Kill or corrupt: Mechanisms of action and drug-resistance of nucleotide analogues against SARS-CoV-2.

Nucleoside/tide analogues (NAs) have long been used in the fight against viral diseases, and now present a promising option for the treatment of COVID-19. Once activated to the 5'-triphosphate state, NAs act by targeting the viral RNA-dependent RNA-polymerase for incorporation into the viral RNA genome. Incorporated analogues can either 'kill' (terminate) synthesis, or 'corrupt' (genetically or chemically) the RNA. Against coronaviruses, the use of NAs has been further complicated by the presence of a virally encoded exonuclease domain (nsp14) with proofreading and repair capacities. Here, we describe the mechanism of action of four promising anti-COVID-19 NAs; remdesivir, molnupiravir, favipiravir and bemnifosbuvir. Their distinct mechanisms of action best exemplify the concept of 'killers' and 'corruptors'. We review available data regarding their ability to be incorporated and excised, and discuss the specific structural features that dictate their overall potency, toxicity, and mutagenic potential. This should guide the synthesis of novel analogues, lend insight into the potential for resistance mutations, and provide a rational basis for upcoming combinations therapies.

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

Identifying Structural Features of Nucleotide Analogues to Overcome SARS-CoV-2 Exonuclease Activity.

With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3' RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2'- and 3'-OH groups were most resistant to ExoN excision, whereas those possessing both 2'- and 3'-OH groups were efficiently removed. We also found that the 3'-OH group in the nucleotide analogues was more critical than the 2'-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.

2'- and 3'-Ribose Modifications of Nucleotide Analogues Establish the Structural Basis to Inhibit the Viral Replication of SARS-CoV-2.

Inhibition of RNA-dependent RNA polymerase (RdRp) by nucleotide analogues with ribose modification provides a promising antiviral strategy for the treatment of SARS-CoV-2. Previous works have shown that remdesivir carrying 1'-substitution can act as a "delayed chain terminator", while nucleotide analogues with 2'-methyl group substitution could immediately terminate the chain extension. However, how the inhibition can be established by the 3'-ribose modification as well as other 2'-ribose modifications is not fully understood. Herein, we have evaluated the potential of several adenosine analogues with 2'- and/or 3'-modifications as obligate chain terminators by comprehensive structural analysis based on extensive molecular dynamics simulations. Our results suggest that 2'-modification couples with the protein environment to affect the structural stability, while 3'-hydrogen substitution inherently exerts "immediate termination" without compromising the structural stability in the active site. Our study provides an alternative promising modification scheme to orientate the further optimization of obligate terminators for SARS-CoV-2 RdRp.

Studying the Key Intermediate of RNA Autohydrolysis by Cryogenic Gas-Phase Infrared Spectroscopy.

Over the course of the COVID-19 pandemic, mRNA-based vaccines have gained tremendous importance. The development and analysis of modified RNA molecules benefit from advanced mass spectrometry and require sufficient understanding of fragmentation processes. Analogous to the degradation of RNA in solution by autohydrolysis, backbone cleavage of RNA strands was equally observed in the gas phase; however, the fragmentation mechanism remained elusive. In this work, autohydrolysis-like intermediates were generated from isolated RNA dinucleotides in the gas phase and investigated using cryogenic infrared spectroscopy in helium nanodroplets. Data from both experiment and density functional theory provide evidence for the formation of a five-membered cyclic phosphate intermediate and rule out linear or six-membered structures. Furthermore, the experiments show that another prominent condensed-phase reaction of RNA nucleotides can be induced in the gas phase: the tautomerization of cytosine. Both observed reactions are therefore highly universal and intrinsic properties of the investigated molecules.

Incorporation efficiency and inhibition mechanism of 2'-substituted nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase.

