Posters P1 P2

Posters
P1
P2
Dihydrofolate reductase
phosphorylation catalyzed by
CK2α kinase and its influence
on enzyme activity
Bisubstrate inhibitors of Pim kinases
Anna Antosiewicz1, Elżbieta Senkara-Barwijuk1,
Patrycja Wińska1, Monika Wielechowska1,
Joanna Cieśla1,2, Maria Bretner1,3
Institute of Chemistry, University of Tartu, Tartu, Estonia
1Warsaw
University of Technology, Faculty of Chemistry,
Institute of Biotechnology, Warsaw, Poland; 2Nencki
Institute of Experimental Biology PAS, Warsaw, Poland;
3Institute of Biochemistry and Biophysics, PAS, Warsaw,
Poland
e-mail: Anna Antosiewicz <[email protected]>
Dihydrofolate reductase (DHFR) plays an essential role
in cell growth and proliferation. In the cellular sole de novo
thymidylate biosynthesis cycle, DHFR and thymidylate
synthase (TS) catalyze sequential reactions, leading to
formation of thymidine monophosphate — a substrate
indispensable for DNA synthesis. Taking into account
that posttranslational modifications, such as phosphorylation, can substantially change protein conformation
and activity, studies of the enzyme’s regulation pathways
are of great importance. It has been determined that
thymidylate synthase (TS) catalytic activity is decreased
due to CK2-catalyzed in vitro phosphorylation, whereas
phosphorylation of dihydrofolate reductase (DHFR) is
yet to be elucidated. CK2 is a constitutively active serine/
threonine kinase that phosphorylates even 20% of residues in eukaryotic cells. Considering the significance of
the thymidylate synthesis cycle, we found it interesting
to undertake studies on dihydrofolate reductase (DHFR)
phosphorylation by CK2 and to examine possible influence of this modification on enzyme activity.
Application of bioinformatics tools allowed in silico prediction of four amino acids on the polypeptide chain
of human DHFR (hDHFR), which could be potentially phosphorylated by CK2. Performed experiments,
involving far-Western techniques, indicated mutual
interactions of human DHFR with the alpha subunit
of human CK2 (CK2α). Kinetic parameters of CK2αcatalyzed phosphorylation of hDHFR were measured,
using a quartz crystal microbalance with dissipation
monitoring. Moreover, isotopic studies confirmed
that hDHFR is a substrate for two variants of CK2α
(the kinase with or without HisTag). However, linear
time-dependence of hDHFR phosphorylation was
observed only with CK2α without the HisTag variant,
whereas the HisTag CK2α variant was inactivated in a
time-dependent manner in the course of the reaction.
Ramesh Ekambaram, Erki Enkvist,
Angela Vaasa, Marja Kasari , Asko Uri
e-mail: Ramesh Ekambaram <[email protected]>
Pim kinases are an exciting class of serine/threonine protein kinases with a structurally unique ATP
binding pocket incorporated in all three isoforms
(Pim-1, Pim-2 and Pim-3). The Pim kinases are key
components of the JAK/STAT signaling pathway
and regulate cancer cell survival, cycle progression,
suppression of apoptosis and angiogenesis, providing an important set of therapeutic targets for the
treatment of various hematological malignancies and
solid tumors. Herein we have developed bisubstrate
inhibitors of Pim kinases-1 as a novel class of Pim
inhibitor that potentially inhibit the Pim kinase-1 with
subnanomolar KD values and exhibit excellent selectivity against a diverse panel of 128 kinases.
7th International Conference: Inhibitors of Protein Kinases, 2012
Posters35
P3
P4
Bisubstrate inhibitors as tools
for studying protein kinases
The interplay between sunitinib
malate crystal packing, charge
density distribution, and proteinligand interactions in sunitinibcontaining biological systems
Erki Enkvist, Asko Uri
Institute of Chemistry, University of Tartu, Tartu, Estonia
e-mail: Erki Enkvist <[email protected]>
Adenosine and oligo-arginine conjugates (ARC-s) are
bisubstrate inhibitors of basophilic protein kinases
that are used as fluorescent ligands and affinity carriers in various biochemical assays. Some of the ARC-type inhibitors have single-digit picomolar affinity
with long residence times for several AGC-kinases.
This kind of high affinity enables aneffective pull
down assay for quantification of protein kinases in
complex mixtures.
