6 on AscI-
HindIII and ligated to pECFP-C1 on XhoI(blunted)-HindIII sites. KRT10 was
subligated from pCMVSPORT6 on EcoRI-NotI to pGEX4T2 and pCMV-HA,
and on EcoRI-SalI to pECFP-C3. Results. The genetic constructs for bacterial
and mammalian expression were created and verified. Conclusions. The
derived vectors are suitable for further protein production and consequent
protein-protein interaction analysis by far-Western blot, Coimmuno-
precipitation and FRET.
7
Interconnection between functional activity and subcellular localization of
mTOR kinase
V. Kosach, A. Khoruzhenko, O. Cherednyk, V. Filonenko
kosach.viktoriia@gmail.com
Introduction. The mammalian target of rapamycin (mTOR) is a highly
conserved serine/threonine kinase. It is a member of several signal transduction
pathways, but the most studied one is PI3K/Akt/mTOR signaling network.
mTOR takes part in the regulation of translation initiation, autophagy, ribosome
biogenesis, transcription, actin reorganization, cell-cycle progression, and cell
survival. mTOR hyperactivation was detected in a number of tumors, namely
the carcinomas of thyroid gland, breast cancer, prostate and stomach
malignancies. Moreover, mTOR inhibitors are regarded as anti-cancer drugs.
Aim. The main goal of our work is to investigate the role of mTOR subcellular
localization in functioning of mTOR signal transduction pathway in normal and
malignant human breast tissues, and in cultured MCF-7 cells.
Methods. The advantage of our research is the use of several types of anti-
mTOR antibodies, generated to different epitopes of mTOR molecule. In our
laboratory antibodies to the N-terminal part, C-terminal part and central region
of mTOR kinase were produced and tested by western blot, and
immunocytochemistry in the presence of mTOR antigenic polypeptide.
Subcellular localization was investigated by double immunofluorescent analysis
and confocal microscopy. For more careful visualization of the nucleoli
immunonucleochemistry was applied.
Results. The immunochemical reaction revealed predominantly cytoplasmic
localization of mTOR. A more sensitive immunofluorescent analysis allowed
detecting additional nucleolar localization of mTOR, which was confirmed by
colocalization with ribosomal S6 protein in the MCF-7 cells and malignant
human breast tissue, but not in the normal samples. The antibodies to the N-
terminal part of kinase detected fibrils-like structure. That is why, co-
localization with tubulin, actin, and cytokeratins was examined, and, as a result,
strong co-localization with cytokeratins was observed in MCF-7 cells, normal
and malignant breast tissue.
Conclusions. The data about the subcellular localization of mTOR kinase in
mammalian cells are incomplete and contradictory. In our project we showed
cytoplasmic localization of mTOR that is in accordance with the results of other
authors. However, for the first time we revealed strong co-localization of
mTOR and cytokeratins in human breast cells, and presence of mTOR in the
nucleoli of malignant breast tissue and MCF-7 cells. It could be explained by
the existence of several splicing isoforms of mTOR kinase. Further research
will help to verify this hypothesis and broaden our knowledge about mTOR
signal transduction pathway.
8
Investigation of protein-protein interactions of the adaptor protein TKS4
V. O. Kryklyva1, S. V. Kropyvko1, T. A. Gryaznova1, L. O. Tsyba1,
L. B. Drobot2, A. V. Rynditch1
1 – Institute of Molecular Biology and Genetics, NAS of Ukraine
2 – Palladin Institute of Biochemistry, NAS of Ukraine
valentyna_kryklyva@ukr.net
The adaptor protein Tks4 is a key protein of invadopodia and podosomes –
actin-rich membrane structures that facilitate cell movement and extracellular
matrix degradation and lead to cell invasion. The aim of our research was to
identify new partners of Tks4 via testing its interaction with potential partner
proteins of the intersectin protein family (ITSN), reorganizators of the actin
cytoskeleton neural Wiskott-Aldrich syndrome protein (N-WASP), and the
verprolin protein family as well as with adaptor protein Tks5.
Methods. The cDNA fragments encoding the SH3 domains of Tks4 were
cloned into the vector for bacterial expression pGEX-4T-2. The recombinant
GST-fused SH3 domains of ITSN1, ITSN2 and Tks4 were expressed in E.coli
XL1Bluetet and purified by affinity chromatography. Interactions of the SH3
domains with proteins overexpressed in HEK293 cells were analyzed using
GST pull-down assay and Western blotting.
Results. We showed that the SH3A, C and E domains of ITSN1 and ITSN2
bind Tks4. The SH3 domains of ITSN effectively precipitated with a wild type
Tks4 and a mutant for the three sites of phosphorylation. The results of pull-
down experiments demonstrated that four SH3 domains of Tks4 bound weakly
to N-WASP, known component of invadopodia and podosomes. Among the
proteins of verprolin family only CR16 (Corticoid Regulated) interacted with
SH3_2 and SH3_3 domains of Tks4. No interaction was observed in pull-down
assays with GST-fused SH3 domains of Tks4, overexpressed WIP (WASP
Interacting Protein) and WIRE (WIP-related).
Tks4 contains several proline-rich motifs. We have found that the SH3
domains of Tks4 could interact with its proline-rich motifs and therefore
mediate inter- or intramolecular interactions of Tks4 molecules. It was also
shown that the SH3 domains of Tks4 bind another member of the Tks protein
family, Tks5. It may be supposed that these interactions can regulate activity of
Tks proteins.
Conclusions. The adaptor protein Tks4 is crucial for the formation of
invadopodia – key structures for cancer invasion. We have shown that Tks4
interacts with scaffolding proteins ITSN1, ITSN2, Tks5 as well as with
reorganizators of the actin cytoskeleton N-WASP and CR16.
This work was partially supported by Joint Project of NAS of Ukraine and
Russian Foundation for Basic Research (16-04-12U) and State Fund for
Fundamental Researches (Project F33.4/001).
9
Нematopoietic progenitor cells from native and cryopreserved placental
tissue and umbilical cord blood: comparative analysis
M. Kuchma1,2, V. Shablii1,2, V. Kyryk3, Anya Onishchenko2, L. Lukash1,
G. Lobitseva2
1Institute of Molecular Biology and Genetics, NAS of Ukraine
2Institute of Cell Therapy,
3State Institute of Genetics and Regenerative Medicine, NAMS of Ukraine
k.maria@ua.fm
Objectives. The main goal of our study was to show the possibility of
obtaining viable hematopoietic progenitor cells (HPCs) from native and
cryopreserved placental tissue and compare their phenotype with phenotype of
HPCs from umbilical cord blood.
Materials. The tissue of human term placenta was cryopreserved by
conventional methods. Native and cryopreserved placental tissues were
enzymatically digested and cells harvested for FACS analyses. FACS was
performed with the next list of antibodies: anti-CD34, anti-CD45, anti-
CD45RA, anti-CD90, anti-CD31, anti-CD235, anti-CD7, anti-CD19, anti-СD33
(all from BD, USA). CFU analysis was performed with culture medium
MethoCult (StemCell Tech., Canada).
