Nucleotide Analogues as Inhibitors of Viral Polymerases

Jingyue Ju; Shiv Kumar; Xiaoxu Li; Steffen Jockusch; James J. Russo

In contrast to sofosbuvir, both the 2'-and 3'-OH groups are unmodified, but a cyano group at the 1' position . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.30.927574 doi: bioRxiv preprint presumably serves to inhibit the RdRp in the active triphosphate form. Additional information on the ProTide Prodrug technology is described by Alanazi et al (2019) . A related prodrug analogue developed by BioCryst Pharmaceuticals, BCX4430, also known as galidesivir (Fig. 5) , has been shown to inhibit RNA polymerases from a broad spectrum of RNA viruses, notably including the filoviruses (e.g., Ebola, Marburg) in rodents and Marburg virus in macaques (Warren et al 2014) . Upon entry into infected cells, BCX4430 is rapidly phosphorylated, and the resulting nucleoside triphosphate serves as an RNA chain terminator. Based on the above background information, we describe here a novel strategy to design and synthesize viral polymerase inhibitors, by combining the ProTide Prodrug approach used in the development of Sofosbuvir with the use of 3'-blocking groups that we have built into nucleotide analogues that function as reversible terminators for DNA sequencing (Ju et al 2003 , Ju et al 2006 , Guo et al 2008 . We reasoned that (1) the phosphate masking groups will allow entry of the compounds into infected cells, (2) the 3'-blocking group on the 3'-OH with either free 2'-OH or modifications at the 2' position will encourage incorporation of the activated triphosphate analogue by viral polymerases but not host cell polymerases, thus reducing any side effects, and (3) once incorporated, further extension will be prevented by virtue of the 3'-blocking group, thereby inhibiting viral replication. These modified nucleotide analogues should be potent polymerase inhibitors and thus active against various viral diseases, including but not limited to the coronaviruses such . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.30.927574 doi: bioRxiv preprint as 2019-nCoV, and the strains causing SARS and MERS. Once incorporated, our newly designed nucleotide analogues containing 3' blocking groups will permanently block further viral genome replication. This is in contrast to other nucleotide analogue-based viral inhibitors that have a free 3' OH group, which have the possibility of allowing further polymerase extension, enabled by viral mutations. For the same reason, at high concentrations, nucleotide analogue-based viral inhibitors with free 3' OH groups have the potential of being incorporated by host polymerases.