Crystal structures of ARC-kinase complexes have
shown diverse binding patterns of oligo-arginine
moieties revealing potential new interaction sites and
modes how protein kinases recognize their substrates.
Crystallization of protein kinases with their substrate
peptides and proteins is often complicated because of
the low affinity of the peptides. High overall affinity
of bisubstrate inhibitors increases the possibility to
crystallize kinases with an occupied peptide substrate
binding region. Strong interactions between kinase
and bisubstrate inhibitors stabilize the protein and
may help to crystallize flexible protein kinases. Several
studies using ARC-type inhibitors as tools for crystallographic studies are currently in progress.
Oligo-arginine is a known cell penetrating peptide
sequence that helps ARC-type inhibitors to cross
the plasma membrane and to use in cellular assays.
Recently we have widened the bisubstrate approach
outside the AGC-family of protein kinases. Unique
binding pockets of CK2 and PIM1 have allowed us to
design very selective bisubtrate inhibitors and appropriate fluorescent ligands to these kinases.
Anna M. Goral1, Maura Malińska1, Katarzyna
N. Jarzembska1, Andrzej Kutner2, Paulina
M. Dominiak 1, Krzysztof Woźniak1
1Department of Chemistry, University of Warsaw, Warsaw,
Poland; 2Pharmaceutical Research Institute, Warsaw,
Poland
e-mail: Anna M. Goral <[email protected]>
Despite the importance of sunitinib malate [N-[2(diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1Hpyrrole-3-carboxamide l-malate, in anti-tumour
therapy, its crystallographic structure has been only
recently published. Well-established molecular geometry constitutes foundation for further energy studies. In turn, a charge density distribution of a crystal
sheds light on more subtle effects and interactions
present in the crystal lattice. A comprehensive charge
density and energy analysis provides valuable information on the molecular binding properties and nature of interactions. Therefore, in this contribution,
we present an experimental charge density study of
sunitinib malate supplemented by deep energy and
crystal packing investigations including Hirshfeld
surface, electrostatic potential and topological analysis. The analysis is additionally combined with the
exploration of the previously mentioned pharmaceutical aspect of sunitinib. For that purpose, four complexes of tyrosine kinase with sunitinib deposited in
the Protein Data Bank have been chosen. The four
analyzed proteins belong to three different protein
tyrosine kinase families, i.e., IL-2-inducible T cell kinase [PDB code 3MIY], human phosphorylase kinase
gamma 2 [PDB code 2Y7J], and KIT kinases [PDB
codes: 3G0E and 3G0F]. Here, we report the quantitative characterization of interactions, confirmed by
energy calculations conducted on the basis of charge
density distribution, reconstructed with the aid of the
UBDB2011 databank. We compared the interactions
in the crystal lattice with the interactions inside the
KIT kinase binding pocket. Interestingly, the oxygen
atom of sunitinib is engaged in the crystal lattice only
in intermolecular hydrogen bond, in opposition to the
additional hydrogen bond formed with one of the highly conserved amino acids in kinases.
Acknowledgements:
These studies were supported by the Ministry of Science
and Higher Education (PBZ-MIN-014/p05/2004) (Poland), the Biocentrum-Ochota project (POIG 02.03.00-00003/09) and Institute of Biochemistry and Biophysics.
7h International Conference: Inhibitors of Protein Kinases, 2012
36Posters
P5
P6
Calpain-mediated activation of
cyclin dependent kinase 5 is critical
for alpha-synuclein toxicity in vitro
AMP activated protein kinase
(AMPK) a molecular link between
mitochondria and the cellular clock
Anna Kaźmierczak, Grzegorz A. Czapski,
Magdalena Gąssowska, Agata Adamczyk
Anna Kaźmierczak1,2, Karen Schmitt2,
Amandine Grimm2, Steven A. Brown3,
Joanna B. Strosznajder1, Anne Eckert2
Department of Cellular Signaling, Mossakowski Medical
Research Centre, Polish Academy of Sciences, Warsaw,
Poland
e-mail: Ania Kaźmierczak <[email protected]>
Dysfunction of cyclin-dependent kinase 5 (Cdk5) has
been demonstrated to play a critical role in neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Cdk5 is an atypical cyclin-dependent
kinase, the activity of which is dependent on binding to p35/p39, and the breakdown of Cdk5/p35 to
cdk5/p25 by calpain-mediated cleavage increases its
kinase activity and cytotoxicity.