Results. The cells populations with phenotype CD34lowCD45low and
CD34hiCD45low were present in placental tissue. The level of CD90 expression
on HPCs from placental tissue was significantly higher than on HPC from cord
blood namely 24.5 % (6,2-49,8) and 2.2 % (0,08-10,5) respectively. The
expression of CD31 on HPCs from placental tissue was higher comparing to
HPCs from cord blood. We demonstrated some differences in the percentage of
micropopulations of erythroid, lymphoid and myeloid progenitor cells derived
from placental tissue and cord blood. In contrast we didn’t observe difference in
the of ratio granulocyte-monocyte, monocyte, granulocyte and erythroid
colonies formations in cell culture of HPCs from umbilical cord blood and
placental tissue. Furthermore we obtained viable HPCs from cryopreserved
placental tissue. The content of the HPCs (CD34+CD45lowSSClow) among all
CD45+ cells of native and cryopreserved placental tissue were 0,66 % (0,36-
1,05) and 1,11 % (0,18-2,82) respectively. CFU analysis of cells revealed the
presence in cryopreserved placental tissues of precursors of granulocytes,
monocytes and erythrocytes.
Conclusion: The placental tissue contains HPCs that differ in expression of
some markers comparing with HPCs from cord blood. Cryopreservation of
placental tissue is prospective method for preservation of HPCs.
10
Temporal gene expression profiling in rat hepatocytes treated with quasi-
physiological dose of interferon alpha
A. Kuklin 1, B. Tokovenko 1, N. Makogon 2, B. Jarząb 3, M. Obolenskaya
1Institute of Molecular Biology and Genetics, NAS of Ukraine
2Institute of Physiology, NAS of Ukraine
3Marie Skłodowska-Curie Cancer Center and Institute of Oncology, Gliwice, Poland.
av.kuklin@gmail.com
Background. Interferon alpha (IFNα) is a pleiotropic multifunctional
cytokine that has a key role in early immune events. It is produced at the
minimal level in healthy liver and up-regulated by viral RNA, LPS and
damage-associated molecules. Knowing how a cell responds to IFNα in
dynamic is important for understanding its function. Herein for the first time we
examined the gene expression profile in primary rat hepatocytes treated during
two time periods - 3 and 6 hours, with quasi-physiological dose of IFNα similar
to that locally produced in regenerating rat liver after partial hepatectomy.
Methods. We incubated primary rat hepatocytes with 250u/ml of rat IFNα.
We used gene expression profiling with Affymetrix rat genome array 230 2.0
(Affymetrix, USA) followed by computational analysis of promoter regions of
differentially expressed genes to identify the signaling pathways that are
engaged by IFNα.
Results. We identified 28 and 124 differentially expressed up-regulated
genes which reveal the cell-specific traits of response and distinctive dynamics.
From the beginning the up-regulation of genes responsible for autophagy,
ISGylation, inhibition of translation and transition of mitochondrial dNTP
synthesis to salvage pathway and chemokine CXCL10 production comes to the
fore. Altogether they manifest the transit from the initial mode of cellular
activity to the resource-conserving one, the usage of preexisting proteins and
the attraction of the cells of putative hepatic environment for the development
of response. The later changes are associated with the expansion of spectrum of
differentially expressed genes, increasing magnitude of response and emerging
interplay between activating and inhibiting factors which regulate the signal
transduction, transcription and the activity of putative cells of hepatic
environment. The classical Jak/STAT/ISGF3, Jak/STAT, PI3K and p38
signaling pathways are engaged in IFNα induced response of hepatocytes.
Conclusion. The short-term treatment of hepatocytes with quasi-
physiological dose of IFNα induces the self-autonomous changes in hepatocytes
and those referring to the hepatocytes potential input to the whole hepatic
response. The interplay between activating and inhibiting factors of immune
response is a characteristic feature of hepatocytes reaction to specified IFNα
treatment.
11
Morphological and growth properties of human cell line 4bl and its stem
potential
V. O. Kushniruk, S. P. Shpilevaya, L. L. Lukash
kushniruk_v_o@ukr.net
Morphology of cells and growth properties are important characteristics of
cell line, so the aim of this research was to study these peculiarities of the cell
line 4BL. Materials and methods. We investigated the original cell
line 4BL, obtained from peripheral blood of healthy donor, which has
successfully passed through the Heyflick limit and was cultivated for more
than 220 passages. For morphological studies cells were vitally stained by
neutral red. To analyze the growth properties cells were seeded on 10 cultural
flasks with 105 cells on each. Every 24 hours, the cells were removed from the
surface of the flask and the total number of cells was calculated. Stem potential
of the cell line 4BL to differentiate into adipogenic, osteogenic and myogenic
types was investigated by growing cells on specific induction mediums.
Results. The cell line 4BL consists of two main morphological types of cell:
fibroblast-like and epithelioid cells. Growth curves are characterized by almost
complete absence of lag-phase, double-step log-phase and double-step decline
phase with the formation of two peaks with maximum number of cells that
resembles the damped oscillations.
The cell line 4BL maintains original cytoarchitectonics in culture at different
passages: the cells are non-random placed on the surface of the culture vessels
and often form circular and half-circular cellular associations. The latter fact
may indicate the stem potential of cell line, which was confirmed by induction
of cells to adipogenic, osteogenic and myogenic differentiation.
Conclusions. Regardless the number of passages, fibroblast-like and
epithelioid cells are two main morphological types of the cell line 4BL,
remaining its ability to form cycle structures at the surface of culture vessels.
Growth curves have graded character. The cell line 4BL has stem potential due
to its ability to differentiate in adipogenic, osteogenic and myogenic types.
12
Green fluorescent protein chimeric mouse model
T. M. Lutsenko, O. V. Okunev, T. P. Gulko, M. V. Dragulyan,
A. U. Zhyvolozhnyi, V .A. Kordium
tamaralutsenko@hotmail.com
The term “microchimerism” (Mc) indicates the coexistence, in the same
organism, of genetically distinct populations of cells or DNA derived from
different individuals. This commonly occurs in patients with transplantations or
transfusions and termed iatrogenic Mc. Naturally there is bi-directional transfer
of cells between mother and child during pregnancy, and the presence of a
mother’s cells in her child and “vice versa” has been termed maternal-fetus Mc.
This phenomenon may play an important role in autoimmune diseases, cancer
and tissue repair.
Aims: The question of sensitive methods of Mc determination and
monitoring after transplantation/transfusion is still important. The biological
significance of harboring Mc in the "host" organism requires further
investigation. Human studies are often limited by the number of samples for
analysis. In this case chimeric mouse model is an important tool in biological
research. We have observed that Green fluorescent protein (GFP)-based
detection used as a reporter for patterns of gene expression and the origin of
cells in transplantation experiments.
Methods: We used FVB.Cg-Tg(CAG-EGFP)B5Nagy/J – transgenic mouse
strain that will allow us to analyze foreign cells in recipient organism using
fluorescent microscope, flow cytometry and western-blot analysis. The method
based on PCR analysis for determination of DNA that codes for the target
protein in mouse strain was also used.
Results: The GFP transgenic (Tg) FVB.Cg-Tg(CAG-EGFP)B5Nagy/J
mouse strain as donor was performed with recipient Balb/c mouse strain. Graft
survival of the donor-derived GFP(+) cells was analyzed by flow cytometry.
The western-blot analysis was used for the GFP gene expression.
