Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase

Steffen Jockusch; Chuanjuan Tao; Xiaoxu Li; Thomas K. Anderson; Minchen Chien; Shiv Kumar; James J. Russo; Robert Kirchdoerfer; Jingyue Ju

Tenofovir alafenamide (TAF, Fig. 1a ), a prodrug form of TFV-DP, is activated by a series of hydrolases to the deprotected monophosphate form, Tenofovir (TFV) (Fig. 1b) , and then by two consecutive kinase reactions to TFV-DP (Fig. 1c) . 18 TAF shows potent activity for HIV and HBV, but only limited inhibition of host nuclear and mitochondrial polymerases. 19,20 TFV-DP is an acyclic nucleotide and notably does not have a 3'-OH group. Upon incorporation by both HIV and HBV polymerase catalyzed reactions, nucleic acid elongation is terminated and viral replication is stopped. 18, 19 In addition, resistance mutations rarely occur in individuals treated with regimens that include TAF. 21 TDF is another prodrug of TFV-DP, differing only in the protective group used to mask the phosphate in comparison to TAF 18 . Given that Tenofovir diphosphate (TFV-DP), which is the common active triphosphate form of TAF or TDF, is much smaller than natural nucleoside triphosphates, we expect that it can easily fit within the active site of SARS-CoV-2 RdRp. As an acyclic nucleotide, TFV-DP lacks a normal sugar ring configuration, thus we reasoned that it is unlikely to be recognized by 3'-exonucleases involved in SARS-CoV-2 proofreading processes, thereby decreasing the likelihood of developing resistance to inhibition by the drug. Based on these analyses, we previously demonstrated the the ability of TFV-DP to inhibit the SARS-CoV-2 RdRp. 11 This inhibitory effect was furthered confirmed in the current study. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.022939 doi: bioRxiv preprint Emtricitabine (FTC) is a 5-fluorocytidine analog containing an oxathiolane ring with an unnatural (-)-b-Lstereochemical configuration (Fig. 1e) . 22 This pro-drug is converted by cellular enzymes, first to a monophosphate (Fig. 1f) then to the active triphosphate form Ec-TP (Fig. 1g ). An alternative 5'-Ophosphoramidate prodrug can also be synthesized (Fig. 1d) . The absence of an OH group at the 3' position ensures that once this nucleotide analogue is incorporated into the primer in polymerase reaction, no further incorporation of nucleotides by the polymerase can occur. Like TAF, FTC is effective against the retrovirus HIV and has also been shown to inhibit HBV in clinical studies. 23 Due to its structural similarity with molecules we previously tested 11 and because FTC is a key component of DESCOVY and TRUVADA, Ec-TP (the active form of FTC) was included in this SARS-CoV-2 RdRp inhibitory study. .