Authors:	Johe, Patrick
Title:	Biochemische und biophysikalische Untersuchung der parasitären Cysteinprotease Rhodesain und deren Inhibition durch kovalent-reversible Inhibitoren
Online publication date:	13-Apr-2021
Year of first publication:	2021
Language:	german
Abstract:	Despite its high mortality rate and the huge number of people at risk, Human African Trypanosomiasis (HAT), also called sleeping sickness, still counts as a neglected tropical disease according to the World Health Organization. HAT is caused by different subspecies of African trypanosomes. These unicellular parasites can be transmitted to a human host during the blood meal of a Tse-Tse-fly. Only six drugs with severe side effects are currently available against HAT for which resistances have already been reported. Therefore, the development of new treatment strategies against HAT is urgent. A promising cellular target is the cathepsin L protease (CathL) of Trypanosoma brucei rhodesiense (TbCathL or rhodesain). This enzyme is essential for the parasite’s survival and is involved in the parasite’s pathogenicity, including its ability to cross the blood-brain barrier and to escape the immune system of the host. Like other lysosomal proteases rhodesain is expressed as an inactive pro-form that requires cleavage of its N-terminal propeptide to yield a catalytically active protease. To elucidate the structural basics of this maturation process and the auto-inhibition of the protease by its propeptide, the structure of pro-rhodesain was first time solved using X-ray crystallography. Intriguingly, the propeptide forms a trypanosome-specific -helix that blocks the active site and undergoes molecular interactions with the catalytic domain that are reminiscent of the covalent cysteine protease inhibitor K11777. Thereby, the crystal structure provides a rational explanation for the efficacy of this inhibitor. The pro-rhodesain crystal structure also provided the basis for the investigation of the pH-dependent auto-activation of the rhodesain zymogen through a combination of molecular dynamic simulations (MD), mutagenesis studies and various spectroscopic measurements. To facilitate these investigations a heterologous expression and purification protocol for rhodesain from E. coli was established. In addition, the binding of rhodesain to different (-fluoro-)vinyl sulfones (VS), which inhibit the enzyme in a covalent reversible or irreversible manner, was thermodynamically and kinetically determined using isothermal titration calorimetry (ITC) and a photoinduced electron transfer-fluorescence correlation spectroscopy (PET-FCS) system. To inhibit rhodesain, the inhibitors must reach the lysosome of the trypanosome, in which the cysteine protease is located. Therefore, synthesis and analysis options of an organelle-specific drug delivery system based on VS-RNA aptamer bioconjugates were examined. This approach was inspired by an RNA aptamer that, after binding to the flagellar pocket of T. brucei, is transported into the lysosome of the parasite. Thus, these VS-RNA aptamer bioconjugates might help minimizing side effects of cysteine protease inhibitors in the long run. In summary, this work provides detailed information on the structure of pro-rhodesain, its pH-dependent auto-activation and the drug-target interactions between rhodesain and vinyl sulfone based inhibitors. These insights as well as methods and strategies that have been developed to examine the protease and its interactions with small molecules enable further investigation and optimization of potential drug candidates.
DDC:	540 Chemie 540 Chemistry and allied sciences 570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-5695
URN:	urn:nbn:de:hebis:77-openscience-df933fc3-3b10-43a9-9d6f-41bf3caee51e2
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	xxii, 233 Seiten, Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen