scholarly journals Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3213
Author(s):  
Alon Ben David ◽  
Eran Diamant ◽  
Eyal Dor ◽  
Ada Barnea ◽  
Niva Natan ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput Screening ◽  
Cell Attachment ◽  
Drug Repurposing ◽  
Heparin Sodium ◽  
Compound Libraries ◽  
Global Pandemic ◽  
Ic50 Values ◽  
Binding Inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

scholarly journals High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

2021 ◽  
Author(s):  
Ryan Choi ◽  
Mowei Zhou ◽  
Roger Shek ◽  
Jesse W. Wilson ◽  
Logan Tillery ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Nonstructural Protein ◽  
Unmet Need ◽  
Drug Repurposing ◽  
Great Promise ◽  
Compound Libraries ◽  
Global Pandemic ◽  
Essential Enzyme

AbstractSARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 μM range in vitro. Furthermore, Exebryl-1, a β-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 μM, in VERO, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.Author summaryDrugs to treat COVID-19 are urgently needed. To address this, we searched libraries of drugs and drug-like molecules for inhibitors of an essential enzyme of the virus that causes COVID-19, SARS-CoV-2 nonstructural protein (nsp)15. We found several molecules that inhibited the nsp15 enzyme function and one was shown to be active in inhibiting the SARS-CoV-2 virus. This demonstrates that searching for SARS-CoV-2 nsp15 inhibitors can lead inhibitors of SARS-CoV-2, and thus therapeutics for COVID-19. We are currently working to see if these inhibitors could be turned into a drug to treat COVID-19.

scholarly journals High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250019
Author(s):  
Ryan Choi ◽  
Mowei Zhou ◽  
Roger Shek ◽  
Jesse W. Wilson ◽  
Logan Tillery ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Unmet Need ◽  
Drug Repurposing ◽  
Great Promise ◽  
Compound Libraries ◽  
Global Pandemic ◽  
Dependent Endonuclease ◽  
High Throughput Assay

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1–20 μM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer’s therapy, was shown to have antiviral activity between 10 to 66 μM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

scholarly journals Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E

2021 ◽  
Vol 22 (16) ◽  
pp. 8577
Author(s):  
Alon Ben David ◽  
Ada Barnea ◽  
Eran Diamant ◽  
Eyal Dor ◽  
Arieh Schwartz ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput Screening ◽  
Lethal Dose ◽  
Screening Assay ◽  
Vitro Assay ◽  
Time To Death ◽  
High Throughput Screening Assay ◽  
Binding Inhibitors

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

scholarly journals A structure-based rationale for sialic acid independent host-cell entry of Sosuga virus

2019 ◽  
Vol 116 (43) ◽  
pp. 21514-21520 ◽  
Author(s):  
Alice J. Stelfox ◽  
Thomas A. Bowden
Keyword(s):  
Sialic Acid ◽  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Cell Entry ◽  
Head Region ◽  
Close Proximity ◽  
Host Cell Attachment ◽  
Host Cell Entry

The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae. The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed β-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.

scholarly journals Molecular insights into the inhibition of plasmepsins by HIV-1 protease inhibitors: Implications for antimalarial drug discovery

2021 ◽  
Author(s):  
Vandana Mishra ◽  
Ishan Rathore ◽  
Anuradha Deshmukh ◽  
Swati Patankar ◽  
Alla Gustchina ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Drug Targets ◽  
Drug Repurposing ◽  
Binding Mode ◽  
Ic50 Values ◽  
Multiple Regions ◽  
Potential Drug Targets ◽  
Hiv 1 ◽  
Micromolar Range

Malaria is a deadly disease, and the worldwide emergence of drug resistance in the Plasmodium parasites demands the development of novel and potent antimalarials. HIV-1 protease inhibitors (HIV-1 PIs) alleviate the Plasmodium pathogenesis during malaria/HIV-1 co-infection plausibly by inhibiting vacuolar plasmepsins (PMs), the hemoglobin degrading proteases from P. falciparum. All five FDA-approved HIV-1 PIs tested against PMII exhibit the Ki values in the low micromolar range of which RTV and LPV display the highest inhibition activity. Both inhibitors impede in vitro growth of P. falciparum at low micromolar IC50 values. We report the first crystal structures of PMII complexed with RTV and LPV that reveal the binding mode and interactions of the inhibitors in the active site as well as elucidate the mechanism underlying their differential potency. The conformational flexibility of the P4 group in RTV allows it to explore multiple regions of the S4 pocket. The present study establishes vacuolar PMs as potential drug targets of HIV-1 PIs. The molecular details explaining the inhibitory mechanism of HIV-1 PIs might be crucial in designing novel and potent antimalarial analogs. This work strengthens the prospect of drug repurposing as an alternative strategy towards antimalarial treatments and provides an opportunity to tackle malaria and HIV-1 co-infection.

