Structure-based design of new antiviral substances against the Venezuelan equine encephalitis virus

Sammanfattning

The Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus responsible for severe outbreaks and epidemics in the Americas. The nonstructural protein 2 (nsP2) cysteine protease plays a fundamental role in the VEEV replication process, and is therefore, an attractive drug target for structure-based drug design using x-ray crystallography. Generating protein crystals of sufficient quality for x-ray crystallography is challenging and time-consuming. This thesis aims to develop methods to obtain such crystals by optimizing crystallization conditions. Moreover, this thesis also investigates interactions between potential inhibitors and the nsP2 protease experimentally by conducting biochemical assays. By screening numerous crystallization conditions with variations in pH, additives, precipitants and protein concentrations, a handful of conditions were found to generate crystals of high quality. Their quality was later confirmed by x-ray crystallography experiments. Furthermore, the overall collected data allowed structures with resolutions below 2 Å to be obtained. Some of these collected data sets are believed to comprise the nsP2 protease in complex with an inhibitor. Biochemical assays provided information about steady-state kinetic parameters of the nsP2 and the potency of three potential inhibitors. Three-dimensional structures of the nsP2-inhibitor-complex together with information about interactions between potential inhibitors and the nsP2, will provide information to further design inhibitors based on structure.