The enzyme O-GlcNAc transferase (OGT) is a key enzyme in the process of N-acetylglucosamination, which means that it is also involved in numerous cellular processes. Its deregulation is associated with many diseases, making it a potential target for patients' treatment. For this purpose, we need an efficient molecular tool that would enable us to study the role of OGT. In recent years, chimeric molecules, also known as PROTAC molecules, have come to the forefront. PROTAC molecules do not only inhibit the activity of target proteins but lead to their degradation by utilizing the ubiquitin-proteasome system for their action.
As a part of this master's thesis, we evaluated the biological effect of twelve chimeric OGT enzyme degraders synthesized at the Department of Pharmaceutical Chemistry (UL FFA). In the design of PROTAC molecules, the inhibitor OSMI-4 and various ligands for E3 ligases (VHL, CRBN and IAP) were used as a ligand for OGT. The MM.1S cell line was used as a cellular model, as it expresses both the target protein OGT and all three used ligases. In the first part, we examined the potential impact of PROTAC molecules on the degradation of the OGT enzyme. We evaluated the OGT protein levels using the western blot method and found that at a concentration of 1 µM, none of the PROTAC molecules caused visible degradation of the enzyme. As chimeric degraders have large molecular weight, we investigated whether the absence of effects might be due to poor cellular permeability. We found that molecules based on ligands for the ligases IAP and CRBN cross the membrane, as we confirmed the autodegradation of cIAP-1 and the degradation of the CRBN neosubstrate IKZF1. Inhibition of OGT is known to lead to compensatory mechanisms that result in increased levels of the enzyme, therefore we furthter tested whether the absence of OGT degradation was due to a feedback loop. To test this we inhibited de novo protein synthesis in our cells by adding cycloheximide and then exposed them to the chimeric inhibitor 12. We found that PROTAC 12 did not cause visible degradation of OGT even in the presence of cycloheximide. We also investigated the possible impact of PROTAC 12 on the secretion of selected inflammatory mediators. In this case as well, we did not observe a noticeable effect on the cell phenotype.
The results thus indicate that none of the twelve compounds leads to the degradation of the OGT enzyme. Therefore, further modifications of the PROTAC molecules or optimization of the experimental models will be necessary in order to achieve a successful degradation of the OGT enzyme to investigate the influence of the enzyme in cellular homeostasis.
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