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The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19. [ABSTRACT FROM AUTHOR]

Highlights: [•] Combinations of rilpivirine, emtricitabine, and tenofovir display anti-HIV-1 synergy. [•] Gel shift assays show dead-end complex (DEC) formation with these drugs. [•] Mechanisms of synergy map to DEC-like complexes. [Copyright &y& Elsevier]

Background: Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds. Results: In this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTCmonophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations. Conclusion: This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti- HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations. [ABSTRACT FROM AUTHOR]

The article reports on the mutations of A62v and S68G in HIV-1 reverse transcriptase and the effects on the replication defect related to K65R mutation. It mentions that the K65R mutation in HIV-1 transcriptase can be selected by drugs and to reduce susceptibility and viral replication. The study examines A62v and S68G in K65R mutation. An overview of the study is provided.

Abstract: 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 (k pol) 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. [Copyright &y& Elsevier]

Amdoxovir [(-)-β-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5′-nucleotidase (5′-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7mM). Human 5′-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG–MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG–DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG–DP. In CEM cells incubated with 5μM DXG for 24h, 0.015pmole/106 cells (~7.5nM) of DXG–TP was detected as the primary metabolite. Our study demonstrated that 5′-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo. [Copyright &y& Elsevier]

Details a study which sought to characterize novel reverse transcriptase and other RNA-associated catalytic activities by human DNA polymerase gamma. Formation of DNA/RNA heteroduplex during mitochondrial DNA replication; Findings that polymerase gamma also incorporates ribonucleotide triphosphate into a DNA primer.

Investigates the use of dioxolone guanosine 5'-triphosphate (DXG-TP) as an alternative substrate inhibitor of wild-type and mutant HIV-1 reverse transcriptase. Inhibition of HIV-1 Rt by DXG-TP; Retention of dGMP incorporation by DXG-TP; Increase in the level of resistance to inhibition by nucleoside analogs.

Examines DNA synthesis by the 3TC-resistant mutant (184v) of HIV reverse transcriptase. Effect of a single amino acid substitution on codon 184 on the incorporation of the natural substrate dCTP; Mechanics of correct incorporation.

Presents information on a study which determined the maximum rate of incorporation of dideoxy nucleoside analogues using HIV-1 reverse transcriptase. Materials and methods; Results and discussion.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS646939 is the active 50 -triphosphate metabolite of a 4ʹ-cyano modified C-adenosine analog phosphoramidate prodrug GS7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS646939. Once incorporated into the nascent RNA strand, GS646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹmodification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 10 -cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹcyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses. [ABSTRACT FROM AUTHOR]

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation. [ABSTRACT FROM AUTHOR]

Antiviral drugs for managing infections with human coronaviruses are not yet approved, posing a serious challenge to current global efforts aimed at containing the outbreak of severe acute respiratory syndrome-coronavirus 2 (CoV-2). Remdesivir (RDV) is an investigational compound with a broad spectrum of antiviral activities against RNA viruses, including severe acute respiratory syndrome-CoV and Middle East respiratory syndrome (MERS-CoV).RDVis a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps). Here, we co-expressed the MERS-CoV nonstructural proteins nsp5, nsp7, nsp8, and nsp12 (RdRp) in insect cells as a part a polyprotein to study the mechanism of inhibition of MERS-CoV RdRp by RDV. We initially demonstrated that nsp8 and nsp12 form an active complex. The triphosphate form of the inhibitor (RDVTP) competes with its natural counterpart ATP. Of note, the selectivity value for RDV-TP obtained here with a steady-state approach suggests that it is more efficiently incorporated than ATP and two other nucleotide analogs. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i + 3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the inhibitor from excision by the viral 3'-5' exonuclease activity. Together, these results help to explain the high potency of RDV against RNA viruses in cell-based assays. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases. [ABSTRACT FROM AUTHOR]

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C- methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity. [ABSTRACT FROM AUTHOR]

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of Coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'- UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease. [ABSTRACT FROM AUTHOR]

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5–7.4) that might not occur in the bladder of rats (urinary pH 7.3–8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions. [ABSTRACT FROM AUTHOR]

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain termination at position i + 3. Here we demonstrate that the S861G mutation in RdRp eliminates chain termination, which confirms the existence of a steric clash between Ser-861 and the incorporated RDV-TP. With WT RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned because of a significant clash with Ala-558. The adjacent Val-557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS-CoV-2 RdRp by RDV is biologically relevant. [ABSTRACT FROM AUTHOR]

