Sustainable synthesis of antiviral and anticancer drugs through chemoenzymatic routes

Nucleoside analogues (NAs) have been in clinical use for almost 50 years and are the cornerstone for the treatment of cancer or viral infections. NAs must undergo three in vivo phosphorylations in a stepwise manner to yield the corresponding active nucleoside triphosphate analogue (NTP), which exerts the therapeutic effect. Unfortunately, NAs suffer from many drawbacks such as poor cellular uptake because of insufficient expression of membrane transporters, premature breakdown, and slow conversion to the triphosphate form due to rate-limiting first phosphorylation step catalysed by nucleoside kinase (NK). Therefore, monophosphorylated NA prodrugs have been extensively studied leading to several FDA-approved medicines. However, some NAs have been reported to suffer from a second or third slow and inefficient phosphorylation step, respectively catalysed by nucleoside monophosphate kinase (NMPK) and nucleoside diphosphate kinase (NDPK). These analogues are often associated with toxicity or low efficacy due to their mono- and diphosphate forms accumulation or metabolic deactivation respectively. In addition, for many other NAs in the literature, the detailed metabolism to yield the NTP is still not known. Interestingly, the design of higher phosphorylated NA prodrugs is relatively underexplored with only few examples reported and currently a drug to bypass the whole phosphorylation cascade does not exist. These new approaches require the synthesis of NMP and NDP, and then reaction with a monophosphorylating reagent to incorporate the last phosphate group bearing the masking groups. In general, the vast majority of nucleoside and nucleotide analogues are synthesized chemically. Despite the progress achieved in the development of chemical methods, their preparation remains a challenging problem. Disadvantages of these multistep synthesis are long reaction times, toxic reagents, low yields, and tedious purification protocols. In addition, limited regioselectivity leads often to the formation of different phosphorylation products, while some nucleotide products harbouring sensitive functional groups are not accessible using these approaches.

In this scenario cascade biocatalysis, which is emerging as a powerful tool to obtain pharmaceuticals from readily available materials using natural or evolved enzymes, could offer a valid alternative to access nucleotides analogues.

Aim

Taking advantage of the recent efforts in enzyme discovery and protein engineering by rational drug design toward nucleosides or nucleotides, this work aims to develop a chemoenzymatic route that gives access to nucleotide analogues.

The project involves the use of natural or engineered nucleoside kinases to prepare mono and diphosphate species then can be converted chemically into their corresponding di- and triphosphate prodrugs. It will explore the possibility to merge NK and NMPK activity by creating a dual-function biocatalyst, capable of performing both phosphoryl-transfer reactions. Finally, it will investigate the possibility to use engineered enzymes for the synthesis of the prodrugs.