Investigating resistance in infectious diseases: Functional and structural characterization of the HIV-1 protease and the fungal lanosterol 14α-demethylase(Erg11p)
|dc.contributor.author||Huschmann, Franziska Ulrike|
|dc.identifier.citation||Huschmann, F. U. (2014). Investigating resistance in infectious diseases: Functional and structural characterization of the HIV-1 protease and the fungal lanosterol 14α-demethylase(Erg11p) (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4924||en|
|dc.description.abstract||Infectious diseases are a worldwide health problem. The lack of effective and affordable medications for infections such as AIDS and systemic fungal infections poses a serious concern in modern medicine. Furthermore, most available drugs are prone to the development of resistance caused by mutations in their protein targets. HIV-1 protease (PR) is one of the targets for the treatment of human immunodeficiency virus (HIV) infections. The small retroviral aspartyl protease is essential for the life-cycle of HIV-1, the retrovirus that causes AIDS. Lanosterol 14α-demethylase (Erg11p or Cyp51) is the target of azole antifungals, the class of antifungal drugs most commonly used to treat patients with life-threatening fungal infections. Erg11p is a membrane-anchored cytochrome P450 enzyme that catalyzes a key step in the biosynthetic pathway of ergosterol, an essential component of the fungal cell membrane. Both AIDS and systemic fungal infections remain deadly diseases, the treatments of which are hampered by drug resistance. The aim of this study was the functional and structural characterization of the two prominent drug targets, HIV-1 PR and Erg11p, in order to establish a platform for the development of improved drugs less susceptible to target-based resistance. HIV-1 PR was expressed in Escherichia coli strain BL-21 (DE3) and recovered from inclusion bodies by cell disruption and sonication. The HIV-1 PR was then purified in a denatured state by size exclusion chromatography and high-performance liquid chromatography. After dialysis, the HIV-1 PR was refolded and crystallized in the presence, and in the absence, of novel inhibitors SZ-1 and 89. Diffraction data sets for the crystals were collected to a resolution of 1.4-1.7 Å at the Australian Synchrotron and programs iMOSFLM, PHASER, REFMAC5 (from the CCP4 suite) and COOT were used to solve the structures. The crystal structures of the HIV-1 PR with and without inhibitor SZ-1 revealed a peptide-like density in the active site. The modelled peptide (Ala-Ala-Asn-Ile-OH) appeared to be a cleavage product of an unknown protein that prevented the inhibitors from binding and was likely to have beenderived from the E. coli expression system. Further studies are required to eliminate interference of the unknown peptide with inhibitor binding. In contrast, crystallographic data revealed binding of inhibitor 89 to HIV-1 PR. Furthermore, flexibility of the norleucine ligand at P2 of the macrocyclic inhibitor in binding to the S2 sub-site of the HIV-1 PR was observed. E. coli was a poor expression host for genetically modified constructs of Erg11p from the major fungal pathogen Candida albicans (CaErg11p). Satisfactory expression of the full-length membrane-anchored CaErg11p was achieved in Saccharomyces cerevisiae. Milligram quantities of stable CaErg11p6xHis suitable for crystallographic studies were obtained by solubilization with n-decyl-β-D-maltopyranoside (DM), Ni-NTA affinity purification and size exclusion chromatography. The same conditions were successfully applied to Erg11p from another fungal pathogen, Candida glabrata (CgErg11p6xHis). Azole binding assays were performed for purified CaErg11p6xHis and CgErg11p6xHis using type II binding that measures the shift caused in the wavelength of the heme peak in the absorbance spectrum, and revealed tight binding of the drugs. Novel compounds designed to occupy additional binding sites to current azoles did not show type II binding. Concurrently, S. cerevisiae ScErg11p6xHis crystals were obtained by Dr. Brian Monk and our collaborators at the University of California, San Francisco (UCSF) Membrane Protein Expression Center, using the above CaErg11p6xHis purification protocol. Data collection was carried out at the Advanced Light Source in Berkeley, California and six ScErg11p6xHis structures (ligand-free, with substrate lanosterol, the pseudosubstrate estriol, and the triazole inhibitors itraconazole, fluconazole and voriconazole) were solved at the University of Otago and the UCSF. The 1.9-2.8 Å ScErg11p6xHis crystal structures are the first structures for any full-length cytochrome P450 enzyme showing the transmembrane anchor. The two N-terminal helices were oriented ~60° to each other and positioned the substrate entry channel of ScErg11p6xHis so that it was facing the lipid bilayer. The crystal structures also indicated a proposed product egress channel. Further they revealed how triazole antifungals bind to ScErg11p6xHis and how they inhibit the reaction. Last but not least, they identified possible interactions that confer triazole resistance. Crystals of His6-tagged Erg11ps from the pathogens C. albicans and C. glabrata were obtained but further refinement is needed to obtain diffracting crystals. In conclusion, this work has generated reproducible methods to obtain HIV-1 PR and Erg11p crystals. People suffering from AIDS are especially susceptible to systemic fungal infections and their lives rely on effective, specific, and mutation-insensitive drugs. This work provides a foundation for drug discovery and development to combat significant viral and fungal diseases.|
|dc.publisher||University of Otago|
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|dc.title||Investigating resistance in infectious diseases: Functional and structural characterization of the HIV-1 protease and the fungal lanosterol 14α-demethylase(Erg11p)|
|thesis.degree.discipline||School of Pharmacy|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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