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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.

Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2',3'- and 2',4'-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 μM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5'-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors., Graphical abstract Image 1

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50= 7 nM) without affecting the activity of human cathepsin L (IC50> 10 μM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10(RNA copies/mg) reduction of the viral RNA copies and 3.7 log10(TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.

BackgroundIn the treatment of HIV, the loose active site of the HIV-1 reverse transcriptase (RT) allows numerous nucleotide analogues to act as proviral DNA ‘chain-terminators’. Acyclic nucleotide phosphonate analogues (ANPs) represent a particular class of nucleotide analogue that does not possess a ribose moiety. The structural basis for their substrate efficiency regarding viral DNA polymerases is poorly understood.MethodsPre-steady-state kinetics on HIV-1 RT together with molecular modelling, were used to evaluate the relative characteristics of both the initial binding and incorporation into DNA of three different ANP diphosphates with progressively increasing steric demands on the acyclic linker: adefovir-diphosphate (DP), tenofovir-DP, and cidofovir-DP.ResultsThe increase of steric demand in ANPs induced a proportional loss of the binding affinity to wild-type HIV-1 RT ( Kdcidofovir-D P>> Kdtenofovir-D P> Kdadefovir-DP∼ KddNTPs), consistent with the lack of HIV-1 inhibitory activity for cidofovir. We show that, starting from adefovir-DP, the steric constraints mainly map to Gln151, as its mutation to alanine provides cidofovir-DP sensitivity. Interactions between the Gln151 residue and the methyl group of tenofovir-DP further increase with the mutation Gln151Met, resulting in a specific discrimination and low-level resistance to tenofovir-DP. This alteration is the result of a dual decrease in the binding affinity ( Kd) and the catalytic rate ( kpol) of incorporation of tenofovir-DP. By contrast, the tenofovir resistance mutation K65R induces a broad ‘ kpol-dependent’ nonspecific discrimination towards the three ANPs.ConclusionsOverall, our results show that the efficiency of ANPs to compete against natural nucleotides as substrates for RT is determined by their close interaction with specific amino acids such as Gln151 within the RT active site. These results should help us to map and predict ANP sensitivity determinants in cellular and viral DNA polymerase active sites for which the understanding of different ANP sensitivity patterns are of medical importance.

Highlights • This review summarizes current knowledge on the norovirus RdRp. • Multiple X-ray structures of norovirus RdRp show important conformational changes. • Norovirus RdRp recognizes specific promotor sequences to initiate RNA synthesis. • Anti-HCV nucleoside analogs such as 2CM-C also inhibit Norovirus RdRp. • Suramin and its analogs act as allosteric non-nucleoside polymerase inhibitors., Noroviruses belong to the Caliciviridae family of single-stranded positive-sense RNA viruses. The genus Norovirus includes seven genogroups (designated GI-GVII), of which GI, GII and GIV infect humans. Human noroviruses are responsible for widespread outbreaks of acute gastroenteritis and represent one of the most common causes of foodborne illness. No vaccine or antiviral treatment options are available for norovirus infection. The RNA-dependent RNA polymerase (RdRp) of noroviruses is a key enzyme responsible for transcription and replication of the viral genome. Here, we review the progress made in understanding the structures and functions of norovirus RdRp and its use as a target for small molecule inhibitors. Crystal structures of the RdRp at different stages of substrate interaction have been determined, which shed light on its multi-step catalytic cycle. The in vitro assays and in vivo animal models that have been developed to identify and characterize inhibitors of norovirus RdRp are also summarized, followed by an update on the current antiviral research targeting different regions of norovirus RdRp. In the future, structure-based drug design and rational optimization of known nucleoside and non-nucleoside inhibitors of norovirus RdRp may pave the way towards the next generation of direct-acting antivirals.