Our previous studies demonstrated that extracellular
alpha-synuclein (ASN) that is a hallmark of Parkinson’s disease, and involved in other neurodegenerative
disorders evokes calcium homeostasis deregulation,
leading to enhancement of NO synthesis, oxidative
stress and apoptotic cell death. The aim of the present study was to investigate the involvement of Cdk5
in the molecular mechanism of extracellular ASN cytotoxicity.
Our data indicate that exogenously added ASN (10
μM) and its toxic fragment, the non-Aβ component
of Alzheimer‘s disease amyloid (NAC), affected Cdk5
in dopaminergic PC12 cells. In this study, ASN/NAC
increased the expression of Cdk5, and Cdk5r1 and
Cdk5r2 genes, coding for p35 and p39 peptides. Moreover, truncated peptide p25 was identified in ASNtreated cells that suggest calpain-dependent cleavage
of p35. In addition, it was found that phosphorylation of Cdk5 on Tyr15 was significantly increased, indicating stimulation of Cdk5 activity in the presence
of ASN. Our results showed that the specific Cdk5
inhibitor (BML-259) and calpeptin, an inhibitor of
calpains, protected dopaminergic cells against ASN/
NAC-evoked cell death.
In summary, our results demonstrate that extracellular ASN, via activation of calcium influx, induces
calpain-dependent proteolytic cleavage of p35 leading to overactivation of Cdk5 and, in consequence to
cell death.
1Mossakowski Medical Research Centre, Polish Academy
of Sciences, Department of Cellular Signaling, Warsaw,
Poland; 2Neurobiology Laboratory for Brain Aging and
Mental Health, Psychiatric University Clinics, Warsaw,
Poland; 3Department of Pharmacology and Toxicology,
University of Zurich, Switzerland
e-mail Anna Kaźmierczak: <[email protected]>
AMPK is a key enzyme in energy metabolism and
its activity is synchronized to the light-dark cycle. It
functions as a sensor of AMP/ATP ratio and is an
important regulator of several transcription factors
involved in mitochondria metabolism and circadian
clock function. Mitochondria were shown to be strategically positioned between circadian rhythm and cell
metabolism. Nevertheless little is known about their
function in controlling the cycle. In our study, we addressed the question of the role of mitochondria in
regulation of AMPK activity related to the circadian
clock.
The study was carried out in primary human fibroblasts, an already established model to investigate molecular clock mechanisms in vitro. We have found that
mitochondria activity showed rhythmic oscillations
within 24 hours. A circadian pattern was detected
for mitochondrial ROS including superoxide anion
production. Of note, a significant 24-hour oscillation was also found for cellular redox state. Furthermore, mitochondrial ATP levels were rhythmic and
the maximum of ATP production paralleled the peak
of mitochondrial ROS level. Circadian rhythm was
also detected for calcium ion concentration in cytosol and mitochondria. Increase of ATP synthesis as
well as changes in calcium and ROS levels may activate AMPK. Our data demonstrate that in primary
human fibroblasts AMPK protein level and activity
fluctuate in an antiphase relationship with rhythmic
ATP production.
Summarizing, we suggest that AMPK might represent the molecular link between mitochondria energy
state and circadian clock machinery.
Acknowlegement:
Supported by grants: Sciex-NMSch to A.K. as well as Swiss
National foundation (SNF #310030_122572) and Synapsis
Foundation to A.E.
7th International Conference: Inhibitors of Protein Kinases, 2012
Posters37
P7
P8
Effect of new CK2 inhibitors and
pentabromobenzylisothioureas
on a KG-1 cell line
Dual activity of human
topoisomerase I and its
interactions with SRSF1
Mirosława Koronkiewicz1, Zygmunt
Kazimierczuk2, Kinga Szarpak1,
Zdzisław Chilmonczyk1
Krystiana A. Krzyśko1,2, Aleksandra Skrajna2,3,
Takao Ishikawa3, Katarzyna Grudziąż3, Karolina
Rokosz3, Bogdan Lesyng1,2, Krzysztof Staroń3
1National Medicines Institute, Warsaw, Poland; 2Institute
of Chemistry, Warsaw University of Life Sciences, Warsaw,
Poland
1Bioinformatics Laboratory, Mossakowski Medical
Research Centre, Polish Academy of Sciences,
Warsaw, Poland; 2Department of Biophysics and CoE
BioExploratorium, Faculty of Physics, University of
Warsaw, Warsaw, Poland; 3Institute of Biochemistry,
Faculty of Biology, University of Warsaw, Warsaw; Poland
e-mail: Mirosława Koronkiewicz <[email protected]>
Protein kinase CK2 (previously known as casein kinase II) is a very pleiotropic serine/threonine protein
kinase whose abnormally high levels have been documented in a number of cancers. Therefore, CK2 inhibitors could be considered as potential anticancer
drugs.
