Evaluation of novel inhibitors targeting SARS-CoV-2 Main Protease; Towards effective Antiviral Drug Discovery.

The SARS-CoV-2 pandemic, which began in 2019, prompted extensive efforts to develop effective therapeutic interventions. Although mRNA vaccines were rapidly created and approved by 2020, the need for alternative treatment strategies remains critical. Computational modeling quickly highlighted a potential vulnerability of the virus: the Main protease (Mpro). This essential protease plays a vital role in processing viral polyproteins into individual proteins required for virion assembly. High-throughput screening identified compounds capable of inhibiting Mpro, suggesting a promising strategy to target a conserved protein pocket and address the challenges posed by the virus's high mutation rate. In this thesis, we investigate the inhibitory potential of two sets of compounds towards SARS-CoV-2 main protease through in vitro screening. First set of compounds are Urolithin derivatives, that are derived from natural urolithin molecules, metabolites produced by gut microbiota through the breakdown of ellagitannins, a type of polyphenol found in fruits like pomegranates and berries. Second set of compounds are M-A23 derivatives, which are modifications of a previously computationally modelled potent inhibitor. This study uses UV-visible spectroscopy for the molecular characterization, fluorescence resonance energy transfer (FRET)-based inhibition/ activity assays, mass spectrometry analysis for studying the bindings of our compounds’ to Mpro. Furthermore, we used Parallel Artificial Membrane Permeation Assay (PAMPA) to predict the potential diffusion of urolithin derivatives across cellular membranes. Our findings indicate that while Urolithin derivatives show potential as Mpro inhibitors, their binding lacks specificity, with evidence of nonspecific interactions with the target. Additionally, these compounds demonstrate poor permeability across cellular membranes, limiting their effectiveness as drug candidates. On the other hand, structural modifications on the computationally modelled M-A23 could lead to improvement in terms of solubility in the assay conditions and their activity towards Mpro inhibition. Despite these advances, several critical questions stay unanswered. Efforts should focus on finding ways to enhance the specificity of potential inhibitors of SARS-CoV-2 Mpro and developing efficient delivery methods to improve their membrane permeability. Moreover, the long-time period balance and behaviour of those compounds below complicated physiological conditions, as well as their interactions with off-goal biomolecules, require further research. Addressing these gaps will not only improve the design of future derivatives but also open the way for broader applications in antiviral drug development.