In the past 30 years there were several studies that have thoroughly characterized the
structure and the physiological function of the ubiquitin proteasome system. This is a
sophisticated 2.5 MDa protein complex named 26S proteasome, which consists of 20S core
particle and two terminal 19S regulatory particles.
The proteasome is a complex multicatalytic enzymatic system that regulates vital
physiological and pathological cellular processes through selective breakdown of the
proteins. Identification and removal of misfolded, damaged and toxic proteins is crucial for
the regulation and maintenance of cell homeostasis.
Proteins that are involved in the processes of carcinogenesis and cancer cell survival are
also substrates for the proteasome. Its inhibition in cancer cells therefore leads to
accumulation of pro-apoptotic proteins and induction of cell death.
The cancer drug discovery puts a significant emphasis on the identification of new and
cancer-specific molecular targets. One of the most recently discovered and very promising
targets is also the immunoproteasome, tightly regulated, highly-specific system that is
responsible for cellular protein turnover. The discovery that cancer cells are more
susceptive to proteasome inhibition than other cells had a strong impact on the discovery
and synthesis of novel therapeutically useful inhibitors. The knowledge and recent findings
associated with the structure and the functions of the ubiquitin proteasome system make
this enzyme machinery a promising target in cancer treatment.
Within this masters degree, we successfully synthesized several potential
imunoproteasome inhibitors. The prepared compounds had a psoralen core scaffold with
methyl group bound at the position 4. With the aim to explore the chemical space, we
introduced several carefully planned substituents on the main psoralen structure.
The synthesis of compounds was performed in several consecutive reaction steps. Firstly,
we synthesized the 7-hydroxycoumarin ring by reacting diethyl 2-acethylsuccinate or
diethyl 2-acetylpentanedionate with resorcinol. Then, different 2-bromoacetophenones
were introduced to properly modify the hydroxyl group. Afterwards, the condensation
reaction was carried out, leading to the formation of the core psoralen ring. Finally, we introduced three different electrophilic moieties at the C-terminus, such as 1-
hydroxypyrrolidine-2,5-dione, 2-aminoacetonitrile or 2-(methylamino)acetonitrile. The
purpose of this last step was to achieve covalent bonding with the catalytic amino acid in
the immunoproteasome active site.
All compounds were biochemically evaluated at the Department of Clinical Biochemistry.
The residual activities of the chymotrypsin-like imunoproteasome subunit after the
addition of compounds were determined. Based on the data, compund 5c showed the most
potent inhibitory activity. On the basis of results from inhibition assays, we can also
conclude that the transformation of the C-terminal carboxylic acid into an activated ester is
crucial for improving inhibition; most probably because of the formation of covalent bonds
within the active site of the immunoproteasome. Introduction of an amide bond and Ncianomethylamides
as electrophiles did not improve inhibitory effect. In addition, bromine
at the para position of the phenyl fragment led to diminished affinity for binding into the
active site of the immunoproteasome.
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