Conclusions: The GFP-based detection technology can be used to locate and
quantify Mc cells in mouse model after transplantation/transfusion and during
pregnancy. So in our investigations FVB.Cg-Tg(CAG-EGFP)B5Nagy/J mouse
strain was used as universal donor of foreign material in GFP chimeric mouse
model. Such animal model is suitable for further studies of Mc.
13
Conformational adaptations of catalytic loop in complexes of M.
tuberculosis tyrosyl-tRNA synthetase with different substrates according to
the molecular dynamics
V. Mykuliak1,2 and A. Kornelyuk 1,2
1Institute of High Technologies, Taras Shevchenko National University of Kyiv
2Department of Protein Engineering and Bioinformatics,
Institute of Molecular Biology and Genetics, NAS of Ukraine
v.mykuliak@gmail.com
Tyrosyl-tRNA synthetase from M. tuberculosis (MtTyrRS) is an enzyme that
belongs to class I of aminoacyl-tRNA synthetases, that catalyzes the attachment
of tyrosine to cognate tRNATyr at the preribosomal protein synthesis step.
MtTyrRS is not able to cross-recognition and aminoacylation of human
cytoplasmic tRNATyr, therefore this enzyme may be a promising target for
development of novel selective inhibitors as new antituberculosis drugs.
Methods: In order to study the conformational mobility of MtTyrRS active
center we have performed 100 ns molecular dynamics (MD) simulations of
pure dimer of the enzyme and its complexes with different substrates.
Crystalline 3D structure of MtTyrRS dimer (PDB code 2JAN) was used to
complete the missing amino acid residues at the A-subunit (M1-M4 and G424)
and B-subunit (M1-M4 and P81-D93) by ModLoop server. The tyrosine in the
MtTyrRS active center was obtained by superposition with crystal E.coli TyrRS
(1X8X), ATP - T. thermophilus TyrRS (1H3E), tyrosyl-adenylate - E.coli
TyrRS (1VBM), and inhibitor SB219383 - S. aureus TyrRS (1JII). All MD
simulations of MtTyrRS dimer were performed at 310 K using GROMACS
program package with the AMBER99SB-ILDN force field. MD simulations
were calculated at the Ukrainian National Grid clusters using the services of
MolDynGrid virtual laboratory (http://moldyngrid.org).
Results: It was found that in the course of MD the KMSKS catalytic loop
fluctuates between open and semi-open conformations in unliganded state and
in complex with the tyrosine. With the ATP in the active site the loop adopts
semi-open conformation interacting with the ligand. In the presence of larger
tyrosyl-adenylate or SB-219383 inhibitor the loop adopts the conformations
more similar to the closed one. Our results are in good correlation with the
previous work devoted to S. aureus TyrRS [Li et.al., 2008].
Conclusion: Fluctuations of MtTyrRS with different substrates studied by
MD simulations show conformational adaptations of the KMSKS catalytic loop
depending on a ligand in the active center.
14
Epigenetic markers in plasma of colorectal cancer patients
A. G. Kondratov, K. Nekrasov, L. V. Lototska, G. V. Panasenko, L. A. Stoliar,
O. O. Kolesnyk, I. B. Shchepotin, A. V. Rynditch and V. I. Kashuba
kanekrasov@gmail.com
Colorectal cancer (CRC) is the third commonly diagnosed cancer that causes
400 000 deaths worldwide. The most sensitive modern diagnostic tool of CRC
is colonoscopy which is painful procedure and can not be recommended for
petient with altered topography of colon. Thus, development of less invasive
tools for screening of CRC is actual problem of the modern oncology.
To detect concentration of cell free circulating DNA in plasma the qRT-PCR
was used. Methylation status of LRRC3B, APC, and FHIT genes in cell free
circulating DNA of samples was determined by using methyl-specific PCR with
subsequent melting curve analysis.
It was shown that mean-value of plasma DNA is statistically higher in CRC
patients than healthy donors (p < 0.01). Thus the mean-value concentration of
cell free circulating DNA in plasma of CRC patients was 17.57 ± 3.43 ng/mL
whereas 7.07 ± 0.84 ng/mL for healthy donors. Accordingly with it the upper
cut-off value of the free circulating DNA concentration was 17.74 ng/mL of
plasma of healthy donors. Therefore there were 8 out of 22 samples of CRC
which fell into criteria as samples with abnormally increased DNA level.
We have revealed hypermethylation of APC, FHIT and LRRC3B genes in in
48 % (10/21), 71 % (15/21) and 67 % (14/21) of tumor samples,
correspondingly. Altogether hypermethylation at least one of the selected genes
was detected in 95 % (20/21) of samples. Using MSP with subsequent melting
curve analysis we have previously detected methylated fragments of APC,
FHIT and LRRC3B genes in plasma of 19 % (4/21), 29 % (6/21) and 14 %
(3/21) of CRC patients, respectively. We have identified hypermethylation at
least in one of the selected genes in 48 % (10/21) of plasma samples for CRC
patients.
We have proposed that two stage verification might be applied for CRC
screening. These stages include measurement of cell-free circulating DNA with
following detection of methylated fragments of APC, FHIT and LRRC3B genes
in plasma of CRC patients. Overlapping of the mentioned approaches allowed
increasing sensitivity of studied panel up to 95 % in CRC detection. An
approach based on APC, FHIT and LRRC3B genes did not show of 100 %
sensitivity in registration of CRC which is essential for prevention of this death
disease. Therefore we suggested that higher sensitivity could be achieved by
further extension of gene panel for identification of methylated DNA in plasma
of CRC patient.
15
4'-hydroxyflavone, as inhibitors of protein kinase CK2. Search,
optimization and biological testing
O. V. Ostrynska, V. G. Bdzhola, A. G. Golub, V. M. Sapelkin,
A. O. Prykhod'ko, A. P. Kukharenko and S. M. Yarmoluk
Institute of Molecular Biology and Genetics, NAS of Ukraine
sovetova@bigmir.net
Aim. CK2 is a pleiotropic serine/threonine protein kinase that plays an
important role in a number of cellular processes. Over expression and hyper
activation of this enzyme associated with many diseases. Thus, development of
new potent and selective CK2 inhibitors is a task of great importance which
provides a powerful tool to extend our knowledge about CK2 function as well
as to regulate its activity in both in case of health and disease. In this work we
developed and researched new synthetic inhibitors of protein kinase CK2
among flavones derivatives.
Methods and Results. To search for new inhibitors of CK2 the receptor-
based virtual screening of 150,000 organic compounds library has been used.
Followed in vitro tests (γ – 32P -ATP method) revealed that 19 flavones out of
49 selected by virtual screening display the ability to inhibit activity of CK2
(IC50 < 30 μM). Structure-activity relationships of flavones derivatives have
been studied and binding mode of this chemical class has been predicted to
realize further compounds chemical optimization. 28 derivatives of 4'-
hydroxyflavone have been synthesized as a result of directed optimization, 80%
of them have sub-micromolar activity against CK2. The most active compounds
were FNH68 (IC50 = 0,01 μM) – 6,8-dichloro-2-(4-hydroxy-3-methoxy-
phenyl)-chromen-4-one and FNH79 (IC50 = 0,004 μM) – 6,8 dybromo-2-(4-
hydroxy-3-methoxy-phenyl)-chromen-4-one.
