An Engineered Cytidine Deaminase for Biocatalytic Production of a Key Intermediate of the Covid-19 Antiviral Molnupiravir

Abstract

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N -hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N -hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5′-acylation using Novozym 435.

A Rapid SARS-CoV-2 Nucleocapsid Protein Profiling Assay with High Sensitivity Comparable to Nucleic Acid Detection

“…Additionally, the relative added N protein concentration in human saliva was calibrated by enzyme-linked immunosorbent assay using the commercial ELISA standard curve. The N protein concentrations were chosen to cover the range suggested by the ELISA kit (5,10,20,50,100, and 200 pg mL −1 , respectively). A linear relationship between optical density at 450 nm and standard N protein concentration was conducted using the kit by weighted least-squares regression analysis (R 2 = 0.999) and the saliva used was diluted 10-fold beyond the requirements for ELISA.…”

Enzyme‐mediated synthesis of Molnupiravir: paving the way for the application of biocatalysis in pharmaceutical industry

“…Recently, Burke et co-workers (Manchester University) have paved the way for a total greener synthesis of Molnupiravir by replacing the chemical hydroxyamination with a simple enzyme-mediated transformation of 3 to 5, catalyzed by an evolved E. coli cytidine deaminase (EcCD*, EC 3.5.4.5) (Scheme 4). [16] This work started from the hypothesis that EcCD could use hydroxylamine as the nucleophile for the deamination reaction instead of water (Scheme 4, route 4). Unfortunately, as expected, 5 was further hydrolyzed and converted to 2 by EcCD.…”

“…The natural diversity represents a wealthy source of extremophilic biocatalysts, which can be mined using metagenomics. [13,19,25] Alternatively, when a starting candidate enzyme is available, protein engineering, [13,16,20] or enzyme immobilization [26] is usually required for tailoring the activity or stability of the abovementioned enzymes. Based on previous findings of the required enzymes, the first goal seems achievable.…”

Strategic Enzymatic Transformations in Modern Natural Product Synthesis

“…In an alternative approach towards the same nucleoside analogue, the groups of Lovelock, Turner and Green employed an engineered cytidine deaminase for the installation of the N-hydroxyl group in cytidine by relying on the promiscuous activity of the enzyme with hydroxylamine. 36 In addition to these examples, our group recently demonstrated the diversification of nucleoside analogues bearing alkylated sugar moieties through the use of hyperthermostable nucleoside phosphorylases. 37 The inherent thermodynamic constrains in such phosphorolysis systems could either be manipulated by reaction engineering or selective product removal through strategies such as in situ formation of borate esters.…”