Search for inhibitors of SARS-CoV 2 PLpro protease by genetic selection in collections of cyclic head-to-tail peptides biosynthesized in bacteria

Developing new drugs is often a long and painstaking process. The coronavirus pandemic made us realize that time is not always an affordable resource. Prior to this project, an innovating attempt to find inhibitors of SARS-CoV 2 Papain-Like protease (PLpro), which is an essential protease involved in viral replication has been designed. It consists in building a combinatorial library of peptides biosynthesized in Streptococcus thermophilus. To improve pharmacological potential, these peptides are cyclized head-to-tail by autocatalytic splicing of a permuted intein. These libraries underwent a Darwinian selection process meant to enrich the library in cyclic peptides harboring an inhibitory activity against PLpro. Unfortunately, false positives arose in the library. The aim of this project is to screen the enriched library in search of true positives. This will be done by attempting to use next generation sequencing (NGS) data obtained from the library to screen out false positives. Then, the selected candidates will be synthesized in a cell-free medium by in vitro transcription and translation and assayed for PLpro inhibition. PLpro will be biosynthesized in E. coli and will be purified through affinity chromatography. A fluorogenic assay will be performed in order to validate the inhibitory potency of peptides mixed with purified PLpro. Although this project did not meet all the initial objectives, we optimized the PCR protocol for preparing the DNA libraries that were sent for NGS. We successfully detected intein activity in cell-free expression mixture of intein-peptide precursors. We also managed to obtain highly pure PLpro in good yields but encountered difficulties in detecting its activity. Although we cannot fully assume that PLpro is active, we identified the inherent limitations. Notably, a careful analysis of the literature indicates that our fluorogenic substrate may not be sensitive enough. Our mixed results indicate the importance of choosing a tailormade specific and sensitive fluorescent probe. Finally, even if the NGS data could not be processed in time as to be included in this thesis, the global project highlights NGS sequencing as a promising way to screen peptide inhibitor libraries.