Kinetic studies of FNH68 and FNH79 showed that they are ATP
competitors. Inhibition constants are 3,5 nM and 1,8 nM, respectively. Initial in
vitro tests of the most active compounds (FNH68 and FNH79) on four
serine/threonine (ASK1, JNK3, Aurora A and Rock 1) and three tyrosine
protein kinases (FGFR1, Met and Tie2) revealed remarkable specificity towards
CK2.
Conclusion. New synthetic inhibitors of protein kinase CK2 among class
flavones have been received by the methods of computer modeling and
chemical optimization. These compounds are more active compared to their
natural analogues. High efficiency of developed inhibitors provides a basis for
their further use in biological research and for development of therapeutic
agents.
16
Metagenomic analysis of domesticated kombucha multi-microbial culture
L. P. Ovcharenko
leonidovp@gmail.com
Aim. To screen microbial community of domesticated kombucha microbial
culture (known as tea “fungus”) using analysis of metagenomic data.
Methods. DNA barcoding based on pyrosequencing data. Phylogenetic and
cultural analyses.
Results. Probiotics occupy a wide niche within this sector because of
keeping gut health and boosting immune system, memory and mental
sharpness. Kombucha beverage is becoming increasingly popular around the
world today as a prophylactic and healing agent. Kombucha culture (KC) is an
example of a multi-microbial community with mutualistic relationships
between its members. Bacterial and fungal barcoding followed by alignment
with sequences from public databases revealed 2 bacterial (Proteobacteria and
Firmicutes) and 1 fungal phyla (Ascomycota) in the KC, and an unprecedented
number of yeast operational taxonomic units (OTUs) were detected. The
acetobacteria of the Gluconacetobacter and Gluconobacter genera dominated in
KC metagenome in both phases of the kombucha micro-system (a pellicle and a
cultural liquid) with a few bacterial species of the Gluconacetobacter genus and
Gluconobacter oxydans. Also there were plenty of unknown but clonal bacterial
and fungal species, meaning that the blast scores were not sufficient to reliably
assign them to specific taxonomic units, but several conserved patterns of blast
hits were observed suggesting that these unidentified reads had originated from
several sources of unknown species not represented in the used reference
databases. However, the core community was represented by the 13 most
abundant OTUs of bacteria and yeast. It was found that the most heavily
sequenced bacteria and yeast in KC were not almost related to the cultured
species
Conclusions. The results of pyrosequence-based DNA barcoding showed
that Ukrainian ecotype of kombucha culture contains uncultivable
representatives of yeast which where not described earlier. As some of they
represented major populations, we may speculate that these species are true
kombucha ‘aborigens’, but are uncultivable and non-identified. In general, our
study exhibited a lower representation of cultured species in respect to whole
members of KC. Cultivable forms of the bacterial community members well
matched with the pyrosequencing data, however, some OTUs were not proven
by traditional cultural analysis.
17
Embrionically induced β-catenin haploinsufficiency in heart inhibits adult
myocardium development and leads to fetal genes expression upregulation.
O. L. Palchevska 1,2, V. V. Balatskyy 1,2, A. O. Andreeva 1,2, L. L. Macewicz 1,
O. O. Piven 1, L .L. Lukash 1
Human Genetics Department, Institute of Molecular Biology and Genetics NAS of Ukraine
Scientific and Educational Center “Institute of Biology”, Taras Shevchenko Kyiv National
University, Kyiv, Ukraine
o.l.palchevska@imbg.org.ua
Aim. Though canonical WNT/β-catenin signaling is inhibited in adult heart
it is well known that its activation is critical for heart adaptation to different
changing conditions. The main aim of our work is to determine the significance
of cardiac ablation of β-catenin during cardiogenesis for adult heart formation.
Methods. Using conditional knock-out approaches we have generated mice
with cardiospecific β-catenin haploinsufficiency. The routine PCR was used for
genotyping of the experimental animals. The 1, 3 and 6 months old mice were
used in our research. For morphological analysis the body weight/heart weight
ratio was estimated. Standard histological (HE- and MT-staining) methods were
implemented to analyze the paraffin sections. The quantitative RT-PCR was
used for analyzing the level of ANP, BNP, β-MHC and α-MHC expression. The
data were normalized to GAPDH gene expression.
Results. In our research we focused on the role of β-catenin in adult
myocardium functioning, hypertrophic response and adaptation to stress and
aging. We have monitored the influence of embrionical β-catenin ablation in
mice cardiomyocytes on heart development and canonical Wnt signaling
activity. As a result we have revealed that β-catenin haploinsufficiency leads to
fetal gene upregulation (ANP, BNP, β-MHC) in mice of 1 and 3 months age but
without morphological abnormality of heart tissue. Meanwhile we have
observed the tendency to heart development attenuation under β-catenin
haploinsufficiency condition in adult mice (3 months). Analyzing the next time
point (6 months) we have registered the decrease in expression of the
hypertrophy response (ANP, BNP) genes in comparison to control mice of the
same age group while β-MHC and α-MHC genes were still upregulated.
Conclusion. We have observed that cardiac ablation of β-catenin during
cardiogenesis leads to attenuation of adult heart development and fetal genes
expression violation.
18
Development of dermal equivalents contained living cells for the treatment
of massive burns of human skin
A. Ye. Papuga, Yu. M. Samchenko, T. A. Ruban, L. L. Lukash
alexander.papuga@gmail.com
Aim. We plan to create new dermal equivalents for therapy of extensive skin
damages, for example massive burns. The skin equivalents will the
bioengineered wound coverings with immobilized human cells. These cultured
cells secrete various growth factors. Besides various additional components in
composition of scaffolds also influence positively the tissue regeneration in the
wound area. So our dermal equivalents will be able to accelerate tissue
regeneration, stimulate the engraftment of donor grafts and have a bactericidal
effect. Methods. As a cell component of the skin equivalents we used: 1) cell
line 4BL, which was obtained in the Department of Human Genetics (IMBG
NASU) from peripheral blood cells; 2) human multipotent mesenchymal
stromal cells isolated from human placenta. In this work we develop dermal
equivalents on the basis of different natural and synthetic materials. We focus
our attention on three types of scaffolds for making new skin equivalents. The
first variant is porous polyacrylamide hydrogels, which are synthesized using
acrylamide and acrylonitrile. We introduce various additional components in
these hydrogels and investigate biocompatibility of obtained specimens and
their influence on cell vital functions and wound healing processes. For that we
use microscopic examinations and cytochemical staining. The second type of
scaffolds consists of two natural compounds: collagen and gelatin. We are
patenting this type of bioconstruction. After that they could be produced at the
conditions of our laboratory for experimental purposes. The third type of the
scaffold developed is the mucous film that is created with a symbiotic complex
of bacteria and yeast named as Medusomyces gisevi (so called tea fungus or
kombucha). This film contains a natural polysaccharide chitosan, which
promotes healing of wounds. Besides this film does not require complicated
equipment for synthesis and can be obtained easily. Now we are carrying out
experiments with laboratory mice. The burns are made on mouse skin and we
are using the bioconstructions “membrane/cells” for treatment of wounds.
