1  Introduction
Emerging RNA viruses are serious threats to public health and have become a worldwide concern. The violent Ebola virus crisis in 2014 [1] (case fatality rate ≈ 50%) and the recurrent outbreaks of Coronaviruses in 2003 (SARS-CoV, case fatality rate ≈ 10% [2]), in 2012 (MERS-CoV, case fatality rate ≈ 36% [3]) and in late december 2019 (SARS-CoV-2) [4] illustrate the critical impact of such viruses. The ongoing COVID-19 pandemic caused by the recently emerged new coronavirus, SARS-CoV-2 [4], is a global health crisis touching every continent (187 countries) with severe economic impact. Most infected people display mild to moderate respiratory illness however patients having co-morbidity factors develop serious illness and pneumonia causing significant mortality [5,6]. This disease endangers 7.7 billion people worldwide with in may 2020 more than 5.3 millions of confirmed cases of infected people and more than 350,000 confirmed deaths, and the number of COVID-19 cases will certainly progress in the next months with a possible rebond of the epidemic [7]. Despite this important public health threat, there are no yet approved chemotherapeutic agents or vaccines that can prevent or cure infections. Therefore, the urgent need of antivirals limiting the coronavirus propagation merits intensive efforts.
Most of single positive strand RNA viruses have evolved strategies in order to decorate the 5′ end of their own genome by a cap structure mimicking the eukaryotic messenger RNA. This structure plays several key biological functions such as protection of RNA from 5′-exoribonucleoases and initiation of the RNA translation into proteins. Moreover the cap is a marker of ‘self’ preventing detection by the cellular innate immunity mechanism. Thus viruses such as flaviviruses and coronaviruses code for RNA capping pathway mimicking that of eukaryotic cells. These viruses produce a subset of enzymatic activities including a RNA 5′-triphosphatase, a guanilyltransferase (GTase) and two RNA cap methyltransferases (MTases) [8]. After the cap (GpppN) is set by the GTase, a first methylation occurs at N7 position of the guanine by a N7-MTase in the presence of methyl donor S-adenosyl methionine (SAM) yielding to a cap-0 (7mGpppN) followed by a further methylation that is achieved by a 2′-O-MTase at 2′- position of the ribose of the first transcribed nucleotide in RNA yielding cap-1 (7mGpppNm) (Scheme 1 ). The N7- and 2′-O-methylation of the viral RNA cap are key events for the viral infection as their inhibition might limit the synthesis of viral proteins and support virus elimination by stimulation of the immune response [9]. Therefore it is now admitted that the viral MTases are considered as attractive targets for the development of antiviral therapy [10,11]. Few viral MTase inhibitors have been developed so far, however SAM-mimetics acting as competitors against the MTase co-substrate merit attention. Indeed SAM analogs such as sinefungin, 5′-methylthioadenosine (MTA) or S-adenosyl homocysteine (SAH) inhibit most of viral MTases with a potent efficiency but with a total lack of specificity [12]. This is certainly due to the high conservation of the shape and location of the SAM binding pocket in the human or different viral RNA MTases which share a common Rossman fold organization [13]. The rarity of specific inhibitors for viral MTases constitutes a stimulating challenge for new antiviral therapy but also for functional studies of these fascinating enzymes.
Scheme 1 Schematic representation of the 2′-O-methylation of the cap structure at nucleoside N1 at 5′-end of mRNA to form Cap-1 RNA. General structure of compounds mimicking the 2′-O-methylation transition state between N1 of mRNA (adenosine in green) and SAM (adenosine in blue).
With the aim of enhancing specificity of MTase inhibitors, we developed an approach of bisubstrate inhibitors that display a suitable linker between two adenosines that mimic the transition state of the methyl transfer reaction at 2′-O position of the RNA cap structure [12]. One adenosine is supposed to target the SAM binding site and another adenosine would target the RNA binding site (Fig. 1 ). Recently, we described the synthesis of a first series of bisubstrate adenine dinucleosides with various sulfur-containing linkers [14]. Unexpectedly, such compounds tested at 50 μM or 200 μM concentration failed to inhibit several RNA 2′O-MTases of SARS-CoV, Zika, West Nile, Dengue and Pox viruses such as vaccinia virus. Their potential of inhibition toward N7-MTases of SARS-CoV and vaccinia virus was also explored and none of the S-linked dinucleosides was active against these N7-MTases. From these data, in the present work we replaced the sulfur atom (S) by a nitrogen atom (N) in the linker between both adenosines. This modification presents the advantage of increasing the chemical stability of the linker and offers a possible variability of compounds due to the N-substitution by any chain susceptible to interact with additional protein residues (Scheme 1). It is noteworthy that bisubstrate dinucleosides with N-containing linkers have already been reported in transition state analogs of DNA methylation [15] and of RNA methylation at N6 position of adenosine [16,17]. However, till our work, none viral 2′O-MTase or N7-MTase have been targeted by bisubstrate adenine dinucleosides. This observation led us to explore the impact of N-linkers on the antiviral activity of bisubstrate SAM analogs.
Fig. 1 Rationale for designing a library of bisubstrate compounds for targeting RNA 2′-O-methyltransferases.
We thus synthesized a new series of transition state analogs of the RNA 2′-O-methylation reaction based on the coupling of a 2′-O-ethyl adenosine to a 5′-amino adenosine. We explored a variety of N-substituted linkers in adenine dinucleosides and their inhibition activity was evaluated against several viral RNA 2′O-MTases as well N7-MTases for specificity purpose. Unexpectedly, none of the N-linked dinucleosides inhibited any 2′O-MTases of flaviviruses or SARS-CoV. However, interestingly some N-nitrobenzenesulfonamide-containing dinucleosides were found to specifically inhibit SARS-CoV N7-MTase nsp14 without inhibiting the cognate human N7-MTase or vaccinia N7-MTase. Such specific inhibition results from a high binding affinity of the most potent inhibitors to N7-MTase nsp14. In addition, computational docking analysis identified some residues of the binding site for the best inhibitor targeting nsp14. As SAM and RNA binding sites of N7-MTase nsp14 are almost completely conserved between SARS-CoV and SARS-CoV-2 (95% sequence homology, Supporting Info Fig. S1) [18], we can forecast that the candidate ligands that are efficient in inhibiting the SARS-CoV functions will be efficient in doing the same for SARS-CoV-2, this emphasizes the interest of the present work.