In this study we examined proapoptotic activity of
the known, as well as newly synthesized, CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzimidazole-2-N,N-dimethylamine (DMAT), 2-(4-methylpiperazin1-yl)-4,5,6,7-tetrabromo-1H -benzimidazole
(TBIPIP),
2-aminoethyleneamino-4,5,6,7-tetra­
bromo-1H-benzimidazole (TBIAEA) against human
acute myelogenous leukemia cell line (KG-1). The
cells were treated with CK2 inhibitors alone and in
combination with other new potential anticancer
agents: S-2,3,4,5,6-pentabromobenzylisothiouronium
bromides (ZKK-3, ZKK-9, ZKK-13). Evaluation
of apoptotic effects on KG-1 cells was carried out
using different cytometric methods. Flow cytometry
analyses were run on a FACSCalibur Flow Cytometer (Becton Dickinson Co., San Jose, CA, USA). All
tested compounds induced apoptosis in KG-1 cells
and synergistic effects of combination treatment. The
observed apoptotic effects were dose-dependent.
These results suggest that the examined compounds
exhibit promising anticancer activity.
Acknowledgement:
This study was supported by the Ministry of Science and
Higher Education (Poland), grant No. N N209 371439.
e-mail: Krystiana A. Krzyśko <[email protected]>
Human topoisomerase I (topo I) is a bifunctional enzyme that catalyses DNA relaxation and also phosphorylates an arginine⁄serine-rich domain (RS) of
SRSF1, which inhibits DNA nicking at the same time.
In vitro, the kinase activity is inhibited by the presence
of DNA. These results suggest that there is some
competition between two types of substrates (DNA
or protein being phosphorylated). Although they interact with topo I through different binding sites, one
substrate may negatively influence the binding of the
other. Therefore, understanding the complex spatial
structure of topoisomerase I and SRSF1 would bring
us closer to clarifying its mechanism of action as a
kinase.
The interaction sites of topo I with SRSF1 have been
identified using in vitro experiments, and on this basis
an in silico model of the topo I complex with SRSF1
was constructed. This led to the identification of
structural properties of both proteins engaged in the
complex. Analysis performed, using in silico and in vitro protein engineering methods, revealed the impact
of the RS domain organization of SRSF1 protein on
phosphorylation processes catalyzed by topo I. Moreover, in vitro assays confirmed that selected SRSF1
amino acid residues, indicated on the basis of the in
silico model, are crucial to the phosphorylation mechanism carried out by topo I.
Acknowledgements:
These studies were supported by the Biocentrum-Ochota project (POIG.02.03.00–00–003⁄09) and BST 1534⁄BF
funds.
7h International Conference: Inhibitors of Protein Kinases, 2012
38Posters
P9
ABI1 phosphatase negatively
regulates the activity of
SnRK2.4 and SnRK2.8
Ewa Krzywinska, Maria Bucholc, Arkadiusz
Ciesielski, Anna Kulik, Anna AnielskaMazur, Grazyna Dobrowolska
Institute of Biochemistry and Biophysics, PAS, Warsaw,
Poland
e-mail: Ewa Krzywinska <[email protected]>
SnRK2 family members are plant-specific protein kinases considered as important positive regulators of
response to drought and salinity. All SnRK2 kinases
(except SnRK2.9) are activated rapidly upon hyperosmotic stress [1, 2]. Kinases belonging to group 3 are
also strongly activated by ABA, whereas members of
group 2 are weakly activated by this hormone. SnRK2
kinases from group 1 are not activated by ABA. Activity of all SnRK2 kinases is regulated by reversible
phosphorylation of specific residues in their activation loop [3, 4]. ABA-activated kinases (group 3) are
dephosphorylated and inhibited by PP2C phosphatases from group A, which, in turn, are deactivated by
ABA-bound PYR/RCAR proteins [5–8]. However,
there is no information concerning kinases or phosphatases regulating the activity of ABA-independent
members of SnRK2. We have tested, using the yeast
two-hybrid system, whether SnRK2.4 (from group 1)
and SnRK2.8 (from group 2) interact with group A
PP2C phosphatases. ABI1 was found to be an interacting partner of both kinases, although the interaction between ABI1 and SnRK2.4 was weak. Binding
between ABI1 and SnRK2.4 was confirmed by pull-down assays and the BIFC method. In vitro studies
showed that ABI1 dephosphorylates and deactivates
both SnRK2.4 and SnRK2.8. To evaluate whether
ABI1 acts as a negative regulator of the ABA-independent SnRK2 kinases in vivo, the phosphatase was
coexpressed with SnRK2.4 or SnRK2.8 in protoplasts. Coexpression resulted in inactivation of the kinases. Our results show that ABI1, belonging to group
A of PP2C phosphatases, is a negative regulator of
ABA-independent SnRK2 kinases.