Results. Microscopic examinations on the cell immobilized on polyacrylamide
hydrogels have shown that the cells are adsorbed on the surface of gel systems
without changing their morphology. It should be noted that the best parameters
for cultured cells are the ratio of the hydrogel monomer units "acrylamide:
acrylonitrile = 5: 3". All three types of the scaffolds obtained are biocompatible
and support cell viability on their surface for some days before application. Our
current experiments with laboratory mice are in progress. Conclusions. The
preliminary data allow us to hope for successful development of new
bioengineered dermal equivalents for the treatment of massive burns or other
extensive damages of human skin.
19
Transcriptional profiling of normal and cancer prostate cell lines
E. E. Rosenberg, G. V. Gerashchenko, V. I. Kashuba
kalirra@mail.ru
Background: Prostate cancer is the fifth most common cancer in the
world. Prostate-specific antigen (PSA) is generally accepted as a clinical
biomarker for this disease. The increased concentrations of PSA can be
detected upon emergence of prostate cancer and also in the development of
prostate adenoma, prostatitis and other inflammatory processes. This
marker alone could not discriminate aggressive tumors from non-
aggressive, however. There is an opinion that some of localized tumors will
never progress, therefore would not need any surgery. Unfortunately, with
the help of existing markers it is difficult to distinguish dormant tumors
from aggressive. The better tools are needed to diagnose different forms of
prostate cancer and to identify patients who will benefit from intervention.
Aim: In the present study the gene expression profiles in androgen-
sensitive non-aggressive and androgen-insensitive aggressive cell lines
were compared with the aim to find the new putative biomarkers of
aggressive prostate tumors.
Methods: Quantitative PCR (Q-PCR) was used to analyzed expression
of 65 genes at the mRNA level in the aggressive (DU145, PC3) and non-
aggressive (LNCaP) prostate cancer cell lines.
Results: An increase of expression of a set of genes (IL8, IL1b, PLCB1,
PREX1, THBS4, SPP1) was observed in more aggressive cell lines. These
genes are involved in the different cellular signaling pathways, such as NF-
kB (IL1b, IL8) and chemokines (PLCB1, PREX1), and also in the processes
of cell and focal adhesion (THBS4, SPP1).
Conclusions: We have found that aggressive and non-aggressive
prostate cell lines differ in cell adhesion, invasion and/or metastasis, and
inflammatory signaling pathways. Genes that showed decreased levels of
expression could be putative tumor suppressor genes. Genes that showed an
elevated expression could be putative oncogenes.
20
Oligoribonucleotides affect the expression of some genes encoding the
innate immunity system components
A. O. Rybenchuk, G. V. Gerashchenko Z. Yu. Tkachuk,
alinarybenchuk@gmail.com
Aim: Natural and synthetic oligoribonucleotides are known to possess
immunomodulating, antiviral and anti-inflammatory properties. In this respect,
the aim of our study was to explore the gene expression of antiviral innate
immunity system components under the influence of oligoribonucleotides.
Methods: In our study RT-PCR method was applied.
Results: Significant increase in the IFNα and IFNβ genes expression was
observed upon the oligoribonucleotides injection into mice for preventive and
treatment purposes, similar to virus infected animals. At the same time, the
alteration of gene expression did not occur in the animals of control group. The
MX1 gene expression increased almost 190 times, OAS1 – 19 times and NOS2 –
7 times after oligoribonucleotides were injected for prevention. When
oligoribonucleotides were injected as a treatment drugs we observed the 150
times increase of MX1 gene expression, OAS1 – 180 times, and NOS2 – 18,5
times. In contrast to these genes, the expression of RNAse L gene decreased 1,4
times when oligoribonucleotides were used as a preventive and 1,6 times when
they were used as a treatment drugs. The similar tendency in expression of
these genes was observed for influenza infected animals.
Conclusion: Thus, for both animals, infected with the influenza virus, and
for animals injected with the oligoribonucleotides, we observed the significant
increase in the expression of some genes of innate immunity system enzymes.
21
Flexible structure and interdomain interactions in human tyrosyl-tRNA
synthetase studied by molecular dynamics simulations
O. V. Savytskyi and A. I. Kornelyuk
Institute of Molecular Biology and Genetics, NAS of Ukraine
savytskyi@moldyngrid.org
Tyrosyl-tRNA synthetase (TyrRS) is the key enzyme of protein biosynthesis,
which catalyzes the aminoacylation of tRNATyr. The full-length TyrRS has no
cytokine activity, but its proteolytic cleavage reveals IL8-like activity of the N-
terminal catalytic module and EMAP II-like activity of non-catalytic C-terminal
domain (Wakasugi and Schimmel, 1999; Kornelyuk et al., 1999). Earlier, it was
found that the ELR-motif (E91, L92, R93) in TyrRS is responsible for IL8-like
cytokine activity (Wakasugi and Schimmel, 1999), but the 3D structure of the
full-length human TyrRS (HsTyrRS) is still unknown. Aim. In this work we
performed the modelling of 3D structure of HsTyrRS and studied its
compactization by molecular dynamics simulations (MD). Methods. All
computations were performed using the grid-services of MolDynGrid virtual
laboratory (http://moldyngrid.org). The model of three-dimensional structure of
HsTyrRS was constructed in Modeller 9.7 using templates (PDB codes: 1N3L,
1NTG and 1OPL for interdomain linker). The best 10 structures were selected
and six independent 100 ns MD trajectories of HsTyrRS were computed using
GROMACS 4.0.5 software and G43a1 force field. Results. Our MD
simulations produced compact final structures of HsTyrRS, where both C-
modules were bound to the dimer of N-modules. There is a pronounced
asymmetry in the binding of C-modules, which explained by their almost
independent motions The main “binding hot spot” on the N-terminal module
contains the residues: E25-K26, E29-R34, G46-K47, Y79-L89, R93, R135,
V139, K154, E157-H158, E175-D180, F192-V208, E227-K231, L235-C250,
K243, F328-P342. The strongest binding energy of ~1000 kJ/mol was observed
for the second C-module in simulation 1 and for the first C-module in
simulation 6. In trajectories 1 and 6, only one of the C-modules binds strongly,
whereas in trajectory 4 both C-modules exhibit almost identical binding
energies of ~400–500 kJ/mol. In contrast, the binding energies in simulations 2,
3 and 5 were much smaller (300–400 kJ/mol). Conclusions. Our data suggest
that HsTyrRS in solution exists in a number of compact asymmetric
conformations, which differ significantly by their general rigidity, mobility of
C-modules and the strength of their binding to the dimer of N-modules. The
orientation of bound C-modules is rather unspecific while there is a pronounced
set of binding hot spots on the surface of N-modules. In our MD simulations we
observed the hydrogen bonds formation between R93 of ELR cytokine motif
and A340 and E479 residues in C-module. These data support the idea that the
full-length TyrRS lacks its cytokine activity due to the interactions between N-
terminal and C-terminal modules and protection of ELR cytokine motif.
Acknowledgments. This work was supported by National Academy of Sciences of Ukraine
(grants 15/2010-2013). Authors would like to thank Dr. Semen O. Yesylevskyy for discussions.
