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1.
ACS Infect Dis ; 8(8): 1533-1542, 2022 Aug 12.
Article in English | MEDLINE | ID: covidwho-1931304

ABSTRACT

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.

2.
SLAS Discov ; 26(9): 1200-1211, 2021 10.
Article in English | MEDLINE | ID: covidwho-1290310

ABSTRACT

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.


Subject(s)
COVID-19/genetics , Exoribonucleases/genetics , Protein Binding/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Antiviral Agents/pharmacology , Binding Sites/genetics , COVID-19/virology , Humans , Methylation , Pandemics , RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Virus Replication/genetics
3.
ACS Infect Dis ; 7(8): 2214-2220, 2021 08 13.
Article in English | MEDLINE | ID: covidwho-1275859

ABSTRACT

In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases (MTases), nsp14. This MTase catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to cap the guanosine triphosphate moiety of the newly synthesized viral RNA, yielding the methylated capped RNA and S-adenosyl-l-homocysteine (SAH). As the crystal structure of SARS-CoV-2 nsp14 is unknown, we have taken advantage of its high homology to SARS-CoV nsp14 and prepared its homology model, which has allowed us to identify novel SAH derivatives modified at the adenine nucleobase as inhibitors of this important viral target. We have synthesized and tested the designed compounds in vitro and shown that these derivatives exert unprecedented inhibitory activity against this crucial enzyme. The docking studies nicely explain the contribution of an aromatic part attached by a linker to the position 7 of the 7-deaza analogues of SAH.


Subject(s)
COVID-19 , Methyltransferases , Exoribonucleases , Humans , Ligands , Methyltransferases/genetics , SARS-CoV-2 , Viral Nonstructural Proteins
4.
SLAS Discov ; 26(6): 757-765, 2021 07.
Article in English | MEDLINE | ID: covidwho-1194439

ABSTRACT

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Subject(s)
Adenosine/analogs & derivatives , High-Throughput Screening Assays , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/chemistry , Adenosine/pharmacology , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Methylation , Methyltransferases , Models, Molecular , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Tritium , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism
5.
SLAS Discov ; 26(5): 620-627, 2021 06.
Article in English | MEDLINE | ID: covidwho-1021348

ABSTRACT

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.


Subject(s)
Antiviral Agents/pharmacology , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/analogs & derivatives , Adenosine/pharmacology , Binding, Competitive , COVID-19/drug therapy , COVID-19/virology , Enzyme Inhibitors/pharmacology , Fluorescence Polarization , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Humans , Methyltransferases , Protein Binding , RNA Caps/antagonists & inhibitors , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Virus Replication
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