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Worldwide, respiratory syncytial virus (RSV) causes severe disease in infants, the elderly, and immunocompromised people. No vaccine or effective antiviral treatment is available. RSV is a member of the non-segmented, negative-strand (NNS) group of RNA viruses and relies on its RNA-dependent RNA polymerase to transcribe and replicate its genome. Because of its essential nature and unique properties, the RSV polymerase has proven to be a good target for antiviral drugs, with one compound, ALS-8176, having already achieved clinical proof-of-concept efficacy in a human challenge study. In this article, we first provide an overview of the role of the RSV polymerase in viral mRNA transcription and genome replication. We then review past and current approaches to inhibiting the RSV polymerase, including use of nucleoside analogs and non-nucleoside inhibitors. Finally, we consider polymerase inhibitors that hold promise for treating infections with other NNS RNA viruses, including measles and Ebola.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Thyroid hormones are important modulators of metabolic activity in mammals and alter cholesterol and fatty acid levels through activation of the nuclear thyroid hormone receptor (THR). Currently, there are several THRβ agonists in clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) that have demonstrated the potential to reduce liver fat and restore liver function. In this study, we tested three THRβ-agonism-based NASH treatment candidates, GC-1 (sobetirome), MGL-3196 (resmetirom), and VK2809, and compared their selectivity for THRβ and their ability to modulate the expression of genes specific to cholesterol and fatty acid biosynthesis and metabolismin vitrousing human hepatic cells andin vivousing a rat model. Treatment with GC-1 upregulated the transcription ofCPT1Ain the human hepatocyte-derived Huh-7 cell line with a dose-response comparable to that of the native THR ligand, triiodothyronine (T3). VK2809A (active parent of VK2809), MGL-3196, and VK2809 were approximately 30-fold, 1,000-fold, and 2,000-fold less potent than T3, respectively. Additionally, these relative potencies were confirmed by quantification of other direct gene targets of THR, namely,ANGPTL4andDIO1. In primary human hepatocytes, potencies were conserved for every compound except for VK2809, which showed significantly increased potency that was comparable to that of its active counterpart, VK2809A. In high-fat diet fed rats, a single dose of T3 significantly reduced total cholesterol levels and concurrently increased liverDio1andMe1RNA expression. MGL-3196 treatment resulted in concentration-dependent decreases in total and low-density lipoprotein cholesterol with corresponding increases in liver gene expression, but the compound was significantly less potent than T3. In conclusion, we have implemented a strategy to rank the efficacy of THRβ agonists by quantifying changes in the transcription of genes that lead to metabolic alterations, an effect that is directly downstream of THR binding and activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic that began in 2019. The coronavirus 3-chymotrypsin-like cysteine protease (3CLpro) controls replication and is therefore considered a major target for antiviral discovery. This study describes the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay. Compared with a traditional FRET readout, the label-free SAMDI-MS assay offers greater sensitivity and eliminates false positive inhibition from compound interference with the optical signal. The SAMDI-MS assay was optimized and validated with known inhibitors of coronavirus 3CLpro such as GC376 (IC50 = 0.060 μM), calpain inhibitors II and XII (IC50 ~20–25 μM). The FDA-approved drugs shikonin, disulfiram, and ebselen did not inhibit SARS-CoV-2 3CLpro activity in the SAMDI-MS assay under physiologically relevant reducing conditions. The three drugs did not directly inhibit human β-coronavirus OC-43 or SARS-CoV-2 in vitro, but instead induced cell death. In conclusion, the SAMDI-MS 3CLpro assay, combined with antiviral and cytotoxic assessment, provides a robust platform to evaluate antiviral agents directed against SARS-CoV-2., Highlights • A novel label-free SARS-CoV-2 3CLpro enzymatic assay was developed using mass spectrometry (SAMDI-MS). • Protease inhibitors active in the SAMDI-MS assay under reducing conditions also have antiviral effects in cells. • Shikonin, disulfiram, and ebselen do not inhibit SARS-CoV-2 3CLpro in the SAMDI-MS assay. • Shikonin, disulfiram, and ebselen do not directly inhibit SARS-CoV-2 but instead are toxic to cells.

