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1.
Nucleic Acids Res ; 49(22): 13019-13030, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1546008

ABSTRACT

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Nucleotidyltransferases/antagonists & inhibitors , RNA, Viral/biosynthesis , SARS-CoV-2/enzymology , Adenosine Triphosphate/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Genome, Viral/genetics , Guanosine/analogs & derivatives , Guanosine/metabolism , Humans , Nucleotidyltransferases/metabolism , RNA Caps/genetics , SARS-CoV-2/genetics , Vaccinia virus/enzymology , Vaccinia virus/metabolism
3.
Int J Mol Sci ; 21(19)2020 Sep 27.
Article in English | MEDLINE | ID: covidwho-1299427

ABSTRACT

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.


Subject(s)
Enzyme Inhibitors/chemistry , Membrane Proteins , Molecular Docking Simulation , Nucleotidyltransferases , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Drug Evaluation, Preclinical , Fluorescence Polarization , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry
4.
mBio ; 12(3): e0142321, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1280400

ABSTRACT

The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Guanosine Tetraphosphate/pharmacology , SARS-CoV-2/drug effects , Adenosine Triphosphate/pharmacology , COVID-19/drug therapy , Catalytic Domain/physiology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Ribosomes/metabolism
5.
Vet Clin North Am Small Anim Pract ; 50(5): 1001-1011, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-827004

ABSTRACT

Feline infectious peritonitis (FIP) is a mysterious and lethal disease of cats. The causative agent, feline coronavirus (FCoV), is ubiquitous in most feline populations, yet the disease is sporadic in nature. Mutations in the infecting virus combined with an inappropriate immune response to the FCoV contribute to the development of FIP. Diagnosis can be challenging because signs may be vague, clinical pathology parameters are nonspecific, and the gold standard for diagnosis is invasive: histopathology of affected tissue. This article discusses the developments in the understanding of this disease as well as the progress in diagnosis and treatment.


Subject(s)
Coronavirus, Feline , Feline Infectious Peritonitis/virology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/therapeutic use , Animals , Antiviral Agents/therapeutic use , Cats , Coronavirus, Feline/genetics , Feline Infectious Peritonitis/diagnosis , Feline Infectious Peritonitis/pathology , Feline Infectious Peritonitis/therapy , Genetic Predisposition to Disease , Pyrrolidines/therapeutic use , Sulfonic Acids
6.
Antiviral Res ; 182: 104899, 2020 10.
Article in English | MEDLINE | ID: covidwho-694239

ABSTRACT

Remdesivir was shown to inhibit RNA-dependent RNA-polymerases (RdRp) from distinct viral families such as from Filoviridae (Ebola) and Coronaviridae (SARS-CoV, SARS-CoV-2, MERS). In this study, we tested the ability of remdesivir to inhibit RdRps from the Flaviviridae family. Instead of remdesivir, we used the active species that is produced in cells from remdesivir, the appropriate triphosphate, which could be directly tested in vitro using recombinant flaviviral polymerases. Our results show that remdesivir can efficiently inhibit RdRps from viruses causing severe illnesses such as Yellow fever, West Nile fever, Japanese and Tick-borne encephalitis, Zika and Dengue. Taken together, this study demonstrates that remdesivir or its derivatives have the potential to become a broad-spectrum antiviral agent effective against many RNA viruses.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Flavivirus/drug effects , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/pharmacology , Antiviral Agents/chemistry , Betacoronavirus/drug effects , Betacoronavirus/enzymology , COVID-19 , Flavivirus/enzymology , Humans , Inhibitory Concentration 50 , Pandemics , RNA Viruses/drug effects , RNA Viruses/enzymology , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2
7.
J Antimicrob Chemother ; 75(10): 2977-2980, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-626863