4. Boudsocq M, Droillard MJ, Barbier-Brygoo H, Lauriere C (2007) Different phosphorylation mechanisms are
involved in the activation of sucrose non-fermenting 1
related protein kinases 2 by osmotic stresses and abscisic
acid. Plant Mol Biol 63: 491–503.
5. Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R,
Park SY, et al. (2009) In vitro reconstruction of an abscisic
acid signaling pathway. Nature 462: 660–664.
6. Park SY, Fung P, Nishimura N, Jensen DR, Hiroaki F,
Zhao Y, et al. (2009) Abscisic acid inhibits PP2Cs via the
PYR/PYL family of ABA binding START proteins. Science
324: 1068–1071.
7. Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, et al. (2009) Type 2C protein phosphatases directly regulate abscisic acid-activated
protein kinases in Arabidopsis. Proc Natl Acad Sci USA 106:
17588–17593.
8. Vlad F, Rubio S, Rodrigues A, Sirichandra C, Belin C,
Robert N, et al. (2009) Protein phosphatases 2C regulate the
activation of the Snf1-related kinase OST1 by abscisic acid
in Arabidopsis. Plant Cell 21: 3170–3184.
References:
1. Boudsocq M, Barbier-Brygoo H, Lauriere C (2004) Identification of nine SNF1-related protein kinase 2 activated
by hyperosmotic and saline stresses in Arabidopsis thaliana. J
Biol Chem 279: 41758–41766.
2. Kobayashi Y, Yamamoto S, Minami H, Kagaya Y, Hattori
T (2004) Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic
stress and abscisic acid. Plant Cell 16: 1163–1177.
3. Burza AM, Pekala I, Sikora J, Siedlecki P, Malagocki P,
Bucholc M, et al. (2006) Nicotiana tabacum osmotic stressactivated kinase is regulated by phosphorylation on Ser-154
and Ser-158 in the kinase activation loop. J Biol Chem 281:
34299–34311.
7th International Conference: Inhibitors of Protein Kinases, 2012
Posters39
P10
Phosphoproteomic aproaches to
identification the candidates for
SnRK2 protein kinases targets
4. Shin R, Alvarez S, Burch AY, Jez JM, Schachtman DP
(2007) Phosphoproteomic identification of targets of the
Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes. Proc Natl Acad
Sci USA 104: 6460–6465.
Justyna Maszkowska, Janusz Dębski, Arkadiusz
Ciesielski, Michał Dadlez, Grażyna Dobrowolska
Institute of Biochemistry and Biophysics PAS, Warsaw,
Poland
e-mail: Justyna Maszkowska <[email protected]>
At the moment when stress signal is recognized plants
trigger specialized signaling pathways in which kinases and phosphatases are key components. The serine
/ threonine kinases SnRK2 (SNF1-related kinases 2)
play a role in plant response to abiotic stresses and
abscisic acid (ABA)-dependent plant development.
Even though SnRK2s are studied thoroughly, only a
few of their specific substrates have been identified. It
was shown that transcription factors bZIP [1, 2] and
ArtbohF NADPH oxidase [3] are phosphorylated by
ABA-dependent SnRK2. Thanks to phosphoproteomic analysis protein 14-3-3 was identified as a specific substrate for SnRK 2.8 from group 2 [4].
Therefore, we applied phosphoproteomic approaches, based on 2-dimensional electrophoresis and
mass spectrometry analysis, in order to identify the
differences in phosphorylation profile between wild
type Arabidopsis thaliana, several insert mutants and
Snrk2.10 over-expressing plants.
