PHOsphorylation TOols for Nucleotides Synthesis – PHOTONS

Nucleoside analogues inhibitors (NAI) play a key role in every approved antiviral therapy in use so far. The situation is unlikely to change drastically in the fight against the SARS-CoV-2 responsible for the COVID-19 pandemic: The NAI Remdesivir is the only approved drug in use so far, but requires careful dosage and is not orally available. NAIs target the nsp12 polymerase, acting either as chain terminators or mutagenic agents. To be active, NAI need to be intracellularly phosphorylated into the corresponding nucleoside triphosphate (NTP). To design new and potent NAI of nsp12, we need to characterize the mode of action of available NAIs in order to guide further design and reach improved NAI properties.
However, once an active NAI is identified, the synthesis of its cognate NTP is an important bottleneck that has to be overcome to perform mode-of-action studies. This is well exemplified, by the significant difficulty and cost to obtain the 5'-triphosphate form of Favipiravir, an active broad spectrum antiviral drug currently evaluated in numerous clinical trials around the world.
The present project aims to give rapid and efficient access to NTPs by innovative and broad spectrum enzymatic method(s).
The project, over a period of 12 months, is divided into 2 work-packages.
• The WP-1 is dedicated to the production and engineering of enzymes that are involved in phosphoryl transfer reactions selected from literature data. The innovation here lies in the choice of "phosphorylases", some of which have never been explored for the phosphorylation of nucleosides. The few methods available, whether chemical or enzymatic, are too specific and the effectiveness depends on the natural or modified target nucleoside. Our wish is to make phosphorylase engineering with an increased tolerance for substrates to develop a broad spectrum method. To assess the effectiveness of enzymes alone or in cocktail, we plan to use the bioluminescent ATP-metry technique which is rapid and high-throughput to test a large number of conditions on a large number of enzymes.
• The WP-2 is dedicated to the application of the best "cocktail of phosphorylases" to drive enzymatic cascade reactions (ECR) on natural nucleosides and the validation on NAI of interest to fight coronavirus nsp12 polymerase. We also plan to transpose the method on immobilized enzymes to facilitate the product purification and enzyme(s) recycling.
NAIs are part of the preferred therapeutic arsenal of antiviral treatments. Our project will provide the scientific community with a quick and easy way to obtain nucleoside triphosphates, allowing progress in the study of their mode of action, and fostering novel discoveries of novel, broad spectrum NAIs.