ABSTRACT

BACKGROUND: Remdesivir is a prodrug of the nucleoside analogue GS-441524 and is under evaluation for treatment of SARS-CoV-2-infected patients. OBJECTIVES: To evaluate the pharmacokinetics of remdesivir and GS-441524 in plasma, bronchoalveolar aspirate (BAS) and CSF in two critically ill COVID-19 patients. METHODS: Remdesivir was administered at 200 mg loading dose on the first day followed by 12 days of 100 mg in two critically ill patients. Blood samples were collected immediately after (C0) and at 1 (C1) and 24 h (C24) after intravenous administration on day 3 until day 9. BAS samples were collected on Days 4, 7 and 9 from both patients while one CSF on Day 7 was obtained in one patient. Remdesivir and GS-441524 concentrations were measured in these samples using a validated UHPLC-MS/MS method. RESULTS: We observed higher concentrations of remdesivir at C0 (6- to 7-fold higher than EC50 from in vitro studies) and a notable decay at C1. GS-441524 plasma concentrations reached a peak at C1 and persisted until the next administration. Higher concentrations of GS-441524 were observed in the patient with mild renal dysfunction. Mean BAS/plasma concentration ratios of GS-441524 were 2.3% and 6.4% in Patient 1 and Patient 2, respectively. The CSF concentration found in Patient 2 was 25.7% with respect to plasma. GS-441524 levels in lung and CNS suggest compartmental differences in drug exposure. CONCLUSIONS: We report the first pharmacokinetic evaluation of remdesivir and GS-441524 in recovered COVID-19 patients. Further study of the pharmacokinetic profile of remdesivir, GS-441524 and the intracellular triphosphate form are required.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Adenosine Triphosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacokinetics , Betacoronavirus , Coronavirus Infections/metabolism , Critical Illness/therapy , Pneumonia, Viral/metabolism , Adenosine Monophosphate/pharmacokinetics , Adenosine Monophosphate/therapeutic use , Adenosine Triphosphate/pharmacokinetics , Adenosine Triphosphate/therapeutic use , Aged , Alanine/pharmacokinetics , Alanine/therapeutic use , Antiviral Agents/therapeutic use , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/drug therapy , Female , Humans , Male , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/drug therapy , Recovery of Function/drug effects , Recovery of Function/physiology , SARS-CoV-2
8.
J Vet Intern Med ; 34(4): 1587-1593, 2020 Jul.
Article in English | MEDLINE | ID: covidwho-343296

ABSTRACT

Feline infectious peritonitis (FIP) is caused by a mutant biotype of the feline enteric coronavirus. The resulting FIP virus (FIPV) commonly causes central nervous system (CNS) and ocular pathology in cases of noneffusive disease. Over 95% of cats with FIP will succumb to disease in days to months after diagnosis despite a variety of historically used treatments. Recently developed antiviral drugs have shown promise in treatment of nonneurological FIP, but data from neurological FIP cases are limited. Four cases of naturally occurring FIP with CNS involvement were treated with the antiviral nucleoside analogue GS-441524 (5-10 mg/kg) for at least 12 weeks. Cats were monitored serially with physical, neurologic, and ophthalmic examinations. One cat had serial magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analysis (including feline coronavirus [FCoV]) titers and FCoV reverse transcriptase [RT]-PCR) and serial ocular imaging using Fourier-domain optical coherence tomography (FD-OCT) and in vivo confocal microscopy (IVCM). All cats had a positive response to treatment. Three cats are alive off treatment (528, 516, and 354 days after treatment initiation) with normal physical and neurologic examinations. One cat was euthanized 216 days after treatment initiation following relapses after primary and secondary treatment. In 1 case, resolution of disease was defined based on normalization of MRI and CSF findings and resolution of cranial and caudal segment disease with ocular imaging. Treatment with GS-441524 shows clinical efficacy and may result in clearance and long-term resolution of neurological FIP. Dosages required for CNS disease may be higher than those used for nonneurological FIP.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/therapeutic use , Feline Infectious Peritonitis/drug therapy , Adenosine Triphosphate/administration & dosage , Adenosine Triphosphate/therapeutic use , Animals , Antiviral Agents/administration & dosage , Cats , Female , Male
9.
J Antimicrob Chemother ; 75(7): 1772-1777, 2020 07 01.
Article in English | MEDLINE | ID: covidwho-154881

ABSTRACT

BACKGROUND: Remdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification. OBJECTIVES: The validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma. METHODS: Remdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 µm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines. RESULTS: Analyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety. CONCLUSIONS: This method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Adenosine Triphosphate/analogs & derivatives , Alanine/analogs & derivatives , Chromatography, High Pressure Liquid/methods , Hemorrhagic Fever, Ebola/drug therapy , Tandem Mass Spectrometry/methods , Adenosine Monophosphate/analysis , Adenosine Monophosphate/blood , Adenosine Monophosphate/pharmacokinetics , Adenosine Triphosphate/analysis , Adenosine Triphosphate/blood , Adenosine Triphosphate/pharmacokinetics , Alanine/analysis , Alanine/blood , Alanine/pharmacokinetics , Betacoronavirus , COVID-19 , Coronavirus Infections/drug therapy , Humans , Pandemics , Pneumonia, Viral/drug therapy , SARS-CoV-2 , Sensitivity and Specificity
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