Introduction: The STING (STimulator of INterferon Genes) protein has been identified as an attractive target for cancer immunotherapy due to its central role in inducing T-cell mediated tumor control. Here we describe the preclinical characteristics of the novel STING agonist, ALG-031048, that demonstrates potent anti-tumoral activity in in vivo mouse models. Methods: Binding to STING was determined using differential scanning fluorimetry (DSF). Cellular activation and cytokine release were assessed using HEK 293T reporter cell lines as well as THP-1 and Raw264.7 cells. In vitro stability was measured in the presence of snake venom phosphodiesterase (SVPD). In vivo anti-tumoral activity was tested in the syngeneic CT26 and B16F10 mouse model. Results: ALG-031048 demonstrated potent binding to the STING R232 protein in the DSF assay with a mean Kd of 3.12 ± 0.12 μM and a thermal shift of 12.8 °C, similar to the natural ligand 2'3'cGAMP (Kd of 2.75 ± 0.78 μM, shift of 16.7°C) or the clinical stage STING agonist, ADU-S100 (Kd of 4.61 ± 0.42 μM, shift of 10.7°C). In the HEK 293T R232 reporter assay, ALG-031048 was more potent than ADU-S100 with a mean EC50 of 55.9 ± 24.1 and 30.3 ± 17.1 nM for the IFNβ and IRF reporter, respectively, compared to ADU-S100 with EC50 values of 233 ± 179 and 88.7 ± 35.6 nM. Cellular activation was confirmed in THP-1 and Raw264.7 cells where ALG-031048 induced the secretion of IFNβ and CXCL-10. ALG-031048 was highly stable and exhibited no degradation for up to 24 hours in the presence of SVPD while ADU-S100 and 2'3'cGAMP were quickly degraded with less than 10% remaining after 24 hours. In vivo, 3 intra-tumoral (IT) doses of 100 μg ALG-031048 q3d caused complete tumor regression in 90% of animals in the CT26 mouse colon carcinoma model, while treatment with 25 μg ALG-031048 IT 3xq3d resulted in survival of 60 % of animals. In contrast, only 10 and 44% of mice showed complete tumor regression when treated IT 3xq3d with 25 or 100 μg ADU-S100, respectively. To study whether induction of a protective immune response was associated with STING agonist treatment, surviving animals were re-challenged with a second inoculation of CT26 cells on the contra-lateral side. All naïve animals formed tumors within 23-30 days. In contrast, 5 out of 5 and 8 out of 9 mice pre-treated with 25 or 100 μg ALG-031048, respectively, did not exhibit any significant tumor growth over 40 days, while 3 out of 4 animals pretreated with 100 μg ADU-S100 showed complete tumor suppression. Initial results using the B16F10 mouse melanoma model confirmed the potent anti-tumoral activity of ALG-031048 observed in the CT26 model. Summary: ALG-031048 is a novel STING agonist combining high stability and excellent in vitro potency. In preclinical models, ALG-031048 demonstrated potent and long-lasting tumor suppression. ALG-031048 is advancing in toxicology studies for further profiling as a clinical candidate. Citation Format: Andreas Jekle, Santosh Thatikonda, Sarah Stevens, Caroline Williams, April Kinkade, Suping Ren, Ruchika Jaisinghani, Qingling Zhang, Dinah Misner, Antitsa Stoycheva, Jerome Deval, Sucheta Mukherjee, Francois Gonzalvez, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt, Leonid Beigelman. Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4520.

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′- C -methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC 50 s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′- C -methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

Respiratory syncytial virus (RSV) is a leading pathogen of childhood and is associated with significant morbidity and mortality. To date, ribavirin is the only approved small molecule drug, which has limited use. The only other RSV drug is palivizumab, a monoclonal antibody, which is used for RSV prophylaxis. Clearly, there is an urgent need for small molecule RSV drugs. This article reports the design, synthesis, anti-RSV activity, metabolism, and pharmacokinetics of a series of 4'-substituted cytidine nucleosides. Among tested compounds 4'-chloromethyl-2'-deoxy-2'-fluorocytidine (2c) exhibited the most promising activity in the RSV replicon assay with an EC50 of 0.15 μM. The 5'-triphosphate of 2c (2c-TP) inhibited RSV polymerase with an IC50 of 0.02 μM without appreciable inhibition of human DNA and RNA polymerases at 100 μM. ALS-8176 (71), the 3',5'-di-O-isobutyryl prodrug of 2c, demonstrated good oral bioavailability and a high level of 2c-TP in vivo. Compound 71 is a first-in-class nucleoside RSV polymerase inhibitor that demonstrated excellent anti-RSV efficacy and safety in a phase 2 clinical RSV challenge study.