The ongoing pandemic caused by SARS-CoV-2 emphasizes the need for effective therapeutics. Inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by nucleotide analogs provides a promising antiviral strategy. One common group of RdRp inhibitors, 2'-modified nucleotides, are reported to exhibit different behaviors in the SARS-CoV-2 RdRp transcription assay. Three of these analogs, 2'-O-methyl UTP, Sofosbuvir, and 2'-methyl CTP, act as effective inhibitors in previous biochemical experiments, while Gemcitabine and ara-UTP show no inhibitory activity. To understand the impact of the 2'-modification on their inhibitory effects, we conducted extensive molecular dynamics simulations and relative binding free energy calculations using the free energy perturbation method on SARS-CoV-2 replication-transcription complex (RTC) with these five nucleotide analogs. Our results reveal that the five nucleotide analogs display comparable binding affinities to SARS-CoV-2 RdRp and they can all be added to the nascent RNA chain. Moreover, we examine how the incorporation of these nucleotide triphosphate (NTP) analogs will impact the addition of the next nucleotide. Our results indicate that 2'-O-methyl UTP can weaken the binding of the subsequent NTP and consequently lead to partial chain termination. Additionally, Sofosbuvir and 2'-methyl CTP can cause immediate termination due to the strong steric hindrance introduced by their bulky 2'-methyl groups. In contrast, nucleotide analogs with smaller substitutions, such as the fluorine atoms and the ara-hydroxyl group in Gemcitabine and ara-UTP, have a marginal impact on the polymerization process. Our findings are consistent with experimental observations, and more importantly, shed light on the detailed molecular mechanism of SARS-CoV-2 RdRp inhibition by 2'-substituted nucleotide analogs, and may facilitate the rational design of antiviral agents to inhibit SARS-CoV-2 RdRp.

The Enzyme-Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid.

Phosphoramidates composed of an amino acid and a nucleotide analogue are critical metabolites of prodrugs, such as remdesivir. Hydrolysis of the phosphoramidate liberates the nucleotide, which can then be phosphorylated to become the pharmacologically active triphosphate. Enzymatic hydrolysis has been demonstrated, but a spontaneous chemical process may also occur. We measured the rate of enzyme-free hydrolysis for 17 phosphoramidates of ribonucleotides with amino acids or related compounds at pH 7.5. Phosphoramidates of proline hydrolyzed fast, with a half-life time as short as 2.4 h for Pro-AMP in ethylimidazole-containing buffer at 37 °C; 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP. Crystal structures of Gly-AMP, Pro-AMP, ßPro-AMP and Phe-AMP bound to RNase A as crystallization chaperone showed how well the carboxylate is poised to attack the phosphoramidate, helping to explain this reactivity. Our results are significant for the design of new antiviral prodrugs.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.

Tenofovir-Amino Acid Conjugates Act as Polymerase Substrates-Implications for Avoiding Cellular Phosphorylation in the Discovery of Nucleotide Analogues.

Nucleotide analogues are used for treating viral infections such as HIV, hepatitis B, hepatitis C, influenza, and SARS-CoV-2. To become polymerase substrates, a nucleotide analogue must be phosphorylated by cellular kinases which is rate-limiting. The goal of this study is to develop dNTP/NTP analogues directly from nucleotides. Tenofovir (TFV) analogues were synthesized by conjugating with amino acids. We demonstrate that some conjugates act as dNTP analogues and HIV-1 reverse transcriptase (RT) catalytically incorporates the TFV part as the chain terminator. X-ray structures in complex with HIV-1 RT/dsDNA showed binding of the conjugates at the polymerase active site, however, in different modes in the presence of Mg2+ versus Mn2+ ions. The adaptability of the compounds is seemingly essential for catalytic incorporation of TFV by RT. 4d with a carboxyl sidechain demonstrated the highest incorporation. 4e showed weak incorporation and rather behaved as a dNTP-competitive inhibitor. This result advocates the feasibility of designing NTP/dNTP analogues by chemical substitutions to nucleotide analogues.

Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

COSMO-RS-Based Descriptors for the Machine Learning-Enabled Screening of Nucleotide Analogue Drugs against SARS-CoV-2.

Chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as COVID-19, correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. We propose novel descriptors based on a COSMO-RS (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ML). The descriptors' performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. The COSMO-RS-based descriptors account for both chemical reactivity and structure, and are more effective for ML-based screening than fingerprints based on molecular structure and simple physical/chemical properties. The descriptors are evaluated using principal component analysis, an unsupervised ML technique. Our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug EIDD-2801 with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. The COSMO-RS-based descriptors could help accelerate drug discovery for the treatment of emerging diseases.

Discovery, Design, Synthesis, and Application of Nucleoside/Nucleotides.

For decades, nucleosides and nucleotides have formed the cornerstone of antiviral, antiparasitic and anticancer therapeutics and have been used as tools in exploring nucleic acid structure and function [...].

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.