High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1–4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

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A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals

10.1101/2020.04.16.044016 ◽

2020 ◽

Cited By ~ 17

Author(s):

Laura Riva ◽

Shuofeng Yuan ◽

Xin Yin ◽

Laura Martin-Sancho ◽

Naoko Matsunaga ◽

...

Keyword(s):

Large Scale ◽

Kinase Inhibitor ◽

Drug Repositioning ◽

Clinical Stage ◽

Severe Pneumonia ◽

Cysteine Protease Inhibitors ◽

Global Pandemic ◽

Drug Registration ◽

Ccr1 Antagonist ◽

Therapeutic Doses

AbstractThe emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

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A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

PLoS ONE ◽

10.1371/journal.pone.0129234 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0129234 ◽

Cited By ~ 11

Author(s):

Lauren Forbes ◽

Katherine Ebsworth-Mojica ◽

Louis DiDone ◽

Shao-Gang Li ◽

Joel S. Freundlich ◽

...

Keyword(s):

Small Molecules ◽

High Throughput ◽

Adenylate Kinase ◽

High Throughput Screening ◽

Cell Lysis ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Adenylate Kinase Release

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In pursuit of the ideal antifungal agent for Candida infections: high-throughput screening of small molecules

Drug Discovery Today ◽

10.1016/j.drudis.2014.06.009 ◽

2014 ◽

Vol 19 (11) ◽

pp. 1721-1730 ◽

Cited By ~ 30

Author(s):

Sarah S.W. Wong ◽

Lakshman P. Samaranayake ◽

Chaminda J. Seneviratne

Keyword(s):

Small Molecules ◽

High Throughput ◽

Antifungal Agent ◽

High Throughput Screening ◽

Candida Infections ◽

The Ideal

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Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00601-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4825-4834 ◽

Cited By ~ 20

Author(s):

Kao-Lu Pan ◽

Jin-Ching Lee ◽

Hsing-Wen Sung ◽

Teng-Yuang Chang ◽

John T.-A. Hsu

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

High Throughput ◽

High Throughput Screening ◽

Reporter System ◽

Viral Protease ◽

Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

Journal of Visualized Experiments ◽

10.3791/55199 ◽

2017 ◽

Cited By ~ 3

Author(s):

Ahmed Fouda ◽

Mahasti Tahsini ◽

Fatemeh Khodayarian ◽

Fatimah Al-nafisah ◽

Moutih Rafei

Keyword(s):

Small Molecules ◽

High Throughput ◽

High Throughput Screening

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Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105278013 ◽

2005 ◽

Vol 10 (7) ◽

pp. 725-729 ◽

Cited By ~ 4

Author(s):

Upasana Singh ◽

Vinita Panchanadikar ◽

Dhiman Sarkar

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Glutamine Synthetase ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Coli Strain ◽

Compound Library ◽

Essential Enzyme ◽

Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

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A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation

Marine Drugs ◽

10.3390/md18040192 ◽

2020 ◽

Vol 18 (4) ◽

pp. 192

Author(s):

Pietro Marchese ◽

Nipun Mahajan ◽

Enda O’Connell ◽

Howard Fearnhead ◽

Maria Tuohy ◽

...

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Chondrogenic Differentiation ◽

Stem Cell Differentiation ◽

Ectopic Calcification ◽

Tissue Calcification ◽

Screening Platform

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

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Application of high-throughput screening to identify a novel αIIb-specific small- molecule inhibitor of αIIbβ3-mediated platelet interaction with fibrinogen

Blood ◽

10.1182/blood-2007-08-105544 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1248-1256 ◽

Cited By ~ 50

Author(s):

Robert Blue ◽

Marta Murcia ◽

Charles Karan ◽

Markéta Jiroušková ◽

Barry S. Coller

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Conformational Changes ◽

High Throughput Screening ◽

Metal Ion ◽

Selective Inhibition ◽

Docking Simulations ◽

Fibrinogen Binding ◽

Molecule Inhibitor ◽

Novel Structures

AbstractSmall-molecule αIIbβ3 antagonists competitively block ligand binding by spanning between the D224 in αIIb and the MIDAS metal ion in β3. They variably induce conformational changes in the receptor, which may have undesirable consequences. To identify αIIbβ3 antagonists with novel structures, we tested 33 264 small molecules for their ability to inhibit the adhesion of washed platelets to immobilized fibrinogen at 16 μM. A total of 102 compounds demonstrated 50% or more inhibition, and one of these (compound 1, 265 g/mol) inhibited ADP-induced platelet aggregation (IC50: 13± 5 μM), the binding of soluble fibrinogen to platelets induced by mAb AP5, and the binding of soluble fibrinogen and a cyclic RGD peptide to purified αIIbβ3. Compound 1 did not affect the function of GPIb, α2β1, or the other β3 family receptor αVβ3. Molecular docking simulations suggest that compound 1 interacts with αIIb but not β3. Compound 1 induced partial exposure of an αIIb ligand-induced binding site (LIBS), but did not induce exposure of 2 β3 LIBS. Transient exposure of purified αIIbβ3 to eptifibatide, but not compound 1, enhanced fibrinogen binding (“priming”). Compound 1 provides a prototype for small molecule selective inhibition of αIIbβ3, without receptor priming, via targeting αIIb.

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An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110375848 ◽

2010 ◽

Vol 15 (8) ◽

pp. 907-917 ◽

Cited By ~ 11

Author(s):

Franck Madoux ◽

Scott Simanski ◽

Peter Chase ◽

Jitendra K. Mishra ◽

William R. Roush ◽

...

Keyword(s):

Cell Cycle ◽

Fusion Protein ◽

Small Molecules ◽

High Throughput ◽

High Throughput Screening ◽

Cell Cycle Progression ◽

Ubiquitin Proteasome Pathway ◽

Subsequent Degradation ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-luciferase fusion protein. To ensure ultra-high-throughput screening (uHTS) compatibility, the assay was scaled to a 1536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogeneous assay demonstrated robust performance, with a calculated Z′ factor of 0.65 ± 0.05. The assay was screened against a publicly available library of ~218,000 compounds to identify Wee1 stabilizers. Nonselective, cytotoxic, and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of an N-cyclin B-luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of 4 unrelated cell-permeable small molecules that showed selective Wee1-luciferase stabilization with micromolar potency. One of these compounds, SID4243143 (ML 118), was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.

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