22
Mesenchymal and trophoblast progenitor cell subpopulations in the
culture of placental multipotent mesenchymal stromal cells
V. A. Shablii1, 2, M. D. Kuchma1,2,V. M. Kyryk3, G. M. Onishchenko²,
Yu. М. Shablii2, І. Ya. Skrypkina1, O. M. Tsupykov3,4, P. P. Klymenko3,
L. L. Lukash1, G. S. Lobintseva2
1 Institute of molecular biology and genetics, NAS of Ukraine,
2 Institute of Cell Therapy,
3 State Institute of Genetics and Regenerative Medicine, NAMS of Ukraine
4 Bogomoletz Institute of Physiology, NAS of Ukraine
v_shabliy@ukr.net
Introduction. Placenta is an attractive source of multipotent mesenchymal
stromal cells (MMSC) for needs of regenerative medicine. The origin of
placental MMSC remains unknown though the hypothesis about perivascular
origin has been recently published. We previously showed that placental
MMSC contain various populations of cells that express trophoblast-specific
markers.
The aim of this study was investigation of immunophenotype and expression
of some genes in the subpopulations of mesenchymal and trophoblast cell in
culture of placental MMSC.
Methods. Placental MMSC were obtained by classical culture methods, the
placental cells were phenotyped by flow cytometry and immunocytochemistry,
gene expression was analyzed by RT-PCR. The expression of vimentin,
chorionic gonadotropin, HER2 and cytokeratin 7 in the placental tissue was
investigated by immunohistochemistry and flow cytometry.
Results. Populations of colony-forming cells with immunophenotype
Vim+CG+HER2+СК7-СD90+ and Vim+CG+HER2+СК7+CD90+ were present in
the culture of placental MMSC. It was established mRNA expression of the
EOMES, CDX-2 genes that is characteristics of trophoblast stem cells, and
mRNA of the POU5F1 gene, which is a marker of embryonic stem cells. In
placental tissue we found the placental MMSC-like populations with
immunophenotype CD90+CD73+CD45-CD34-CD14-- and CD90-CD73+CD45-
CD34-CD14- but we did not observe the vimentin-positive stromal cells, which
express both chorionic gonadotropin and cytokeratin 7.
Conclusions. Placental MMSC simultaneously express mesenchymal and
trophoblast markers and contain populations of trophoblast and mesenchymal
stem cells.
23
Development of method of cultivation of mesenchymal stem cells from
umbilical cord matrix with preservation of the cells with reduced
attachment rate
N. S. Shuvalova, O. G. Deryabina, V. A. Kordium
riyena@yandex.ru
Aim: The aim of present work was to develop the method of optimization of
mesenchymal stem cells from umbilical cord matrix (UC-MSC) cultivation
with preserving the spontaneously detached cells, which evaded the impact of
the standard passing procedure on the surface apparatus, and to test this method
for cultivation of the UC-MSC under physiological oxygen tension. Cultivation
of mesenchymal stem cells (MSC) from various sources is associated with the
loose of the features relevant for their use in clinical practice. One of the
reasons is the damaging of the cell surface proteins during standard passing
procedures. Recent studies have developed various variants of cultivating MSC
without obligatory detachment. It is also known that during the specific phase
of the cell cycle, prior to the division, cell's attachment to the substrate is
decreased. Preserving these non-attached cells may appear a helpful addition to
the accepted passing procedures. Methods: UC-MSC were cultivated up to 3
passages under CO2 incubator conditions. When the monolayer confluence
reached ~70 %, the cells of the control groups were passed using the mixture of
trypsin (0.25 %) and versene (0.02 %) (1:1). In the experimental group, while
replacing the cultural medium with the fresh portion, the medium where the
culture was maintained (probably containing spontaneously detached cells) was
collected, transferred into a new flasks with the addition of fetal bovine serum
and FGF2. Two days after the first detection of attached cells the medium was
replaced by the fresh portion. The same was done for UC-MSC cultivated under
nitrogen-based and argon-based gas mixtures containing 3 % oxygen. Cell
numbers and morphology were analyzed at each passage. Results: Two days
after the medium transfer 10–20 cell’s “attachment places” can be noticed on
the bottom of the culture flask. Approximately two weeks after the first
detection of cells, the clones reached the size and confluence sufficient to be
passed using standard methods. The morphology of the cells in these cultures
was typical for UC-MSC of initial passage. The degree of surface markers
expression was similar to that in cultures, replated by the standard method.
The method was also tested for UC-MSC cultivated under nitrogen-based
and argon-based gas mixtures containing 3 % oxygen. The cells obtained from
cultures maintained in both gas mixtures had a slightly higher proliferation rate.
Conclusion: The method of keeping the medium with spontaneously
detached cells helps to preserve MSC population which avoided the negative
effect of the standard passing procedure. Cultivation under physiological
oxygen concentrations appeared to enhance the proliferative potential of
cultures obtained by this method.
24
Design of protein kinase CK2 inhibitors based on the 3-alkyl substituted 2-
quinolone scaffold
A. R. Synyugin, M. O. Chekanov, O. P. Kukharenko, V. G. Bdzhola,
S. M. Yarmoluk
Institute of Molecular Biology and Genetics, NAS of Ukraine
asinugin@gmail.com
Aim. The activity of protein kinase CK2 (casein kinase 2) increased in a
wide range of tumors. It inhibits cell apoptosis and is used by many viruses for
their own protein phosphorylation. Therefore, the potent and selective inhibitors
of CK2 would be important compounds for the development of clinical agents.
Recently some 4-quinolone derivates as effective CK2 inhibibitors have been
developed [1]. Therefore we have chosen their structure analogs – 2-quinolone
derivatives for investigation of inhibitory activity against CK2. Methods and
Results. In order to discover CK2 inhibitors we have performed flexible
docking of virtual library containing 3000 derivatives of 2-quinolone in CK2
ATP-binding site. The most promising 112 compounds have been selected for
chemical synthesis. These compounds were divided into four general sub-
classes: amides of quinolone-3-ylacetic- and quinolone-3-ylpropanoic- acids,
amides and sulfonamides of quinolone-3-ylmethane- and quinolone-3-ylethane-
amines. Synthesis of 3-alkyl substituted 2-quinolone derivatives was based on
the 2-chloro quinoline obtaining method. Treatment of aliphatic carboxylic
acids anilides with Vilsmeer-Haak complex in phosphorus oxychloride leads to
intramolecular cyclization with 2-chloroquinoline ring formation. As we
previously reported some new 3-alkyl substituted 2-quinoline derivatives were
synthesized by optimization of this method in high yields [2, 3]. Series of new
3-alkyl substituted 2-quinolone derivatives were obtained by chlorine atom
hydrolyses in 2 position of quinoline with water acetic acid treatment. Building
blocks were synthesized after removing protected groups. Resulting screening
compounds were obtained by high-throughput combinatorial synthesis.
Synthesized derivatives of 3-alkyl substitute 2-quinolone were taken for the
kinase assay study. In vitro experiments revealed four compounds with
inhibitory activity toward CK2 in the micromolar range. Several important
structural features of 2-quinolone derivatives were identified as a result of SAR
analysis. It was found that the increasing of the side-chain length in 3 position
of 2-quinolone derivatives leads to the decreasing of inhibitory activity against
CK2. Conclusion. Some novel CK2 inhibitors have been developed on the base
of the 3 – alkyl substituted 2- quinolone scaffold.