Highlights • New therapies for infections caused by positive-strand RNA viruses are needed. • Novel nucleoside and nucleotide analogs that inhibit HCV have been developed. • Some of these molecules also inhibit other positive-strand RNA viruses. • Optimization of antiviral potency and/or target delivery is necessary., A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. One exception is the chronic infection caused by hepatitis C virus (HCV), against which new generations of potent inhibitors are being developed. One of the main molecular targets for anti-HCV drugs is the viral RNA-dependent RNA polymerase, NS5B. This review summarizes the search for nucleoside and nucleotide analogs that inhibit HCV NS5B, which led to the FDA approval of sofosbuvir in 2013. Advances in anti-HCV therapeutics have also stimulated efforts to develop nucleoside analogs against other positive-strand RNA viruses. Although it remains to be validated in the clinic, the prospect of using nucleoside analogs to treat acute infections caused by RNA viruses represents an important paradigm shift and a new frontier for future antiviral therapies.

Current therapy for chronic hepatitis C virus (HCV) infection constitutes a combination of pegylated interferon alfa-2a or alpha-2b and ribavirin. Although successful for many patient populations, this regimen has numerous limitations, including non-response, relapse, poor tolerability and long duration of treatment. To address these shortcomings, new small molecule agents are advancing in clinical development. Most of the current clinical candidates act by directly inhibiting key enzymes in the viral life-cycle: the NS5B polymerase, or the NS3/4A protease. Less well-studied, the non-structural 4B (NS4B) protein has recently emerged as an alternative target for Direct-acting Antiviral Agents (DAAs). NS4B is a 27-kDa membrane protein that is primarily involved in the formation of membrane vesicles--also named membranous web--used as scaffold for the assembly of the HCV replication complex. In addition, NS4B contains NTPase and RNA binding activities, as well as anti-apoptotic properties. This review summarizes the current understanding of the structure and functions of NS4B, an essential component of the replication machinery of HCV. In this literature and patent review, we report the recent developments in anti-NS4B drug discovery. These advances open the possibility for future combination therapies with other DAAs.

Dengue virus (DENV) infects 50-100 million people worldwide per year, causing severe public health problems. DENV RNA-dependent RNA polymerase, an attractive target for drug development, catalyzes de novo replication of the viral genome in three phases: initiation, transition, and elongation. The aim of this work was to characterize the mechanism of nucleotide addition catalyzed by the polymerase domain of DENV serotype 2 during elongation using transient kinetic methods. We measured the kinetics of formation of the elongation complex containing the polymerase and a double-stranded RNA by preincubation experiments. The elongation complex assembly is slow, following a one-step binding mechanism with an association rate of 0.0016 ± 0.0001 μM(-1) s(-1) and a dissociation rate of 0.00020 ± 0.00005 s(-1) at 37 °C. The elongation complex assembly is 6 times slower at 30 °C and requires Mg(2+) during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a K(d) of 275 ± 52 μM and k(pol) of 18 ± 1 s(-1). The fidelity of the polymerase is 1/34,000, 1/59,000, 1/135,000 for misincorporation of UTP, ATP, and CTP opposite CMP in the template, respectively. The fidelity of DENV polymerase is comparable with HIV reverse transcriptase and the poliovirus polymerase. This work reports the first description of presteady-state kinetics and fidelity for an RNA-dependent RNA polymerase from the Flaviviridae family.

Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses., Author summary RNA viruses replicate and transcribe their genomes using complex enzymatic machines known as RNA-dependent RNA polymerases. The chemical reactions driving nucleotide addition are shared among nucleic acid polymerases but the underlying mechanisms of RNA biosynthesis and the complex polymerase structures are diverse. Of these RNA viruses is the paramyxovirus family, which includes major human pathogens. Paramyxoviruses have common biological and genetic properties but little is known about their replication machinery. Insights into the structure, function, and mechanisms of RNA synthesis of one paramyxovirus polymerase will likely extend to the entire virus family. An emerging, highly pathogenic paramyxovirus is Nipah virus (NiV), which causes encephalitis in humans. We have purified NiV polymerase, probed its enzymatic and biophysical properties and developed it as a model paramyxovirus polymerase. We investigated template strand sequence elements driving RNA biosynthesis for NiV polymerase and obtained a snapshot of NiV polymerase molecular organization using electron microscopy to provide the first structural information on a paramyxovirus polymerase. This work extends previous knowledge by producing the first recombinant paramyxovirus polymerase and using this protein in enzymatic assays to highlight key functional and structural characteristics for the design of new medicines.

