4th EucheMs chemistry congress

Poster Session 1
s892
chem. Listy 106, s587–s1425 (2012)
Poster session 1 - life sciences
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deveLoPMent of new no-ASA-derivAtiveS for
the treAtMent of ChroniC LyMPhoCytiC
LeuKeMiA (CLL)
A. BerKeSSeL 1 , M. KrüGer 1
1 Institute of Organic Chemistry, Department of Chemistry,
Köln, Germany
no-ASA was originally developed by NicOx to treat human
colon cancer. [1] It was found later that this Aspirin-derivative is
effective for treating many other human cancers, such as prostate,
pancreatic, lung, skin, breast, and bladder cancers, and is also
suitable for the treatment of type-2-diabetes and the
cardioprotection of animal hearts. [3] Kreuzer et al. reported that
NO-ASA is also very effective against chronic lymphocytic
leukemia (CLL). [3] According to the current mechanistic scenario
NO-ASA is initially metabolized to different active compounds.
The key compounds are quinone methide (QM) and nitrate anion.
QM contributes the major part to the anticancer effect of
NO-ASA, due to the fact that it is a very reactive Michael
acceptor. Various studies point out that quinone methide reacts
with many nucleophiles, alkylates the DNA and proteins (like
glutathion). The nitrate anion effects S-nitrosylation and tyrosine
nitration. All reactions lead to modifications in the cell-signaling
pathways and ultimately result in the apoptosis of cells. [2,4] Based
on the promising results achieved with NO-ASA, we decided to
syn-the-s-ise several new NO-ASA-de-rivatives. Our efforts
resulted in the discovery of some more potent NO-ASA-derivatives.
Several in vitro assays and in vivo xenograft nude mice models
showed sig-nificantly improved and sele-ctive cytotoxicity of some
of our com-pounds in comparison to NO-ASA. For future,
mechanistic studies, we are intending to label our compounds in
particular with regard to bio-orthogonal reactions and mass
spectroscopy.
references:
1. P. Del Soldato, WO 02/30866 A1, PCT/EP01/11664, 2002.
2. K. Kashfi, B. Rigas, Biochem. Soc. Trans. 2005, 33,
701–704.
3. R. Razavi, I. Gehrke, K.-A. Kreuzer et al., Clin. Cancer
Res. 2011, 17, 286–293.
4. J. L. Williams, P. Jia, N. Ouyang et al., Exp. Cell Res.
2011, 10, 1359–1367.
Keywords: Cancer; Synthesis design; Bioorganic chemistry;
Biotransformations; Cytotoxicity;
4 th EucheMs chemistry congress
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StruCturAL ASSeMBLy of hexoSAMinidASe
CoMPLex
z. KuKACKA 1 , P. PoMPACh 2 , P. MAn 2 , P. novAK 2
1 Faculty of Science, Department of Biochemistry, Prague,
Czech Republic
2 Institute of Microbiology, Laboratory of Molecular Structure
Characterization, Prague, Czech Republic
Background: Fungal β-N-acetylhexosaminidases
EC 3.2.1.52) are inducible extracellular glycosidases involved in
many biological processes. The native enzyme from Aspergillus
oryzae CCF 1066 is composed of two propeptides (each 10 kDa)
and two catalytic units (each 65 kDa). The propeptides are
noncovalently associated with the catalytic units and are essential
for the enzyme activity.
objectives: In this study we would like to uncover the
position where the propeptide is associated with the catalytic unit.
Methods: β-N-acetylhexosaminidase was purified from the
medium of the producing organism. For EDC (1-Ethyl-3-(3-
-dimethylaminopropyl) carbodiimide) experiment the enzyme
was transferred by gel filtration to 50mM Pyridine pH 5.5,
150mM NaCl and for DSG (disuccinimidyl glutarate) experiment
to 50mM Triethylamine carbonate pH 7.5. After the cross-linking
reaction was over, the products of enzyme were separated by SDS
electrophoresis. In gel digestion was performed and the resulting
peptides were analyzed by LC-ESI FT MS (Agilent 1200,
APEX-Ultra)
results: To obtain good reproducibility and high yields of
cross-linking reactions, different types and concentrations of
cross-linking reagents together with optimization of enzyme
concentrations in the reaction was performed. The best results
were achieved by using DSG and EDC reagents. Enzyme
concentration was set to 0.5 mg/ml. The ratio of cross-linking
agents over the enzyme was 100 fold access of EDC and 50 fold
access of DSG. By LC-ESI FT MS analysis were found several
intermolecular (A80-S96 with I347-E365) and few intramolecular
cross-links (A102-K114 with D409-Y434). Obtained results are
in a good agreement with X-ray diffraction data.
Conclusions: Combinating chemical cross-linking and high
resolution mass spectrometry we uncover the region of
propeptide-catalytic unit ineraction. Moreover we revealed that
the structural changes of the catalytic unit depend on the
presence/absence of the propeptide molecule.
Acknowledgement: This work was supported by grants
P207/10/1040 and P207/10/1934 of the Czech Science
Foundation.
Keywords: Glycoproteins; Enzyme models; Mass spectrometry;
X-Ray diffraction;
AUGUst 26–30, 2012, PrAGUE, cZEcH rEPUbLIc