Design, Synthese und Testung potentieller Inhibitoren der Golgi-α-Mannosidase II
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Abstract
The enzyme Golgi-α-mannosidase II (GMII) plays a crucial role in the N-glycosylation pathway, which is involved in the metastatic process of several types of cancer. It provides essential precursors of glycosyl chains which are neccesary for cell recognition through interaction with selectins on the cell surface. In different cancer cells, modified glycosylation patterns were found. Their origin is believed to be the misregulation of protein expression which leads to unnatural high concentration of glycosly transferases. In clinical trials, the reduction of metastasis and tumor growth by inhibition oft he GMII has been shown. Hence the inhibition of this enzyme represents a promising target in cancer therapy.
As a new class of active substances covalent reversible inhibitors are to be developed and tested. These should combine the advantages of covalent inhibitors omitting their disadvantages. These inhibitors require extremely precise adaption of the ligand structure to its receptor and in the reactivity of the inhibitor in order to form a covalent reversible adduct with the enzyme. To predict potential inhibitors molecular docking methods and quantum mechanics/molecular mechanics (QM/MM) methods were used. The main task of this thesis was the synthesis of these structures as well as the synthesis of fluorescent substrates to establish new assays with α-mannosidases and β-glucosidases. In total, 41 substances were synthesized. An overview of the individual subjects is shown in figure 1.
In a docking assisted de-novo-design a structure (L039) was identified, to meet all identified requirements for a covalent reversible inhibition of GMII in QM/MM-calculation. In further docking studies, several structures from the substance class of monocyclic N,O-acetals were identified as potential covalent reversible inhibitors. Furthermore, investigations on a spiroacetal were carried out.
In the context of this work, 25 monocyclic N,O-acetals, two derivatives of azamannose as potential building block, isofagomine and its 5-isomer, four potential inhibiors of a potential allosteric site of GMII and fragments of L039 were synthesized, purified and analysed spectrometroscopically. None of those compounds showed significant inhibition of dGMII.
To investigate the activity of potential inhibitors, enzyme assays were carried out. For this task, two substrates were prepared in four- and five-steps synthesis, as well as a reference inhibitor of β-glucosidases (isofagomine) in a six-step synthesis to validate the assay systems.