[1]-Golub AG, Yakovenko OY, Bdzhola VG, Sapelkin VM, Zien P, Yarmoluk SM. Evaluation
of 3-carboxy-4(1H)-quinolones as inhibitors of human protein kinase CK2.// J Med Chem.-2006. №
49 (22) –P. 6443-6450. [2] M.O. Chekanov, А.R. Synyugin, S.S. Lukashov, S.M. Yarmoluk.
Synthesis of 3-(aminomethyl)- and 3-(2-aminoethyl)-quinolin-2-ones// Ukrainica Bioorganica
Acta - 2009.-Vol.2.-P 59-63. [3]А.R. Synyugin, M.O. Chekanov, S.S. Lukashov, S.M. Yarmoluk.
Synthesis of 3-(quinoline-2-on-3-yl)propanoic acids// Ukrainica Bioorganica Acta. – 2010. – № 2. –
Р. 58-62.
25
2'-5'-linked oligoadenylates alter the conformation of S100A1 and S100B
proteins
O. Yu. Skorobogatov, I. Yu. Zhukov, Z. Yu. Tkachuk
skorobogatov.alx@gmail.com
Aim. In order to broaden the understanding of biological action of
dephosphorylated 2'-5'-linked triadenylates (2'-5'A3), this study was aimed at
searching for additional biological targets being under their regulating impact.
We showed previously, that 2'-5'A3 binding to Calmodulin – multifunctional
Calcium ions transducer – caused considerable (~ 3 orders of magnitude)
increase of its Ca2+ affinity (Tkachuk et al., 2011). Therefore, we suggested a
possibility of 2'-5'A3 interaction with other Ca2+-binding proteins – S100A1 and
its close structural homologue – S100B.
Methods. For these purposes we used the Circular Dichroism Spectroscopy
method, as it provides valuable structural data. CDNN deconvolution software
was used for the extraction of protein’s secondary structure content.
Results. In this study we have obtained the evidence for interaction between
2'-5'A3 and Ca2+-binding proteins in both apo- and holo-form. It was proved by
the occurrence of conformational changes within protein’s secondary structure
upon interaction with both 2'-5'A3 and its epoxy-modified analogue. It was
shown, that in both cases of S100A1 and S100B interaction with 2'-5'A3, 10 %
of their helical structure content converts to randomly coiled elements. This
value may be considered as insignificant, but, on the other hand, it may serve as
an evidence for the existence of concrete binding site for 2'-5'A3.
Conclusion. Our earlier proposed suggestions about the possibility of
interaction between 2'-5'A3 and Ca2+-binding proteins – S100A1 and S100B –
was proved by the experimental data obtained. It turned out, that such binding
may occur even in Ca2+-free solution, though the interaction of S100A1 and
S100B with target peptides/inhibitors was shown to occur only in the presence
of Ca2+ according to the literature sources. This important feature indicates, that
2'-5'A3 are capable of regulating the Ca2+-signal transducing proteins in both
Ca2+-free and Ca2+-saturated states.
References.
1. Tkachuk et al. (2011) The effect of 2'-5'-oligoadenylates on calcium
binding to Calmodulin, 17-th International Symposium on Calcium-Binding
Proteins and Calcium Function in Health & Disease, (July 16-21, Beijing,
China), Book of Abstracts, p. 41.
2. Böhm, G., Muhr, R., and Jaenicke, R. (1992) CDNN: quantitative analysis
of protein far UV circular dichroism spectra by neural networks, Protein Eng.,
5, 191–195.
26
Constitutive expression of CHI3L1 oncogene promotes chromosome
instability in immortalized 293 cells
A. A. Stepanenko, S. V. Andreeva, D. A. Mikitenko, N. Huleyuk, L. L. Lukash,
V. V. Dmitrenko, V. M. Kavsan
a.a.stepanenko@gmail.com
Multiple genetic rearrangements, including whole chromosome and gene
copy number gains and losses, chromosome translocations, gene mutations are
necessary for establishing the malignant cell phenotype. Previously, we and
others have demonstrated that increased CHI3L1 gene expression stimulated
cellular mitogenic and proliferative properties, enhanced migration, invasion,
radio- and chemoresistance of tumor cells, capacity for anchorage-independent
growth in soft agar, promoted tumor growth, and angiogenesis. The aim of this
study was to establish the link between transforming properties of oncogene
CHI3L1 and changes in karyotype of 293 cells with stable expression of
CHI3L1.
Conventional cytogenetics and array comparative genome hybridization
(aCGH) were used to describe chromosome abnormalities in cell lines.
We found that constitutive expression of CHI3L1 promoted chromosome
instability (CIN) in 293 cells. Modal number of chromosomes in 293_CHI3L1
cells was distinct from that in transfected control 293_pcDNA3.1 and parental
293 cells. Interline whole chromosome heterogeneity was manifested. A
number of new distinct marker chromosomes were observed in CHI3L1-
expressing cells in two independent experiments. aCGH revealed significant
difference between the spectra of cytoband gains and losses in 293_CHI3L1 and
control cells.
Here it was established the link between transforming properties of
oncogene CHI3L1 and changes in karyotype of 293 cells with stable expression
of CHI3L1. Deregulation of oncogenic pathways triggers and collaborates with
CIN during transformation and tumorigenesis. Relationship between CIN and
cancer genes explains why such large number of cancer genes was identified
(Cancer Gene Census) and why hundreds of oncogenes with diverse functions
are characterized by the same ability to transform a cell or aggravate
tumorigenicity.
27
Construction and expression of deletion mutants of translation elongation
factors eEF1Bα, eEF1Bβ, eEF1Bγ.
T. V. Trosiuk, V. F. Shalak
chafran@mail.ru
In higher eukaryotes, translation elongation factor 1B (eEF1B) exist as a
stable high molecular weight complex comprising three different subunits:
alpha, beta and gamma. The part of this complex involves also tightly bound
valyl-tRNA synthetase. eEF1Bα and eEF1Bβ subunits catalyze GDP-GTP
recycling on eEF1A, whereas eEF1Bγ is believed to be a structural constituent.
Up to date, the structure of the complex is unknown and the mode of interaction
between the subunits was not studied in details.
One of the purposes of this work is to explain how all three subunits interact
with each other to form the complex. For that, we created series of deletion
mutants for all subunits and expressed them in bacteria. Further, all
recombinant truncated proteins will be purified and their interactions will be
studied in vitro to define domains responsible for the complex formation.
Conservative protein domains in the structures of all three subunits were
delineated by multiple sequences alignment using ClustalW. Corresponding
DNA primers were generated to amplify respective fragments of eEF1Bα,
eEF1Bβ and eEF1Bγ coding regions. DNA fragments encoding truncated
mutants of eEF1Bα and eEF1Bβ were cloned into pGEX6P1 vector in frame
with GST-tag. To produce eEF1Bγ deletion mutants with N-terminal His-tag
the respective DNA fragments were cloned into pET28a(+) vector. The
sequences of all obtained constructs were verified. All recombinant eEF1Bα
and eEF1Bβ mutants were found to be soluble when produced in BL21Gold
bacterial strain at 37°C. In contrast, production of soluble eEF1Bγ mutants was
efficient only in Rosetta strain at 20°C. eEF1Bγ deletion derivatives were
purified by affinity chromatography on Ni-NTA resin and their aggregation
state was analyzed by gel filtration on a Superose 6 column. Two from four
deletion mutants of eEF1Bγ were found to form soluble aggregates, while two
others behaved as monomers judging by gel filtration technique.
