Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 26
Filter
2.
Antiviral Res ; 204: 105364, 2022 08.
Article in English | MEDLINE | ID: covidwho-1894784

ABSTRACT

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.


Subject(s)
Antiviral Agents , Arenavirus , Exoribonucleases , SARS-CoV-2 , Animals , Antiviral Agents/pharmacology , Arenavirus/drug effects , Chlorocebus aethiops , Exoribonucleases/antagonists & inhibitors , Fluorescence Polarization , SARS-CoV-2/drug effects , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors
3.
J Med Chem ; 65(8): 6231-6249, 2022 04 28.
Article in English | MEDLINE | ID: covidwho-1867997

ABSTRACT

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , COVID-19/virology , Exoribonucleases/antagonists & inhibitors , Exoribonucleases/chemistry , Humans , Methyltransferases , Molecular Docking Simulation , RNA, Viral/genetics , S-Adenosylmethionine , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry
4.
J Virol ; 96(8): e0012822, 2022 04 27.
Article in English | MEDLINE | ID: covidwho-1765079

ABSTRACT

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.


Subject(s)
COVID-19 , Furin , SARS-CoV-2 , Serine Endopeptidases , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/pathology , COVID-19/virology , Furin/metabolism , HeLa Cells , Humans , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
5.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320611

ABSTRACT

SARS-CoV-2 is a new human coronavirus (CoV), which emerged in China in late 2019 and is responsible for the global COVID-19 pandemic that caused more than 59 million infections and 1.4 million deaths in 11 months. Understanding the origin of this virus is an important issue and it is necessary to determine the mechanisms of its dissemination in order to contain future epidemics. Based on phylogenetic inferences, sequence analysis and structure-function relationships of coronavirus proteins, informed by the knowledge currently available on the virus, we discuss the different scenarios evoked to account for the origin - natural or synthetic - of the virus. The data currently available is not sufficient to firmly assert whether SARS-CoV2 results from a zoonotic emergence or from an accidental escape of a laboratory strain. This question needs to be solved because it has important consequences on the evaluation of risk/benefit balance of our interaction with ecosystems, the intensive breeding of wild and domestic animals, as well as some lab practices and on scientific policy and biosafety regulations. Regardless of its origin, studying the evolution of the molecular mechanisms involved in the emergence of pandemic viruses is essential to develop therapeutic and vaccine strategies and to prevent future zoonoses. This article is a translation and update of a French article published in M{é}decine/Sciences, Aug/Sept 2020 (http://doi.org/10.1051/medsci/2020123).

6.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323380

ABSTRACT

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains furin-like cleavage sites absent the other SARS-like viruses. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In this study, we used structure- based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti-fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS- CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

7.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-307686

ABSTRACT

A worldwide effort is ongoing to discover drugs against the Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2), which has so far caused >3.5 million fatalities (https://covid19.who.int/). The virus essential RNA-dependent RNA polymerase complex is targeted by several nucleoside/tide analogues whose mechanisms of action and clinical potential are currently evaluated. The guanosine analogue AT-527, a double prodrug of its 5'-triphosphate AT-9010, is currently in phase III clinical trials as a COVID19 treatment. Here we report the cryo-EM structure at 2.98 Å resolution of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 complex with RNA showing AT-9010 bound at three sites of nsp12. At the RdRp active-site, one AT-9010 is incorporated into the RNA product. Its 2'-methyl group prevents correct alignment of a second AT-9010 occupying the incoming NTP pocket. The 2'-F, 2'-methyl 3'-OH ribose scaffold explains the non-obligate RNA chain-termination potency of this NA series for both HCV NS5 and SARS-CoV RTCs. A third AT-9010 molecule 5'-diphosphate binds to a coronavirus-specific pocket in the nsp12 N-terminus NiRAN domain, a SelO pseudo-kinase structural and functional homologue. This unique binding mode impedes NiRAN-mediated UMPylation of SARS-CoV-2 nsp8 and nsp9 proteins. Our results suggest a mechanism of action for AT-527 in line with a therapeutic use for COVID19.

8.
Molecules ; 27(3)2022 Feb 04.
Article in English | MEDLINE | ID: covidwho-1686900

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Heterocyclic Compounds/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Chlorocebus aethiops , High-Throughput Screening Assays , Microbial Sensitivity Tests , Structure-Activity Relationship , Vero Cells
9.
Nat Commun ; 13(1): 621, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1671551

ABSTRACT

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Guanosine Monophosphate/analogs & derivatives , Phosphoramides/chemistry , Phosphoramides/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/enzymology , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , COVID-19/virology , Cryoelectron Microscopy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Guanosine Monophosphate/chemistry , Guanosine Monophosphate/pharmacology , Humans , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Proteins/genetics
10.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Article in English | MEDLINE | ID: covidwho-1541316

ABSTRACT

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.