Effective treatments for coronavirus disease 2019 (COVID- 19) are urgently needed to control this current pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Replication of SARS-CoV-2 depends on the viral RNA-dependent RNA polymerase (RdRp), which is the likely target of the investigational nucleotide analogue remdesivir (RDV). RDV shows broad-spectrum antiviral activity against RNA viruses, and previous studies with RdRps from Ebola virus and Middle East respiratory syndrome coronavirus (MERSCoV) have revealed that delayed chain termination is RDV's plausible mechanism of action. Here, we expressed and purified active SARS-CoV-2 RdRp composed of the nonstructural proteins nsp8 and nsp12. Enzyme kinetics indicated that this RdRp efficiently incorporates the active triphosphate form of RDV (RDV-TP) into RNA. Incorporation of RDV-TP at position i caused termination of RNA synthesis at position i+3. We obtained almost identical results with SARS-CoV, MERS-CoV, and SARS-CoV-2 RdRps. A unique property of RDV-TP is its high selectivity over incorporation of its natural nucleotide counterpart ATP. In this regard, the triphosphate forms of 2'-Cmethylated compounds, including sofosbuvir, approved for the management of hepatitisCvirus infection, and the broad-acting antivirals favipiravir and ribavirin, exhibited significant deficits. Furthermore, we provide evidence for the target specificity of RDV, as RDV-TP was less efficiently incorporated by the distantly related Lassa virus RdRp, and termination ofRNAsynthesis was not observed. These results collectively provide a unifying, refined mechanism of RDV-mediated RNA synthesis inhibition in coronaviruses and define this nucleotide analogue as a direct-acting antiviral. [ABSTRACT FROM AUTHOR]

Abstract Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts. Graphical abstract Unlabelled Image Highlights • CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM). • The molecule KN-93 is the most widely used inhibitor of CaMKII. • We employed SPR, NMR, ITC, and enzymology to characterize the inhibition mechanism. • We show that KN-93 binds directly to Ca2+/CaM and not to CaMKII. • We offer new insight into how in vitro and in vivo KN-93 data should be interpreted. [ABSTRACT FROM AUTHOR]

Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency. [ABSTRACT FROM AUTHOR]

Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PAN) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PAN, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed Km (150 ± 11 nM) and kcat [(1.4 ± 0.2) x 10-3s-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC50 values of 10–20 nM, demonstrating the utility of this system for future high throughput screening. [ABSTRACT FROM AUTHOR]

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [Nature 2016, 531, 381-385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors. [ABSTRACT FROM AUTHOR]

Novel 4′-substituted β- d -2′-deoxy-2′-α-fluoro (2′d2′F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4′-substitution onto 2′d2′F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4′-substitution and not readily predictable. Combining the most potent and selective 4′-groups from N -nucleoside analogs onto a 2′d2′F C -nucleoside analog resulted in the identification of β- d -2′-deoxy-2′-α-fluoro-4′-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV. [ABSTRACT FROM AUTHOR]

The first synthesis of 1′- C -CN, 2′-F, 2′- C -Me pyrimidines is described. Anti-HCV activity was assessed and compared to the 1′- C -CN, 2′- C -Me as well as the 2′-F, 2′- C -Me pyrimidines. A phosphoramidate prodrug of the cytidine derivative showed activity in the low micromolar range against HCV replicons. [ABSTRACT FROM AUTHOR]

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane--anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. [ABSTRACT FROM AUTHOR]

Abstract: The first synthesis of 1′-cyano-2′-C-methyl pyrimidine nucleosides is described. Anti-HCV activity of these nucleosides and their nucleotide phosphoramidate prodrugs was assessed and compared to the 1′-unsubstituted counterparts and to the related 1′-cyano-2′-C-methyl C-nucleoside parent of GS-6620. [Copyright &y& Elsevier]

R4278 --> 658.6 --> Remdesivir (RDV) is a direct‐acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). RDV exhibits broad‐spectrum antiviral activity against positive‐sense RNA viruses, e.g. SARS‐CoV‐2 and hepatitis C virus (HCV) and non‐segmented negative‐sense RNA viruses, e.g. Nipah virus (NiV), while segmented negative‐sense RNA viruses such as influenza (Flu) virus or Crimean‐Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA‐dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV‐triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV‐TP, (ii) the effect of the incorporated RDV‐monophosphate (MP) on primer extension, and (iii) the effect of RDV‐MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV‐TP. Inhibition in primer extension reactions is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template‐dependent inhibition of UTP incorporation opposite the embedded RDV‐MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'‐cyano group of RDV‐MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'‐modification. C.J.G, H.W.L, and E.P.T contributed equally to this work [ABSTRACT FROM AUTHOR]