As a result, we created and produced in bacteria a set of soluble mutants of
eEF1Bα, eEF1Bβ and eEF1Bγ subunits. eEF1Bγ deletion mutants were purified
and can be now used to test their interactions with full length eEF1Bα and
eEF1Bβ proteins in vitro. Purification of eEF1Bα and eEF1Bβ mutants is in
progress.
28
Ammonium ion nanocomposite sensor fabricated at copper-
plated/nafion/polyaniline screen-printed carbon electrodes
M. T. Zhybak, E. Dempsey, Y. I. Korpan
m.zhybak@gmail.com
Aim. Modification of the screen-printed carbon electrode with a new,
easy-to-handle and inexpensive nanocomposite layers for highly
sensitive and selective detection of ammonium ions.
Methods. Electrodeposition, electropolymerization, cyclic
voltammetry, chronoamperometric detection.
Results. For the first time, we propose and report amperometric ammonium
ions selective sensor based on Copper/Polyanyline (PANI) nanocomposites,
which were fabricated using Layer-by-Layer electrodeposition techniques. The
NH4
+ ions diffuse into the PANI layer and provoke the reduction of PANI on
the Copper-plated screen-printed carbon electrodes at a potential of about
−450 mV (vs Ag). Moreover, electrodeposited Copper displays catalytic
properties towards ammonium ions at the same reduction region as for PANI
and leads to the substantial improvement of the fabricated sensor
electrochemical response. The experimental result reveals that the
Cooper/PANI nanocomposite sensor exhibits much higher sensitivity
(325 nA/(per 0.1 mM mm2)) than that of PANI and Cooper alone, and shows
approximate linearity over a range of target analyte concentration from 5 to 100
µM with the limit of detection equal to 1×10−6 M of NH4Cl (signal/noise ratio =
3, n = 10).
Conclusions. A simple and inexpensive Copper/Polyaniline-based
chemosensor for ammonium determination with excellent electrochemical
properties has been developed.
29
Investigation of amino acids of the human cytochrome P450 2E1
active center critical for interaction with substrates
V. Kitam
Aim. The microsomal ethanol-induced cytochrome P450 2E1 (CYP2E1)
belongs to the superfamily of hem-containing monooxygenases that are
responsible for the biotransformation of many endogenous compounds and
xenobiotics. This enzyme catalyzes oxidative metabolism and metabolic
activation of wide range exogenous organic substrates including drugs,
environmental toxicants, procarcinogenic and carcinogenic chemicals. The
major part of them are low molecular weight compounds. Using computational
methods of docking of ligands into cytochrome P450 2E1 active site we have
identified some amino acids that specifically interact with substrates or
inhibitors (Kitam and Chashchyn, 2010). In order to verify obtained data we
performed computational site-directed mutagenesis of the enzyme by changing
one of the identified critical for protein-ligand interactions amino acids Phe298
for Ala (alanine screening) in its structure.
Methods. Computational methods of molecular dynamics (NAMD program,
CHARMM forcefield, standard NVT-ensemble, 310K, 0,15mmole KCl) were
used for modeling of possible crucial changes in CYP2E1 spatial structure
caused by such aminoacid replacement. In order to investigate the influence of
this amino acid substitution on the enzyme’s catalytical activity experimentally
and compare obtained results with computational data we introduced codon
amino acid Ala instead of codon amino acid Phe in the human CYP2E1 cDNA
coding region in position 298 using PCR site-directed mutagenesis method and
cloned this modified cDNA in E. coli expression vector pCWOri+ (Gillam, Guo
and Guengerich, 1994).
Results. Molecular dynamic of this mutated protein and its complexes with
p-nitrophenol (substrate) and 4-methylpirasol (inhibitor) showed that such
amino acid substitution did not affect the overall protein structure, slightly
influenced on its interaction with inhibitor and strongly influenced on its
binding with substrate. This data confirmed previous information obtained from
docking studies. The evaluation of catalytic activity of the expressed in E.coli
DH5α cells recombinant protein have shown 2-fold decrease in its p-
nitrophenol hydroxylation activity related to the unmodified protein.
Conclusions. The experimentally obtained results showed close agreement
with our computational data revealing the significant role of Phe298 in
interaction of CYP2E1 active site with substrates thus evaluating and verifying
the quality of the computational methods used in our work and demonstrating
the reliability of our modeling experiments.
30
Synthesis of 2-amino-3-(1H-benzimidazol-2-yl)-1-aryl-4, 5-dihydro-
4-oxopyrroles derivatives as potential FGFR1 kinase inhibitors
K. V. Levchenko, A. A. Gryshchenko, V. G. Bdzhola and S. M. Yarmoluk
levchenko.kostia@gmail.com
Aim. The fibroblast growth factor receptor kinase (FGFR1) is member of a
family tyrosine receptor proteinkinase. The main role of FGFR1 in postnatal
life is regulation of angiogenesis process, which is necessary for tumor growth.
Therefore kinase inhibitors can be appropriated for effective treatment of a
whole number of diseases, including oncogenic.
There have been described development of FGFR1 inhibitors among 2-
amino-3-(1H-benzimidazol-2-yl)-1-aryl-4, 5-dihydro-4-oxopyrroles derivatives,
which have been found in a virtual screening.
Methods. Receptor-based virtual screening has been used in search for
FGFR1 kinase inhibitors. Consequently, 2 compounds with activity IC50 less
than 25 μM have been obtained. The number of close structural analogs have
been synthesized for the purpose of optimization and tested for activity of
FGFR1 enzymatic inhibition. Eventually, 27 compounds were chosen, obtained
and tested. Two compounds shown the activity IC50 less than 1 μM.
All 2-amino-3-(1H-benzimidazol-2-yl)-1-aryl-4,5-dihydro-4-oxopyrroles
derivatives were synthesized according to the next schema. Firstly,
benzimidazol-2-yl acetonitriles have been obtained through cyclocondensation
reaction of o-phenylendiamine and ethyl cyanoacetate. Then active methylene
group of benzimidazol-2-yl acetonitrile was acylated with chloroacetyl chloride
and product was used to obtain 2-amino-3-(1H-benzimidazol-2-yl)-1-aryl-4, 5-
dihydro-4-oxopyrroles derivatives as desired products.
Results. Structure-activity relationship study showed that the greatest effect
had meta-substitution of aryl fragment especially with hydroxyl group. At the
same time, para-substitution of phenyl appreciably reduced the FGFR1
inhibitory activity of our compounds. Cause of this reducing can be related to
steric obstacles, which para-substitutes made. The compound with methylated
Nitrogen atom of benzimidazolyl has shown low-activity inhibitor, as well. This
fact gave to understand the necessity of availability of hydrogen bond donor
here.
Conclusions. Accordingly, 2-amino-3-(1H-benzimidazol-2-yl)-1-aryl-4,5-
dihydro-4-oxopyrroles derivatives were synthesized and tested in vitro as
human FGFR1 kinase inhibitors firstly.
|