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

The nucleic acid binding channel of the hepatitis C virus RNA polymerase remains to be defined. Here we employed complementary footprinting techniques and show that the enzyme binds to a newly synthesized duplex of approximately seven to eight base pairs. Comparative analysis of surface topologies of free enzyme versus the nucleoprotein complex revealed certain lysines and arginines that are protected from chemical modification upon RNA binding. The protection pattern helps to define the trajectory of the nucleic acid substrate. Lys(81), Lys(98), Lys(100), Lys(106), Arg(158), Arg(386), and Arg(394) probably interact with the bound RNA. The selective protection of amino acids of the arginine-rich region in helix T points to RNA-induced conformational rearrangements. Together, these findings suggest that RNA-protein interaction through the entire substrate binding channel can modulate intradomain contacts at the C terminus.

We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed “hot spots” for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3′ end.

The multi-drug resistance HIV-1 genotype A62V/V75I/F77L/F116Y/Q151M is associated with resistance to many nucleoside reverse transcriptase inhibitors including AZT, ddI, ddC, d4T, abacavir, and 3TC. In this study, we evaluated the antiviral activity of (−)-β- d -1′,3′-dioxolan guanine (DXG) towards mutant HIV-1 containing V75I/F77L/F116Y/Q151M (V75Icomplex) and A62V/V75I/F77L/F116Y/Q151M (A62Vcomplex) in MT-2 cells. We further investigated the mechanism of resistance by studying the incorporation of DXG 5′-triphosphate (DXG-TP) during DNA synthesis by mutant enzymes containing single mutations at Q151M or A62V, and the V75Icomplex and A62Vcomplex using pre-steady state kinetic analysis. Our studies showed that mutant virus containing V75Icomplex and A62Vcomplex were both more than 23-fold resistant to DXG, and this correlated with the 68- and 20-fold resistance changes observed in the enzymatic assay. Compared to the wild-type enzyme, DXG-TP was incorporated 39- and 21-fold less efficiently by the mutant enzyme containing V75Icomplex and A62Vcomplex, mainly due to decreases in the rate of incorporation. The A62V mutation significantly increased the rate of incorporation (kpol) for both dGTP (3-fold) and DXG-TP (7.9-fold), while the binding affinity of A62V HIV-1 RT for DXG-TP was decreased 14-fold. At the enzyme level, the addition of the A62V mutation to V75I/F77L/F116Y/Q151M moderately (3.4-fold) reversed the resistance to DXG-TP.

International audience; Alpha-boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5'-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3-) group renders the incorporation of the analogue independent from amino-acid substitutions in RT. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analogue

Nucleoside analogues play a key role in the fight against HIV-1. Unfortunately, under therapeutic pressure, HIV-1 inevitably develops resistance to these inhibitors. This resistance correlates with specific pol gene mutations giving rise to specific substitutions in reverse transcriptase that are responsible for the loss of efficacy of the corresponding analogue. This work is an overview of the molecular mechanisms of HIV-1 drug resistance as judged by the analysis of chemical reactions at play at the reverse transcriptase active site. One class of mechanism involves nucleotide analogue discrimination either at the binding step or at the catalytic step, the latter being by far the most common mechanism. The other class of mechanism involves repair of the analogue-terminated DNA chain. The mechanisms were elucidated using purified reverse transcriptase and biochemical assays aimed at correlating resistant HIV-1 phenotypes to enzymatic data. The elucidation of these molecular mechanisms of drug-resistant reverse transcriptase is important for effective and rational combination therapies as well as for the conception of second-generation drugs that do not confer nucleotide resistance to reverse transcriptase or are active against pre-existing resistant viruses.

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2′F-2′C-Me-UTP was efficiently incorporated by HCV polymerase with apparent K d (equilibrium constant) and k pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s −1 , respectively, giving an overall substrate efficiency ( k pol / K d ) of 0.0059 ± 0.0015 μM −1 s −1 . We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2′ position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH 2 > F-C-Me > C-Me > N 3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2′F-2′C-Me-, 2′C-Me-, and 2′ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2′-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2′-modified NTP analogs against HCV polymerase.