Abstract: Ribonucleoside phosphonate analogues containing 2′-α-fluoro modifications were synthesized and their potency evaluated against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent inhibition of the HCV polymerase in the range of 1.9–2.1μM, but only modest cell-based activity in the HCV replicon. Pro-drugs of the parent nucleoside phosphonates improved the cell-based activity. [Copyright &y& Elsevier]

Abstract: The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T–6PEG–TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA. [Copyright &y& Elsevier]

A correction to the article "Nucleoside Diphosphate Kinase and the Activation of Antiviral Phosphonate Analogs of Nucleotides: Binding Mode and Phosphorylation of Tenofovir Derivatives," that was published in volume 28 issue is presented.

Abstract: A series of 2′-C-methyl branched purine and pyrimidine C-nucleosides were prepared. Their anti-HCV activity and pharmacological properties were profiled, and compared with known 2′-C-Me N-nucleoside counterparts. In particular, 2′-C-Me 4-aza-7,9-dideazaadenosine C-nucleoside (2) was found to have potent and selective anti-HCV activity in vitro as well as a favorable pharmacokinetic profile and in vivo potential for enhanced potency over the corresponding N-nucleoside. [Copyright &y& Elsevier]

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines were prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model. [ABSTRACT FROM AUTHOR]

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg72 were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg72 were stacked in two different rotameric conformations in TFV-DPand dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations. [ABSTRACT FROM AUTHOR]

Abstract: The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2′-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61–109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC50) regardless of genotype or subtype. Importantly, we observed a significant correlation (p <0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested. [Copyright &y& Elsevier]

Abstract: A series of 1′-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R=CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate. [Copyright &y& Elsevier]

Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC 50 studies) or following a time course where inactivation kinetics were measured. Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency k inact /K i was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a k inact /K i value of 2.4 ± 0.6 × 104 M−1 s−1 for BTK with selectivity against important off-targets. For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases. • The inactivation kinetics of four clinically-relevant BTK inhibitors on BTK and related kinases are characterized. • Kinetics were measured for tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib. • Selectivity between kinases was determined based on the inactivation kinetics. • Tirabrutinib irreversibly and covalently binds to the Cys-481 residue of BTK and is a potent and selective BTK inhibitor. [ABSTRACT FROM AUTHOR]

The genetically diverse Orthocoronavirinae (CoV) family is prone to cross species transmission and disease emergence in both humans and livestock. Viruses similar to known epidemic strains circulating in wild and domestic animals further increase the probability of emergence in the future. Currently, there are no approved therapeutics for any human CoV presenting a clear unmet medical need. Remdesivir (RDV, GS-5734) is a monophosphoramidate prodrug of an adenosine analog with potent activity against an array of RNA virus families including Filoviridae , Paramyxoviridae , Pneumoviridae , and Orthocoronavirinae , through the targeting of the viral RNA dependent RNA polymerase (RdRp). We developed multiple assays to further define the breadth of RDV antiviral activity against the CoV family. Here, we show potent antiviral activity of RDV against endemic human CoVs OC43 (HCoV-OC43) and 229E (HCoV-229E) with submicromolar EC 50 values. Of known CoVs, the members of the deltacoronavirus genus have the most divergent RdRp as compared to SARS- and MERS-CoV and both avian and porcine members harbor a native residue in the RdRp that confers resistance in beta-CoVs. Nevertheless, RDV is highly efficacious against porcine deltacoronavirus (PDCoV). These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV. • In vitro antiviral assays were developed for human CoV OC43 and 229E and the zoonotic PDCoV. • The nucleoside analog RDV inhibited HCoV-OC43 and 229E as well as deltacoronavirus member PDCoV. • RDV has broad-spectrum antiviral activity against CoV and should be evaluated for future emerging CoV. [ABSTRACT FROM AUTHOR]

A series of novel 1′-substituted analogues of 4-aza-7,9-dideazaadenosine C-nucleoside are prepared and tested in antiviral assays. [ABSTRACT FROM AUTHOR]