Because of strong activation SnRK2.10 during the
salt stress, we analyzed the phosphorylation profiles
in roots of hydroponically cultured wild type plants,
snrk2.10 insert mutants, and Snrk2.10 — over-expressing plants, in control conditions and after 30 min
of salinity stress.
We also compared the phosphorylation profiles in siliques of wild type plants and snrk2.2/2.3 double insert
knock-out as the phenotype in seeds is known for this
mutant.
These results allow us to identify several proteins as
candidates for SnRK2 substrates.
References:
1. Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida
R, Shinozaki K, Yamaguchi-Schinozaki K (2006) Abscisic
acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc Natl Acad Sci
USA 103: 1988–1993.
2. Sirichandra C, Davanture M, Turk BE, Zivy M, Valot
B, Leung J, Merlot S (2010) The Arabidopsis ABA-activated
kinase OST1 phosphorylates the bZIP transcription factor
ABF3 and creates a 14-3-3 binding site involved in its turnover. PLoS ONE 5: e13935.
3. Sirichandra C, Gu D, Hu HG, Davanture M, Lee S, Djaoui
M, Valot B, Zivy M, Leung J, Merlot S, Kwak J-M (2009)
Phosphorylation of the Arabidopsis ArtbohF NADPH oxidase by OST1 protein kinase. FEBS Lett 583: 2982–2986.
7h International Conference: Inhibitors of Protein Kinases, 2012
40Posters
P11
P12
Pyridine caffeic acid benzyl ester
(CABE) analogues target the ATP
pocket of atypical RIO kinases
Suppression of apoptosis in
prostate cancer PC3 cells is not a
consequence of phosphorylation
of Bid protein by the kinase CK2
Marcin Mielecki1, Krzysztof Krawiec2, Nicole
La Ronde-LeBlanc3, Borys Kierdaszuk2, Wiesław
Szeja4, Włodzimierz Zagórski1, Krystyna
Grzelak1, Waldemar Priebe5, Bogdan Lesyng2,6
1Polish
Academy of Sciences, Institute of Biochemistry
and Biophysics, Protein Biosynthesis Department,
Warsaw, Poland; 2University of Warsaw, Faculty of Physics,
Department of Biophysics, Warsaw, Poland; 3University of
Maryland, Department of Chemistry and Biochemistry,
USA; 4Silesian University of Technology, Faculty of
Chemistry, Gliwice, Poland; 5The University of Texas, MD
Anderson Cancer Center, Houston, USA; 6Polish Academy
of Sciences, Medical Research Center, Bioinformatics
Laboratory, Warsaw, Poland
e-mail: Marcin Mielecki <[email protected]>
Some features of atypical Rio kinases make them especially attractive as possible targets of anti-cancer
drugs. Rio proteins control cellular processes, such as
cell cycle and ribosome biogenesis. The intensity of
the latter one is significantly elevated in cancer cells.
Overexpression of Rio1 and Rio3 was detected in
some neoplasms. The crucial difference between atypical and “typical” kinases is based on unique structural elements. This fact creates new opportunity for
designing inhibitors with high selectivity towards Rio
kinases. The Rio1 protein from Archaeoglobus fulgidus
was selected as a case study to analyze the interaction of twelve newly synthesized caffeic acid benzyl
ester (CABE) derivatives with Rio kinases. Our results indicate that some analogues interact with Rio1
protein with low micromolar dissociation constants
and inhibit autophosphorylation activity with similar
concentration, ranges in a manner strongly dependent on ATP. In contrast, tyrphostin binding was not
ATP-dependent. Molecular docking studies revealed
two possible binding sites with the lowest inhibition
constants: the ATP-binding site and a flexible-loop
pocket. The first binding mode was justified by X-ray
diffraction analysis. Important structural elements of
the inhibitor molecules were recognized, and the main
mechanism of inhibition proposed. Selected inhibitors are capable of imitating the ATP conformation
in the Rio1 pocket. Our studies of these new derivatives revealed a new opportunity for designing novel
effective inhibitors of atypical protein kinases, using
the CABE scaffold.
