Guo, J; (2017) Fragment-based drug discovery and structural biology of norovirus 3CL protease and other targets. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

X-ray crystallography has been widely used for determination of protein structures for many years. Protein structures are essential in advancing our understanding of their function, thus providing useful information in many areas, such as drug discovery. Seven different proteins have been studied by X-ray crystallography and other methods, and the information has been gained with the intention of applying it to real-life challenges in the biomedical sphere. The outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes a 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential to act as a target for antiviral drugs. The ligand-free crystal structure of the Southampton norovirus 3CLpro (SV3CP) had been determined to 1.3 Å and a system for growing high-quality crystals has been established. This allowed for crystal-based fragment screening to be performed with non-covalent fragments, which identified several hits that will guide drug discovery for SV3CP. Screening with covalent fragments is on-going. Three mutants of Bacillus megaterium porphobilinogen deaminase affecting a key catalytic residue have been analysed. Comparison with the wild-type enzyme shows significant domain movements and suggests that the enzyme adopts ‘open’ and ‘closed’ conformations in response to substrate binding. Potato cathepsin D inhibitor (PDI) is a glycoprotein composed of 188 amino acids which inhibits both the aspartic protease cathepsin D and the serine protease trypsin. The first crystal structure of PDI has been determined to a resolution of 2.1 Å, revealing that PDI adopts a typical β-trefoil fold with several protruding inhibitory loops, as is typical of the Kunitz-family protease inhibitors. The family B DNA polymerase from Pyrobaculum calidifontis (Pc-polymerase) is distinct from other homologues (e.g. those from T. gorgonarius, T. Kodakarensis and P. furiosus) with a low amino acid sequence identity of 37%. The crystal structure of Pc-polymerase has been refined to a resolution of 2.8 Å and several unique features have been identified which may account for its high processivity and thermostability. A complex model with the primer-template duplex of DNA suggests that large movements of the thumb domain occur upon DNA binding. The type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities and has many potential applications in the industrial food processing sector. The crystal structure of TK-PUL represents the first type III pullulan hydrolase to be analysed, revealing N-terminal and C-terminal domains with differences from homologous structures. The crystal structure of the L-asparaginase from Thermococcus kodakarensis (TkA) has been determined at 2.2 Å, revealing a number of distinctive structural features. The enzyme has many applications in food processing and chemotherapy. Finally, on-going studies of juvenile hormone diol kinase (JHDK) are described and a summary of the structures presented in earlier chapters of the thesis is given.

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