Document text
Principal Investigator: Nadya Tarasova
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2022
Award: $22,698
Funding agency: National Cancer Institute
The reliability, and consequently success of virtual screening depends heavily on the accuracy of the x-ray structure depiction of the target protein structure in solution. Crystal packing can distort conformation of flexible parts of the protein thus leading to misrepresentation of the protein structure. Thus, the first step in all screening procedures is optimization and evaluation of the structure used in the screen. Structural biology of COVID-19 proteins has been developing with an explosive speed since the start of 2020 with dozens of structures being deposited to Protein Data Bank every week. We have evaluated suitability of structures of SARS-CoV-2 NSP7, NSP8, NSP9, NSP10, NSP14, NSP15, NSP16, nucleocapsid RNA-binding domain and spike protein receptor-binding domain for virtual screening. All structures have undergone energy minimization procedures before the screens. Initially, they have been screen-tested using a diversity library of 1.92 million compounds that were available for 2-week delivery from Enamine LTD and a library of all drugs marketed in US. The screens were run on NIH High Performing Computing system Biowulf using ICM-Pro docking software from MolSoft and optimized docking protocols developed by us that allow for highly reliable predictions of the binders. Typically, we have been running 2000 parallel docking jobs that allowed screening about 10 million compounds a day for a single binding site in a fast screening mode. In total, we have evaluated 30 x-ray SARS-CoV-2 protein structures in test screens. 4-5 hits from the test libraries have been purchased for each target structure and tested for binding using recombinant COVID-19 proteins and nano differential scanning fluorometry (DSF). Structures of NSP7, NSP8 and NSP14 did not produce hits with binding scores below the threshold. For all other proteins (NSP9, NSP10, NSP15, NSP16, nucleocapsid RNA-binding domain and spike protein receptor-binding domain) we were able to select x-ray structures allowing for accurate prediction of binding, 10 structures in total. Significant efforts have been spent on generation of highly diverse virtual libraries of compounds for the screens. Novel libraries, Synthetically Accessible Virtual Inventory (SAVI) contain compounds that can be synthesized in one easy step from readily available blocks. Current database that was made public in April 2020 (https://cactus.nci.nih.gov/download/savi_download/) was built using 53 robust synthetic procedures or transforms and contains 1,748,464,003 products. Screening databases of that size currently is impractical. That is why we have generated SAVI diversity sets of 2,955,416 and 15 million compounds that were used for identification of initial leads. The leads identified from screening SAVI and Enamine diversity sets have been used for preparation of libraries of structurally similar compounds typically containing hundreds of thousands or 2-3 million compounds from entire SAVI library. We have used machine learning tools (MNA, Multilevel Neighborhoods of Atoms and QNA, Quantitative Neighborhoods of Atoms) generated by our collaborator Prof. Vladimir Poroikov and his colleagues from the Institute of Biomedical Chemistry, Moscow, Russia for generation of libraries of compounds with electronic structures similar to the leads. These libraries were subjected to screening by docking allowing for identification of compounds with improved docking scores. The later have been synthesized, and binding to the target proteins evaluated by NanoDifferential Scanning Fluorometry, fluorescence spectroscopy and Microscale Thermophoresis. Overall, over 200 compounds have been tested for binding. Currently, we have identified compounds that bind to NSP9, NSP10, NSP16, nucleocapsid RBD and spike protein RBD with micromolar affinity. Lead compounds bind to exonuclease NSP15 with sub micromolar affinity. Major focus currently is on the inhibitors of the exonuclease not only because it is essential for viral replication but also because the active site that we are targeting is conserved in all corona viruses. Consequently, newly developed inhibitors are likely to help fighting not only current, but also future pandemics. The leads for other proteins will be developed at later time as resources will permit.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Active Sites><Affinity><Binding><Binding Sites><COVID-19><COVID-19 therapeutics><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID19><COVID19 therapeutics><COVID19 therapy><COVID19 treatment><COVID19 virus><CV-19><CV19><Cactaceae><Cactus><Cell Body><Cells><Chemical Structure><Chemicals><Chemistry><CoV-2><CoV2><Combining Site><Computer Systems><Computer software><Coronaviridae><Coronavirus><Crystallization><Data><Data Bases><Databases><Deposit><Deposition><Development><Diversity Library><Docking><Drug Screening><Drug Therapy><E coli><E. coli><Eligibility><Eligibility Determination><Equipment and supply inventories><Escherichia coli><Evaluation><Exonuclease><Fluorescence Spectroscopy><Fluorometry><Future><Generations><Goals><High Throughput Assay><Institutes><Inventory><Jobs><Lead><Libraries><Machine Learning><Methods><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Moscow><NIH><National Institutes of Health><Neighborhoods><Nucleocapsid><Occupations><Pb element><Pharmacotherapy><Preparation><Procedures><Professional Positions><Property><Proteins><Protocol><Protocol Screening><Protocols documentation><Putative RNA-Binding Region><RNA Binding Domain><RNA Recognition Motif><RNP Domain><RNP Motif><RNP-1 Signature><Reactive Site><Receptor Protein><Recombinants><Research Resources><Resources><Roentgen Rays><Running><Russia><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 therapeutics><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 therapeutics><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Scanning><Screening procedure><Severe Acute Respiratory Coronavirus 2><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-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 therapeutics><Severe acute respiratory syndrome related corona virus 2><Software><Speed><Structure><Testing><Time><United States National Institutes of Health><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virus><Virus Diseases><Virus Replication><Wuhan coronavirus><X-Radiation><X-Ray Radiation><X-ray><Xray><base><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><computing system><conformation><conformational state><corona virus><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 therapeutics><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease-19><coronavirus disease-19 virus><coronavirus infectious disease-19><data base><developmental><drug market><drug treatment><electronic structure><fighting><flexibility><flexible><hCoV19><heavy metal Pb><heavy metal lead><high throughput screening><improved><in silico><inhibitor><machine learned><nCoV2><nano><novel><pandemic><pandemic disease><physical state><pre-clinical study><preclinical study><protein data bank><protein databank><protein structure><protein structures><proteins structure><receptor><receptor binding><receptor bound><screening><screening tools><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><small molecule><structural biology><success><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><viral infection><viral multiplication><viral replication><virtual><virtual library><virtual screening><virtual screenings><virus infection><virus multiplication><virus protein><virus-induced disease>