Understanding the viral protein structures of clinically relevant arboviruses and fragment-based screening for antivirals

Abstract

Arboviruses are viruses that are transmitted to the human hosts by virus-infected arthropods. In the past decade, the unprecedented emergence of arboviral diseases transmitted by Aedes mosquitoes have caused debilitating illnesses that has affected millions of people worldwide. Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes a debilitating musculoskeletal inflammatory disease that could persist for months to years after the virus is cleared. To date, there are no specific therapeutics available to prevent or mitigate the disease. In recent years, antiviral drug discovery has increasingly relied on the use of high-resolution structural information of important viral proteins and have led to the development of potent inhibitors. CHIKV nonstructural protein 2 (nsP2) protease structure is routinely targeted due to its essential polyprotein processing function and although the crystal structure of CHIKV nsP2 helicase has recently been determined, the dynamics between the two domains and overall structure of nsP2 remains unclear. Thus, the first objective of this thesis was to gain structural and functional insights into the global architecture and dynamics of the full length nsP2 by integrating several techniques including Small-angle X-Ray Scattering (SAXS), negative stain electron microscopy (EM) and X-ray crystallography as well as biochemical activity assays. This could give a more holistic view of the protein’s function and mechanism, and open new avenues for antiviral development in the future. Another arthritogenic alphavirus, O’nyong’nyong virus (ONNV), most closely related to CHIKV, has been proposed to share similar nsP2 protease structural folds due to its high sequence homology, but this has never been shown. Therefore, another aim of this study was to reveal its structure through X-ray crystallography. The second objective of this thesis was to discover new chemical motifs that could effectively target alphavirus nsP2 protease. Inhibition of this protein was frequently found to be effective in suppressing viral replication in vitro, while the efficacy is limited by drug dissemination to viral replication sites in vivo. Herein, fragment-based screening was applied to CHIKV nsP2 protease to identify novel start points for drug design. Biochemical and biophysical assays were employed to determine the inhibition modes of hits and in vitro antiviral assays were performed to investigate their efficacies. Another aspect of this study was to evaluate the fragment-protein complex structure and the information could be useful for the optimization of these hits into high affinity leads in the future. Overall, results obtained from this thesis could be used as a platform for the design of novel therapeutics specifically targeting alphavirus nsP2.