Subject(s)
Exoribonucleases/chemistry , Models, Molecular , Protein Conformation , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Catalytic Domain , Conserved Sequence , Exoribonucleases/genetics , Exoribonucleases/metabolism , Microbial Viability , Nucleotide Motifs , RNA, Viral/chemistry , RNA, Viral/genetics , RNA-Binding Proteins , Structure-Activity Relationship , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/genetics
12.
Virologie (Montrouge) ; 25(3): 42-46, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1308206
13.
Eur J Med Chem ; 224: 113683, 2021 Nov 15.
Article in English | MEDLINE | ID: covidwho-1293756

ABSTRACT

The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5-5.3 µM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 µM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Flavivirus/drug effects , Orthomyxoviridae/drug effects , Purines/chemistry , Purines/pharmacology , SARS-CoV-2/drug effects , Molecular Targeted Therapy , Virus Replication/drug effects
14.
Trends Biochem Sci ; 46(11): 866-877, 2021 11.
Article in English | MEDLINE | ID: covidwho-1283592

ABSTRACT

With sizes <50 kb, viral RNA genomes are at the crossroads of genetic, biophysical, and biochemical stability in their host cell. Here, we analyze the enzymatic assets accompanying large RNA genome viruses, mostly based on recent scientific advances in Coronaviridae. We argue that, in addition to the presence of an RNA exonuclease (ExoN), two markers for the large size of viral RNA genomes are (i) the presence of one or more RNA methyltransferases (MTases) and (ii) a specific architecture of the RNA-dependent RNA polymerase active site. We propose that RNA genome expansion and maintenance are driven by an evolutionary ménage-à-trois made of fast and processive RNA polymerases, RNA repair ExoNs, and RNA MTases that relates to the transition between RNA- to DNA-based life.


Subject(s)
RNA Viruses , Amino Acid Sequence , Genome Size , Methyltransferases , RNA Viruses/genetics , RNA, Viral/genetics
17.
Environ Chem Lett ; : 1-17, 2021 Feb 04.
Article in English | MEDLINE | ID: covidwho-1070871

ABSTRACT

SARS-CoV-2 is a new human coronavirus (CoV), which emerged in China in late 2019 and is responsible for the global COVID-19 pandemic that caused more than 97 million infections and 2 million deaths in 12 months. Understanding the origin of this virus is an important issue, and it is necessary to determine the mechanisms of viral dissemination in order to contain future epidemics. Based on phylogenetic inferences, sequence analysis and structure-function relationships of coronavirus proteins, informed by the knowledge currently available on the virus, we discuss the different scenarios on the origin-natural or synthetic-of the virus. The data currently available are not sufficient to firmly assert whether SARS-CoV2 results from a zoonotic emergence or from an accidental escape of a laboratory strain. This question needs to be solved because it has important consequences on the risk/benefit balance of our interactions with ecosystems, on intensive breeding of wild and domestic animals, on some laboratory practices and on scientific policy and biosafety regulations. Regardless of COVID-19 origin, studying the evolution of the molecular mechanisms involved in the emergence of pandemic viruses is essential to develop therapeutic and vaccine strategies and to prevent future zoonoses. This article is a translation and update of a French article published in Médecine/Sciences, August/September 2020 (10.1051/medsci/2020123). SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10311-020-01151-1) contains supplementary material, which is available to authorized users.

18.
19.
NAR Genom Bioinform ; 2(1): lqz022, 2020 Mar.
Article in English | MEDLINE | ID: covidwho-824972

ABSTRACT

The order Nidovirales is a diverse group of (+)RNA viruses, classified together based on their common genome organisation and conserved replicative enzymes, despite drastic differences in size and complexity. One such difference pertains to the mechanisms and enzymes responsible for generation of the proposed viral 5' RNA cap. Within the Coronaviridae family, two separate methytransferases (MTase), nsp14 and nsp16, perform the RNA-cap N7-guanine and 2'-OH methylation respectively for generation of the proposed m7GpppNm type I cap structure. For the majority of other families within the Nidovirales order, the presence, structure and key enzymes involved in 5' capping are far less clear. These viruses either lack completely an RNA MTase signature sequence, or lack an N7-guanine methyltransferase signature sequence, obscuring our understanding about how RNA-caps are N7-methylated for these families. Here, we report the discovery of a putative Rossmann fold RNA methyltransferase in 10 Tobaniviridae members in Orf1a, an unusual genome locus for this gene. Multiple sequence alignments and structural analyses lead us to propose this novel gene as a typical RNA-cap N7-guanine MTase with substrate specificity and active-site organization similar to the canonical eukaryotic RNA-cap N7-guanine MTase.

20.
Nat Commun ; 11(1): 4682, 2020 09 17.
Article in English | MEDLINE | ID: covidwho-779999

ABSTRACT

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.


Subject(s)
Amides/pharmacology , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Betacoronavirus/genetics , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Pyrazines/pharmacology , Amides/pharmacokinetics , Animals , Antiviral Agents/pharmacokinetics , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/virology , Coronavirus RNA-Dependent RNA Polymerase , Models, Molecular , Mutagenesis/drug effects , Pandemics , Pneumonia, Viral/virology , Pyrazines/pharmacokinetics , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2 , Sequence Analysis , Vero Cells , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
SELECTION OF CITATIONS
SEARCH DETAIL