Hepatitis C virus (HCV) is believed to have infected approximately 175 million individuals worldwide, with an estimated two to four million new infections each year. The primary mode of transmission of HCV is via exposure to infected blood, including transfusions from infected donors, and through intravenous use of illicit drugs. Every single step of HCV replication has been targeted for therapeutic intervention with small molecules, three major proteins are emerging as the most attractive targets. The development of a broadly useful HCV subgenomic replicon system came as a major breakthrough after HCV research and drug discovery had been hampered for a long time by the inability to efficiently propagate the virus in tissue culture. In the case of HCV, chimpanzees are the only animal species that are known to be susceptible to virus replication. Nucleoside and nonnucleoside analogs have been used to treat HCV-infected chimpanzees.

Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC 50 s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo -initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity ( K d ) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.

Dengue virus (DENV), an emerging pathogen from the Flaviviridae family with neither vaccine nor antiviral treatment available, causes a serious worldwide public health threat. In theory, there are several ways by which small molecules could inhibit the replication cycle of DENV. Here, we show that the nucleoside analogue beta- d -2′-ethynyl-7-deaza-adenosine inhibits representative strains of all four serotypes of DENV with an EC 50 around or below 1 μM. Using membrane-associated native replicase complex as well as recombinant RNA polymerase from each DENV serotype in enzymatic assays, we provide evidence that beta- d -2′-ethynyl-7-deaza-adenosine triphosphate (2′E-7D-ATP) targets viral replication at the polymerase active site by competing with the natural nucleotide substrate with an apparent K i of 0.060 ± 0.016 μM. In single-nucleotide incorporation experiments, the catalytic efficiency of 2′E-7D-ATP is 10-fold lower than for natural ATP, and the incorporated nucleotide analogue causes immediate chain termination. A combination of bioinformatics and site-directed mutagenesis demonstrates that 2′E-7D-ATP is equipotent across all serotypes because the nucleotide binding site residues are conserved in dengue virus. Overall, beta- d -2′-ethynyl-7-deaza-adenosine provides a promising scaffold for the development of inhibitors of dengue virus polymerase.

Nonobligate chain terminators, such as 2′-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3′-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2′-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2′-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2′-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2′-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

Recent studies have highlighted the need of investigating the in vivo role of ribonuclease H (RNase H) in nucleoside reverse transcriptase inhibitors (NRTIs) resistance. The prevalence of RNase H mutations in naive and in NRTI pre-treated patients in regimen failure were compared and some specific associations between NRTI resistance mutations and RNase H mutations were determined. Four positions were mutated more frequently in pre-treated patients than in naive patients: L469T/I/M/H, T470P/S/E/K, A554T/L/K, and K558R/G/E. Mutations at position K558 were also associated with the number of thymidine analog mutations (TAMs). These results suggest that these mutations could play a role in NRTI resistance. J. Med. Virol. 79:207–211, 2007. © 2007 Wiley-Liss, Inc.

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.

A dipeptide insertion between codons 69 and 70 together with the amino acid substitution T215Y in the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1 (HIV-1) strains are known to confer phenotypic resistance to zidovudine (AZT) and stavudine (d4T). Phenotypic resistance correlates with an increased ATP-dependent phosphorolytic activity. Nucleoside alpha-boranophosphate diastereoisomers derived from AZT and d4T were tested as substrates of a multidrug-resistant HIV-1 RT (designated as SS RT) bearing a Ser-Ser insertion at codons 69-70 and other drug resistance-related mutations, in DNA polymerization assays and ATP-mediated excision reactions. Using pre-steady-state kinetics, we show that SS RT can incorporate both R(p) and S(p) diastereoisomers, although R(p) is the preferred isomer. Chirality at the internucleotidic linkage formed upon incorporation of nucleoside alpha-boranophosphate did not affect ATP-mediated excision. As reported for AZT and d4T-terminated primers, substituting Thr, Asn or Ser for Tyr215 abrogates the ATP-dependent phosphorolytic activity on primers terminated with alpha-boranophosphate derivatives of thymidine analogues. However, unlike in the case of AZT, eliminating the dipeptide insertion in SS RT had no effect on the ATP-mediated excision of primers terminated with alpha-boranophosphate derivatives of d4T. Studies with ATP analogues showed that exchanging a non-bridging oxygen atom at the gamma-phosphate group for sulfur causes a significant reduction of the ATP-dependent phosphorolytic activity of SS RT. Interestingly, SS RT's excision activity is completely eliminated upon phosphorothioate substitution at the 3' end of primers terminated with AZT. These results suggest that phosphorothioate derivatives of currently approved drugs could be useful against excision-proficient HIV-1 strains.

