Apoptosis is a type of programmed cell death and is essential for the normal functioning of an organism. Both the absence of apoptosis and excessive cell death are associated with several pathological conditions. Tumor cells can avoid apoptosis, whereas excessive apoptosis is associated with neurodegenerative and autoimmune diseases and often with the side effects of chemotherapeutics. An irreversible step that leads to apoptosis is permeabilization of the outer mitochondrial membrane, where the Bcl-2 family of proteins plays a key role. In this master’s thesis, we will focus on the Bax protein. Inhibitors of Bax can target either its conformational activation, translocation to the mitochondrial membrane, or subsequent oligomerization. Although the inhibition of Bax protein by small molecules represents a promising strategy for the development of apoptosis modulators, the lack of defined active site requires utilization of modern modulation methods. One such strategy is the so-called proteolysis targeting chimeras (PROTACs) approach. PROTAC molecules consist of three key parts: a ligand for the target protein, a ligand for the E3 ubiquitin ligase, and a linker that connects the two ligands. If the PROTAC molecule has the appropriate properties, the target protein may undergo proteolytic degradation via the ubiquitin-proteasomal system after the formation of the 'target-PROTAC-E3 ligase' ternary complex.
Based on the previously described Bax inhibitor, i.e. compound DAN004, we synthesized five structurally similar molecules. With the method of 'migration of the bromine atom' and its subsequent replacement with a hydroxyl group, we wanted to improve the inhibitory activity and investigate the relationship between the structure of molecules and their inhibitory activities. Two potential degraders (PROTAC molecules) of the Bax protein were also prepared from the two inhibitors. The resynthesized compound DAN004, the new inhibitors and PROTAC molecules were evaluated at the enzyme level by a liposomal permeabilization assay. At the cellular level, their ability to inhibit triggered programmed cell death was assessed. Only one of the compounds showed a slight improvement in inhibition compared to DAN004, whereas the others had an affinity for the Bax protein in the same concentration range as the parent compound DAN004. The position of the bromine and its replacement by a hydroxyl group thus had no significant effect on Bax inhibition. None of the compounds rescued the cells from induced apoptosis under the selected test conditions on the three cancer cell lines. Evaluation of the ability to degrade the target is currently underway by colleagues in the United States. Nevertheless, the obtained experimental data represent a valuable guidance for future research in the field of inhibition and degradation of Bax protein with compounds of this structural class.
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