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Significance of the S1 subsite of rhomboid intramembrane proteases for catalysis and inhibitor design
Kučerová, Jolana ; Stříšovský, Kvido (advisor) ; Hodek, Petr (referee)
This thesis focuses on the development of specific inhibitors of rhomboid intramembrane proteases. These inhibitors are needed for the cell-biological investigation of rhomboid proteases and their potential pharmacological targeting, as rhomboid proteases have been associated with various diseases, such as malaria, Parkinson's disease, cancer or toxoplasmosis. The thesis advisor's laboratory has recently discovered the first such group of compounds, the peptidyl ketoamides. To exploit them fully, it is necessary to examine their properties and the possibilities of their modifications. In this work, synthetic fluorogenic substrates and enzyme kinetics were used to examine the possibilities of exploiting the S1 subsite in the rhomboid active site for rhomboid inhibitor design. Furthermore, using variants of these substrates modified by unnatural amino acids in the P1 position, the mechanism of water transfer to the rhomboid active site was investigated. Comparison of cleavage rates of ten fluorogenic substrates modified in the P1 position revealed that the E. coli rhomboid protease GlpG strongly prefers side chains -CH2-CH3 and -CH3 in the P1 position (i.e. binding into the S1 subsite). This trend was apparent also with peptidyl ketoamide inhibitors. The present substrate and inhibitor study suggests...
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