The coronavirus disease 2019 emerged in December 2019 and has caused millions of deaths to date. Current treatments include antiviral drugs, antibodies, and anti-inflammatory drugs; however, only a limited number of direct-acting drugs are available on the market. SARS-CoV-2 Mpro is a promising therapeutic target, with nirmatrelvir being the most potent inhibitor. However, due to its unfavorable metabolism, it requires co-administration with ritonavir, a CYP3A4 inhibitor, to enhance bioavailability. The urgent need for new antivirals calls for more efficient drug discovery strategies. Traditional drug development approaches, such as high-throughput screening, are costly and time-consuming. Fragment-based drug discovery offers an alternative for accelerating antiviral development, and protein-templated fragment ligation represents a powerful advancement by combining fragment ligation and detection through bioassays. In the first part of this study, a series of small molecular open-chain α-ketoamides, derived from active isatin derivatives, were synthesized to enhance efficacy, and mitigate the toxicity associated with isatin scaffold. The most potent compound 8, exhibited an IC50 value of 75.6 μM, comparable to the unsubstituted isatin derivative 1, and represents a promising starting point for further optimization. Structure-activity relationship studies suggested introducing a hydrophobic substituent at position 4 to fit into the S4 pocket, though an appropriate linker is still required. Starting from the structure of nirmatrelvir, in-situ Strecker reactions were explored as an alternative approach to generate α-aminonitriles, offering greater structural flexibility. The reactions were systematically analyzed in aqueous buffers under physiological conditions. As the Strecker reaction required strongly alkaline conditions (pH 9-10), which are not compatible with the protease assay, protein-templated fragment ligation could not be conducted for this reaction. Instead, an in-situ screening method was developed that efficiently generated diverse α-aminonitriles from an amine library. The hit compound 26 was successfully resynthesized; however, due to spontaneous readdition, it could not be isolated. Acylation of the free amine group can enhance the stability. Further investigations focused on the acylation of the intermediary Strecker product 23 to synthesize nirmatrelvir in-situ, offering an alternative protein-templated synthetic route. The protein-templated effect was demonstrated through the acylation of compound 23 using pentafluorophenol-activated acid, where enhanced inhibition in the FRET assay and increased product formation in HPLC-QToF-MS confirmed the reaction’s efficiency in the presence of protease. A protein-templated fragment ligation screening method could be construct under these conditions, enabling efficient structural variation. Ultimately, nirmatrelvir and its analogs were synthesized via Strecker reaction, isolated, and evaluated for their inhibitory activity. The covalent binding between nirmatrelvir and Mpro was confirmed by protein MS. These results highlight the potential of in-situ Strecker reactions and protein-templated acylation as an efficient method for the discovery of enzyme inhibitors as demonstrated here for SARS-CoV-2 Mpro, enabling the generation and screening of α-aminonitrile libraries. Further efforts should focus on applying this methodology to alternative drug targets which are likely to be inhibited by α-aminonitriles, including cysteine and serine proteases.
