Periodic Reporting for period 1 - CHIKV-FBDD (Structural investigation into functionality of Chikungunya virus nsP2 in replication complex and screening of small molecules for its inhibition)
Période du rapport: 2016-06-07 au 2018-06-06
Work Package 2 was focused of expression and purification of large polyproteins, which benefited from using codon-optimised genes, introducing different mutations to prevent proteolytic processing and prevent aggregation. In parallel individual CHIKV proteins nsP1 and nsP3 were expressed and purified. Attempts to generate complexes of nsP2 protease and N174 domains with nsP1 and nsP3 were performed using mixed purified components and analytical size exclusion chromatography. Purified nsP1, nsP3 and fusion of nsP1 with N174 or Pro with nsP3 were submitted to crystallization trials and SAXS analysis. The obtained deliverables include: purified polyproteins CHIKV P12, P123, purified proteins nsP1, nsP1-N174, nsP3 and Pro-334 and their SAXS analysis. Importantly, thanks to the development of cryo-EM technique the obtained protein preparations of P12 and P123 can now be analysed using in-house cryo-EM microscope commissioned in April 2018.
In Work Package 3 after optimization of conditions thermal shift assay was performed using CHIKV Pro and in-house library of fragments encompassing 960 chemical compounds. This led to identification of several molecules that act at increasing protein stability, suggesting their binding to the target protein, and large number of compounds potentially destabilising protease domain, possibly binding at the interface between two subdomains of CHIKV Pro and promoting protein unfolding therein. These compounds will be used in the future rounds of validation by complimentary biophysical techniques such as ligand-based NMR.
Finally, Work package 4 dealt with the assay development, whereas protease activity assay based on measurements of changes in fluorescence polarization upon substrate cleavage was created.
The obtained preliminary results have already been used to apply for additional round of funding and it is also expected that novel structural information for N174 domain of CHIKV nsP2 will provide with new ideas regarding its possible role in of CHIKV replication, possibilities for its inhibition and will constitute the basis for future research proposal. The resources and methods developed during this project are expected to push the CHIKV research field further and should also be useful in other research projects dealing with difficult proteins. The data obtained within this project will be made publicly available by different means. In particular, sets of expression vectors developed in this study are being deposited to Addgene non-profit plasmid repository and will be described in the corresponding section of the research group website (http://hyvonen.bioc.cam.ac.uk/). After structure validation the corresponding data will be deposited to PDB. Results regarding novel structure of CHIKV N-terminal domain will be published as scientific publication and presented at future scientific meetings.