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

The results of the MALDI-TOF MS analysis of the primer extension products are shown in Fig. 2 . The observed peaks generally fit the predictions above; however, additional peaks from products assigned to intermediate stages of the extension reaction were also observed. In the case of TFV-DP (Fig. 2a) , a peak with the expected molecular weight representing incorporation of two Us and one TFV-DP was obtained (7196 Da observed, 7193 Da expected). As anticipated, partial extension products were also observed for a single U (6621 Da observed, 6618 Da expected) and for two Us (6927 Da observed, 6924 Da expected). Most importantly, once the TFV-DP was incorporated, there was no further extension, which confirms that it is a permanent terminator. These results are similar to our previous primer extension reactions with UTP and TFV-DP. 11 However, we previously observed significant amounts of misincorporation products (3 Us) due to the higher concentration of UTP. Under the current conditions, misincorporations were insignificant (Fig. 2a) . In the case of Ec-TP (Fig. 2b) , an expected peak indicating extension by three Us, four As and one Ec-TP was observed (8863 Da observed, 8855 Da expected), but shorter extension products also occurred, such as the extension by one U (6626 Da observed, 6618 Da expected), or a longer primer extension product containing the sequence Primer-UUAAAAU-3' but excluding the incorporation of an Ec-TP (8556 Da observed, 8546 Da expected). The extra peak at the right (9191 Da) could be explained by misincorporation events, such as a longer primer extension product containing the sequence Primer-UUAAAAUUA-3' (9181 Da expected) in which a U (italicized) was incorporated instead of a C, followed by the incorporation of an A. The occurrence of misincorporation indicates that SARS-CoV-2 RdRp has low fidelity, a property similar to other RNA viruses. 5, 26 In summary, these results demonstrate that the nucleotide analogues TFV-DP and Ec-TP are permanent terminators for the SARS-CoV-2 RdRp catalyzed reaction. Their prodrug versions (TAF or TDF and Emtricitabine or Emtricitabine-5'-O-phosphoramidate that can be readily synthesized using the ProTide prodrug approach 27 ) can be used as lead compounds for COVID-19 therapeutics development. More importantly, since these molecules are contained in the FDA-approved combination drugs TRUVADA and DISCOVY, our results provide a molecular basis to further evaluate them in SARS-CoV-2 virus inhibition and animal models to test their efficacy for the development of potential COVID-19 medications. These current and future studies may also provide potential options for preventing COVID-19 infection in a manner similar to PrEP for HIV prevention. 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 Fig. 2 Incorporation of TFV-DP and Ec-TP by SARS-CoV-2 RdRp to terminate the polymerase reaction. The sequences of the primer and template used for these extension reactions, which are at the 3' end of the SARS-CoV-2 genome, are shown at the top of the figure. Polymerase extension reactions were performed by incubating (a) UTP + TFV-DP, and (b) UTP + ATP + Ec-TP with pre-assembled SARS-CoV-2 polymerase (nsp12, nsp7 and nsp8), the indicated RNA template and primer, and the appropriate reaction buffer, followed by the detection of reaction products by MALDI-TOF MS. The detailed procedure is described in the Met