The COVID-19 pandemic highlighted the urgent need for novel therapeutic strategies against viral infections, particularly in cases where conventional antiviral drugs are ineffective. The main protease of SARS-CoV-2 (Mpro) is a well-established therapeutic target, as it is essential for the proteolytic processing of viral polyproteins required for viral replication. Its catalytic activity is strictly dependent on homodimerisation, making the dimerisation interface an attractive alternative target for inhibition. In this master’s thesis, we established and validated a novel bacterial selection system, LexKan, which enables the phenotypic selection of affibodies based on the survival of E. coli cells in the presence of kanamycin. Using control proteins, we optimised the selection conditions and confirmed the functionality of the system in both liquid and solid media. A library of affibodies was generated by random mutagenesis of 13 surface-exposed amino acid residues, followed by the selection of candidates capable of inhibiting Mpro dimerisation. Six affibodies and one affipeptide were successfully expressed, purified, and characterised. Experimental characterisation by size-exclusion chromatography and mass photometry demonstrated that Mpro exists predominantly as a homodimer at lower concentrations, while higher-order oligomeric species are formed at elevated concentrations. The presence of affibody 4 resulted in a concentration-dependent decrease in the dimeric fraction, indicating partial destabilisation of homodimerisation. Subsequent bioinformatic analysis using AlphaFold, PDBePISA, and HADDOCK suggested that affibody 4 most likely binds to the Mpro dimerisation interface and exhibits the most favourable interactions among the analysed candidates. These results indicate that affibody 4 represents a promising candidate for further optimisation as a potential inhibitor of Mpro and, consequently, SARS-CoV-2 replication.
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