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

Abstract

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.

Popular Abstract

Generating high-quality protein crystals is essential for structure determination when using x-ray crystallography. In this study, several crystallization conditions (pH values, chemicals, salts, etc.) were screened to find the most optimal conditions for generating protein crystals of high quality.

Viral diseases have been a threat to human health for thousands of years and with traces of the Covid-19 pandemic still lingering after two years, the severeness of new and reemerging viral infections and their impact on human health, should not be understated. The Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne virus that is endemic to the Americas and has caused hundreds of thousands of human cases. VEEV was developed as a bioweapon during the cold war, and thus, it poses a threat to human health as a naturally emerging pathogen as well as a potential terrorist agent. As of today, no available treatments such as vaccines and antivirals are available against VEEV.

The nonstructural protein 2 (nsP2) is an enzyme that is essential for the VEEV replication machinery. By inhibiting the nsP2, the replication process stops and consequently halts the spread of the virus. Structural information about the active site in the nsP2, for example, what amino acids are involved in the recognition and cleavage of the substrate and what the binding pocket looks like, will allow chemists to design an inhibitor appropriate in size, shape, and charge to improve protein-inhibitor interactions.

X-ray crystallography is a commonly used technique for structure determination of proteins and other macromolecules. A protein crystal causes incident beams of x-rays to diffract into a unique diffraction pattern, from which information about the placements of the atoms in the crystal can be calculated and visualized. To obtain information with a high enough resolution, protein crystals of high quality are needed. The bottleneck in x-ray crystallography is getting the protein to form crystals. Several crystallization conditions (a solution comprised of chemicals, salts, a set pH value, etc. that induces protein crystallization) needs to be screened to find the right set of conditions that forces the protein to form ordered crystals of high quality.

In this study, numerous crystallization conditions were screened and optimized. More than 15 conditions were found to generate high-quality nsP2 crystals, which were sent to MAX IV in Lund for x-ray crystallography experiments. Several crystals generated data that were used to visualize the active site with an inhibitor bound to one of the amino acids. The structural information about the protein-inhibitor complex will help medicinal chemists to further improve the design of inhibitors to eventually produce antivirals against VEEV.

• author: Öberg, Elin
• supervisor: Cedric Dicko
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: x-ray crystallography, protein crystallization, Venezuelan equine encephalitis virus, structure-based drug design, applied biochemistry
• language: English
• id: 9090349
• date added to LUP: 2022-06-23 09:52:06
• date last changed: 2022-06-23 09:52:06