Document text
Principal Investigator: Steven Gary Van Lanen
Organization: UNIVERSITY OF KENTUCKY
Fiscal Year: 2021
Award: $417,474
Funding agency: National Institute of Allergy and Infectious Diseases
SARS-CoV-2 is a positive-sense, single-stranded RNA [(+)ssRNA] virus that relies on its RNA-dependent RNA
polymerase (RdRp) for survival. Within the months of April and May, five groups have independently reported
the production of recombinant SAR-CoV-2 RdRp and a preliminary activity assessment of this essential enzyme.
Included in these studies was direct evidence that the triphosphate version of the nucleoside analogue
remdesivir, which has received Emergency Use Authorization from the Food and Drug Administration to treat
SARS-CoV-2, is incorporated in place of ATP into the growing RNA oligonucleotide, ultimately leading to chain
termination. Other nucleobase and nucleoside analogues including EIDD-1931 and favipiravir, the latter of which
has been approved in Japan to treat the (-)ssRNA influenza virus, have demonstrated promise as therapeutic
agents against SARS-CoV-2 by likely interfering with RNA metabolism via inhibition or processing as alternative
substrates for RdRp. This and other data suggest that RdRp is an excellent target for the discovery and
development of novel SARS-CoV-2 therapeutics. However, one of the major bottlenecks in exploiting RdRp as
a drug target is the relatively low throughput activity-based assays that are costly, prone to interference, and/or
lack flexibility in the experimental design. The primary objective of this proposal is to develop a broadly applicable
RdRp activity-based assay that will be used for antiviral drug discovery efforts. Our specific aim is to establish a
novel, real-time assay using a five-enzyme coupled system with a colorimetric readout. The new assay will be
directly compared to the traditional polyacrylamide gel electrophoresis and a liquid scintillation proximity end-
point assay, and further validated with high resolution mass spectrometry. It is expected that, by accomplishing
this aim, the assay will enable a thorough biochemical characterization of SARS-CoV-2 RdRp and, for the first
time, enable the testing of synthetic compound and natural product libraries to identify inhibitors, alternative
substrates, modulators, or effectors of SARS-CoV-2 RdRp activity in a high throughput screening format. Notably,
the strategy implemented herein is expected to complement on-going structural-based anti-SARS-CoV-2
discovery efforts. Finally, the activity-based assay can be readily adapted for RdRp from other (+)ssRNA and (-
)ssRNA viruses in an effort to identify therapeutics against a broad spectrum of pandemic-causing viruses.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Acute Poliomyelitis><Address><American><Antiviral Agents><Antiviral Drugs><Antivirals><Assay><Bioassay><Biochemical><Biochemistry><Biologic Assays><Biological Assay><Biological Chemistry><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 assay><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 outbreak><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 therapeutics><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID19 assay><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 outbreak><COVID19 pandemic><COVID19 public health crisis><COVID19 therapeutics><COVID19 therapy><COVID19 treatment><COVID19 virus><Cessation of life><Chemicals><Clinical><Clinical Trials><CoV-2><CoV2><Communicable Diseases><Communities><Complement><Complement Proteins><Complex><Coronaviridae><Coronavirus><Coupled><DNA-Dependent RNA Polymerase I><Data><Death><Detection><Development><Diphosphates><Drug Design><Drug Targeting><EBOV><EC 2.7.7.48><Ebola virus><Ebola-like Viruses><Egypt 101 virus><Emergencies><Emergency Situation><End Point Assay><Endpoint Assays><Enterovirus><Enzyme Gene><Enzymes><Evaluation><Event><Experimental Designs><FDA EUA><FDA Emergency Use Authorization><FDA approved><Flavivirus><Food and Drug Administration EUA><Food and Drug Administration Emergency Use Authorization><Foundations><Future><GS-5734><Gene Transcription><Genetic Materials><Genetic Transcription><Goals><Group B Arbovirus><H1N1><H1N1 Virus><High Throughput Assay><In Vitro><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Influenza A><Influenza A Virus, H1N1 Subtype><Influenza A virus><Influenza Virus><Influenza Viruses Type A><Influenzavirus A><Infrastructure><Japan><Libraries><Life><Liquid substance><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Methods><Molecular><Mutate><NIAID><Names><National Institute of Allergy and Infectious Disease><Natural Products><Non-Polyadenylated RNA><Nucleotides><Oligo><Oligonucleotides><Orthomyxovirus Type A><Patients><Polio><Poliomyelitis><Polyacrylamide Gel Electrophoresis><Polyacrylamide Gel Fractionation><Polymerase><Price><Production><Proteins><Pyrophosphates><RNA><RNA Expression><RNA Gene Products><RNA Polymerase A><RNA Polymerase I><RNA Replicase><RNA metabolism><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><Recombinants><Recovery><Reporting><Research><Research Resources><Resolution><Resources><Rhinovirus><Ribonucleic Acid><SARS corona virus 2><SARS-CoV-2><SARS-CoV-2 assay><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 outbreak><SARS-CoV-2 pandemic><SARS-CoV-2 therapeutics><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-CoV2 epidemic><SARS-CoV2 outbreak><SARS-CoV2 pandemic><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-coronavirus-2 therapeutics><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 outbreak><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome coronavirus 2 therapeutics><Severe acute respiratory syndrome related corona virus 2><Structure><System><Testing><Therapeutic><Therapeutic Agents><Time><Transcription><Type A Influenza><Vaccines><Veklury><Viral Diseases><Virus><Virus Diseases><WNV><West Nile><West Nile virus><Wuhan coronavirus><Yellow Fever><anti-viral agents><anti-viral drugs><anti-virals><base><catalyst><combat><corona virus><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019 assay><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 outbreak><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 therapeutics><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><cost><developmental><drug discovery><ebolavirus><emergency use authorization><expectation><flexibility><flexible><flu virus pandemic><fluid><hCoV19><high throughput screening><in vivo><influenza virus pandemic><influenzavirus><inhibitor><inhibitor/antagonist><interest><intravenous injection><liquid><nCoV2><novel><nucleobase analog><nucleoside analog><oligos><pandemic><pandemic disease><pandemic flu><pandemic influenza><pandemic strain of influenza><polymerization><pricing><reconstitute><reconstitution><remdesivir><scaffold><scaffolding><screening><severe acute respiratory syndrome coronavirus 2 assay><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><side effect><therapeutics against COVID-19><therapeutics against COVID19><therapeutics against SARS-CoV-2><therapeutics against SARS-coronavirus-2><therapeutics against Severe acute respiratory syndrome coronavirus 2><therapeutics against coronavirus disease 2019><therapeutics for novel coronavirus><tool><treat COVID-19><treat COVID19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><triphosphate><tripolyphosphate><viral infection><virus infection><virus-induced disease>