Emilia Orzechowska, Krzysztof Staroń,
Joanna Trzcińska-Danielewicz
Department of Molecular Biology, Institute of
Biochemistry, University of Warsaw, Warsaw, Poland
e-mail: Emilia Orzechowska <[email protected]>
Protein kinase CK2 is a ubiquitous and highly conserved serine/threonine kinase. Many types of cancer cells, including prostate cancer, present elevated
CK2 activity. Phosphorylation of apoptotic proteins
by protein kinase CK2 is considered to be the mechanism responsible for suppression of apoptosis triggered by either extrinsic or intrinsic pathways.
Herein we present studies on the effect of phosphorylation of the Bid protein on induction of apoptosis; in prostate cancer cells. An experimental system
allowing to sensitise PC3 cells to TRAIL-mediated
apoptosis was developed through the introduction of
exogenous Bid protein fused with a Tat sequence. It
was assumed that, if suppression of apoptosis is associated with phosphorylation of proteins by protein
kinase CK2, the use of mutated Bid proteins which
are incapable to be phosphorylated, should cause a
greater sensitization effect. We observed that the effect of the mutated proteins did not differ with respect to the wild variant. This means that suppression of apoptosis in PC3 cells is not a consequence
of phosphorylation the Bid protein by protein kinase
CK2, but probably is caused; by the low levels of endogenous proteins in the cell.
Acknowledgements:
These studies were supported by the Ministry of Science
and Higher Education (PBZ-MIN-014/P05/2004) (Poland), the Biocentrum-Ochota project (POIG 02.03.00-00003/09) and Institute of Biochemistry and Biophysics.
7th International Conference: Inhibitors of Protein Kinases, 2012
Posters41
P13
P14
Responsive nonmetal probes
for mapping activity of protein
kinases in living cells using timegated luminescence microscopy
Bisubstrate fluorescent probe
for protein kinase CK2
Angela Vaasa1, Kadri Ligi1,
Lawrence W. Miller2, Asko Uri1
1Institute of Chemistry, University of Tartu, Tartu, Estonia;
2Department of Chemistry, University of Illinois at
Erki Enkvist1, Kaido Viht1, Nils Bischoff 2, Jürgen
Vahter1, Siiri Saaver 1, Gerda Raidaru1, OlafGeorg Issinger3, Karsten Niefind2, Asko Uri1
1Institute of Chemistry, University of Tartu, Tartu, Estonia;
2Institut für Biochemie, Universität zu Köln, Köln,
Germany; 3Institut for Biokemi og Molekylær Biologi,
Chicago, Chicago, IL, USA
Syddansk Universitet, Odense, Denmark
e-mail: Angela Vaasa <[email protected]>
e-mail: Kaido Viht <[email protected]>
Protein kinases (PK) and their signalling pathways
have been the main targets for cancer drug development during the last decades. Therefore, the development of sensitive, selective and reliable methods for
monitoring localization and activity of PKs in living
cells for understanding complex signalling cascades
and explaining the roles of kinases in human diseases
are of great importance.
We have recently developed a novel and sensitive
method based on the application of responsive smallmolecular photoluminescent probes (ARC-Lums) for
monitoring the activity of basophilic protein kinases
in living cells. ARC-Lum probes are bisubstrate-analogue inhibitors labelled with a fluorescent dye and, if
bound to a protein kinase upon excitation by near-UV
light (λ<375 nm) emit red light with long lifetime (20–
250 µs) and can therefore be applied for time-gated
luminescence (TGL) measurements. As ARC-Lum
probes are cell plasma membrane permeable, stable
in intracellular milieu, and bind to PKs with high affinity (Kd < 100 pM towards PKAc) they are suitable
for mapping of PK activity in living cells using TGL
microscopy. We have shown that ARC-Lum probes
taken up by MDCKII cells specifically associate with
active kinases, resulting in a long lifetime signal that is
well separable from the fluorescence background of
the cells. To study the formation of the TGL signal
of ARC-Lum/kinase complexes in cells, the PKA was
activated or inhibited and the dynamics of the TGL
signal monitored. Activation of PKA resulted in a
rapid formation of TGL signal both in the cytoplasm
and nucleus, hence, such probes are of interest for
high-content analysis of cellular responses to different extracellular and intracellular stimuli, but also for
signal transduction studies.