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is an RNA- and DNA-dependent DNA polymerase capable of copying the viral genome before it gets integrated into the human host DNA. Hence, HIV-1 RT plays a major role in viral replication and represents a key target for anti-AIDS treatments. Amongst the eleven licensed drugs that inhibit RT, eight are chain-terminating nucleoside analogues (NRTIs) that compete with their natural counterparts during the DNA polymerization process. Unfortunately, under therapeutic pressure, the HIV-1 inevitably develops resistance to these inhibitors by accumulating mutations in the viral pol gene encoding RT. Mechanisms for this resistance can be sorted in two categories, depending on the nature of the drug and the selected mutations. The first category includes mutations involving a specific alteration of the discrimination between natural nucleotides and NRTIs. The second category includes mutations able to promote the removal of the incorporated NRTI and thus repair the nascent DNA chain. This review summarizes the modes of inhibition of HIV-1 RT with NRTIs, and describes the mechanisms of resistance to these drugs, based on enzymatic data correlated to crystal structures and molecular models involving HIV-1 RT. We also give insights into different aspects of resistance such as antagonistic mutations, replication capacity, and the implications for a rational, structure-based drug design.

Resistance to zidovudine (3′-azido-3′-deoxythymidine, AZT) by the human immunodeficiency virus, type 1, requires multiple amino acid substitutions such as D67N/K70R/T215F/K219Q in the viral reverse transcriptase (RT). In this background of AZT resistance, additional “suppressive” substitutions such as Y181C restore sensitivity to AZT. In order to characterize the mechanism of this AZT resistance suppression, the Y181C substitution was introduced into both wild-type and AZT-resistant reverse transcriptase. The introduction of the Y181C substitution suppresses the increased repair (or unblocking) of the AZTMP-terminated primer provided by the AZT resistance substitutions in RT using either DNA or RNA templates, independently from the RT RNase H activity. Contrary to wild-type RT, the low level of unblocking activity is not due to inhibition by the next correct nucleotide binding to the RT/AZTMP-terminated primer complex. When Y181C is added to the AZT resistance substitutions, ATP binds with less affinity to the AZTMP-terminated primer-RT binary complex. These results provide an insight into one possible molecular mechanism of re-sensitization of AZT-resistant viruses by suppressive substitutions.

Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k(pol): 18-fold (from 7 to 0.3 s(-1)), 13-fold (from 1.9 to 0.14 s(-1)), and 12-fold (from 13 to 1 s(-1)) in the case of ddATP, ddCTP, and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using alpha-boranophosphate nucleotide analogues. Using A62V/V75I/F77L/F116Y/Q151M RT, k(pol) increases up to 70- and 13-fold using alpha-boranophosphate-ddATP and alpha-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constant k(pol).

Dengue viral RNA polymerase replicates its positive single-stranded RNA genome in a primer-independent manner. The slow and inefficient initiation during replication masks the elongation mode. The aim of this work was to further characterize the mechanism of elongation towards an increased understanding of how the enzyme selectively recognizes different nucleotides. Transient kinetic methods were used to measure the microscopic rates of the reaction pathway comprised of enzyme and RNA binding followed by nucleotide binding and incorporation. After extended pre-incubation of the enzyme with double stranded RNA (12-mer primer with a 26-mer template), addition of a correct nucleotide resulted in a burst of single nucleotide incorporation, and the amplitude of the reaction was used to monitor the time course of RNA binding. The kinetics of enzyme and RNA binding followed a two-step mechanism: an initial binding was followed by a conformational change. The enzyme and RNA binding proceeded to equilibrium about six times faster at 37 oC than at 30 oC, suggesting the conformational change involved in RNA binding is temperature sensitive. Following incubation of RNA and enzyme to form an active complex, we used chemical quench-flow methods to measure the ground state binding Kd and the incorporation rate of a correct nucleotide. The fidelity of the polymerase was determined by measurement of the incorporation of incorrect nucleotides. Using these methods, we characterized the Dengue polymerase in its elongation mode by measuring the kinetics of RNA and polymerase binding and the kinetics for nucleotide binding and incorporation. Based on these data, we built a working model for studying the selectivity of Dengue polymerase.

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3'-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.

The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5′ end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.