Protein kinase CK2 is an acidophilic serine/threonine kinase, which regulates a number of cellular processes. The activity of CK2 is involved in cell growth,
proliferation, angiogenesis and suppression of apoptosis, making the kinase a potential target for cancer
chemotherapy. The characterization of potential drug
candidates and evaluation of the structure and functioning of CK2 require fluorescent probes that bind
to the active site of CK2 with high affinity and selectivity. We have developed bisubstrate inhibitors for
CK2 by conjugating two fragments, a moderately potent and selective ATP-competitive inhibitor of CK2
4,5,6,7-tetrabromo-1H-benzimidazol (TBBi) and
peptide substrate mimicking oligoaspartate. The fragments were conjugated via hydrophobic linkers with
different lengths. The most potent inhibitor with subnanomolar Ki was labelled with a fluorescent dye to
yield a very selective fluorescent probe with subnanomolar affinity towards both the free catalytic subunit
and holoenzyme of CK2. The probe was used in a
binding assay with fluorescence anisotropy detection
to measure the concentration of active CK2 and to
characterize ligands binding to the active site of CK2
in displacement experiments.
7h International Conference: Inhibitors of Protein Kinases, 2012
42Posters
P15
P16
In silico design and in vitro evaluation
of novel ASK1 inhibitors
Discovery of novel chemotypes
of kinase inhibitors by structurebased virtual screening
Galyna Volynets, Volodymyr Bdzhola,
Andriy Golub, Oleksandr Kukharenko, Pavel
Areshkov, Vadym Kavsan, Sergiy Yarmoluk
Institute of Molecular Biology and Genetics of NAS of
Ukraine, Kyiv, Ukraine
Hongtao Zhao, Danzhi Huang, Amedeo Caflisch
Department of Biochemistry, University of Zurich, Zurich,
Switzerland
e-mail: Hongtao Zhao <[email protected]>
e-mail: Galyna Volynetes <[email protected]>
Elevated activity of apoptosis signal-regulating kinase
1 (ASK1) is associated with a number of human disorders, and inhibitors of ASK1 may become important compounds for pharmaceutical application.
To discover ASK1 inhibitors we performed a screening program using both in silico and in vitro approaches. The virtual screening experiments were carried
out targeting the ATP binding site of ASK1 with a
library of 156 000 organic compounds. AutoDock
and DOCK software were used to conduct receptorligand flexible docking. The best-scored compounds
of different chemical classes were taken for evaluation in the kinase assay.
The in vitro tests revealed that compound ethyl 2,7-dioxo-2,7-dihydro-3H-naphtho[1,2,3-de]quinoline1-carboxylate (NQDI-1) inhibits ASK1 with a Ki of
500 nM.
We reported on the discovery of novel chemotypes
of kinase inhibitors with targets including EphB4,
Abl1, JAK2, SYK and ZAP70, based on an in silico
structure-based screening platform, consisting of an
accurate flexible docking tool, an accurate scoring
function, an in silico fragment-based drug discovery
approach, and molecular dynamics-based manipulation of protein structures. One lead compound derived from a 300 nM EphB4 inhibitor identified in
silico shows cellular activity with an EC50 below 6 nM,
good solubility and a good selectivity profile when
screened on a panel of 139 kinases. Antiproliferative
screening on a panel of cancer cell lines conducted in
NIH indicates that it is potent for renal cancers of 8
screened cell lines, two colon cell lines (COLO 205
and HT29), one melanoma cell line (LOX IMVI) and
one breast cancer cell line (MDA-MB-231/ATCC).
It was also found that derivatives of 2-thioxo-thiazolidin-4-one possess inhibitory activity towards ASK1.
The most active compound of this class inhibits
ASK1 with IC50 = 2 µM. In-depth study of these
compounds using a pre-selected library of 2-thioxothiazolidin-4-one derivatives led to identification of
2-{5-[5-(3,4-dichloro-phenyl)-furan-2-ylmethylene]4-oxo-2-thioxo-thiazolidin-3-yl}-propanoic
acid
(PFTA-1) inhibiting ASK1 with a Ki of 340 nM.
Our preliminary studies on specificity of these compounds demonstrated that NQDI-1 and PFTA-1 are
selective inhibitors of ASK1. At the next step, these
compounds were investigated in HEK293 cells during
apoptosis induced by CHI3L2 to determine whether
they are also active in cellular systems. In these experiments inhibitors demonstrated considerable cytoprotective effect.
Structural optimization and further biological evaluation of identified ASK1 inhibitors are under way.
7th International Conference: Inhibitors of Protein Kinases, 2012