scholarly journals Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors

2012 ◽  
Vol 18 (4) ◽  
pp. 388-399 ◽  
Author(s):  
Syed Ahmad ◽  
Mark A. Hughes ◽  
Gary L. Johnson ◽  
John E. Scott
Keyword(s):  
Atpase Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Selective Inhibition ◽  
Mass Spectrometry Analysis ◽  
Activity Assay ◽  
Spectrometry Analysis ◽  
Compound Libraries ◽  
Atpase Assay

The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small-molecule inhibitors of MEKK2 activity.

scholarly journals Screening of the NIH Clinical Collection for inhibitors of HIV-1 integrase activity

2018 ◽  
Vol 114 (3/4) ◽  
Author(s):  
Shaakira Abrahams ◽  
Salerwe Mosebi ◽  
Muhammed Q. Fish ◽  
Maria A. Papathanasopoulos ◽  
Raymond Hewer
Keyword(s):  
Unmet Need ◽  
Epigallocatechin Gallate ◽  
Drug Repurposing ◽  
Strand Transfer ◽  
Vast Number ◽  
Drug Candidates ◽  
Transfer Activity ◽  
Ic50 Values ◽  
Hiv 1

Drug repurposing offers a validated approach to reduce drug attrition within the drug discovery and development pipeline through the application of known drugs and drug candidates to treat new indications. Full exploitation of this strategy necessitates the screening of a vast number of molecules against an extensive number of diseases of high burden or unmet need and the subsequent dissemination of the findings. In order to contribute to endeavours within this field, we screened the 727 compounds comprising the US National Institutes of Health (NIH) Clinical Collection through an HIV-1 (human immunodeficiency virus type 1) integrase stand transfer inhibition assay on an automated scintillation proximity assay platform. Only two compounds were identified within the initial screen, with cefixime trihydrate and epigallocatechin gallate found to reduce integrase strand transfer activity at IC50 values of 6.03±1.29 μM and 9.57±1.62 μM, respectively. However, both cefixime trihydrate and epigallocatechin gallate retained their low micromolar inhibitory activity when tested against a raltegravir-resistant integrase double mutant (FCIC50 values of 0.83 and 0.06, respectively), were ineffective in an orthogonal strand transfer ELISA (less than 30% inhibition at 100 μM) and produced negligible selectivity index values (less than 1) in vitro. While no useful inhibitors of HIV-1 integrase strand transfer activity were found within the NIH Clinical Collection, the identification of two assay-disrupting molecules demonstrates the importance of consideration of non-specific inhibitors in drug repurposing screens.

scholarly journals Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

2007 ◽  
Vol 408 (2) ◽  
pp. 173-180 ◽  
Author(s):  
Paul W. Bowyer ◽  
Ruwani S. Gunaratne ◽  
Munira Grainger ◽  
Chrislaine Withers-Martinez ◽  
Sasala R. Wickramsinghe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Scale ◽  
Expression And Purification ◽  
Purification System ◽  
Pure Protein ◽  
Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

scholarly journals Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo

2020 ◽  
Author(s):  
Stuart Weston ◽  
Christopher M. Coleman ◽  
Rob Haupt ◽  
James Logue ◽  
Krystal Matthews ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rapid Development ◽  
Drug Repurposing ◽  
Ic50 Values ◽  
Human Use ◽  
Approved Drugs ◽  
Fda Approved Drugs

AbstractSARS-CoV-2 emerged in China at the end of 2019 and has rapidly become a pandemic with roughly 2.7 million recorded COVID-19 cases and greater than 189,000 recorded deaths by April 23rd, 2020 (www.WHO.org). There are no FDA approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA approved drugs. Rapid development and human testing of potential antivirals is greatly needed. A quick way to test compounds with potential antiviral activity is through drug repurposing. Numerous drugs are already approved for human use and subsequently there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV. We found that 17 of these inhibit SARS-CoV-2 at a range of IC50 values at non-cytotoxic concentrations. We directly follow up with seven of these to demonstrate all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we have evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found both drugs protect mice from clinical disease.

scholarly journals Potential Therapeutic Agents for Feline Calicivirus Infection

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 433 ◽  
Author(s):  
Tulio M. Fumian ◽  
Daniel Enosi Tuipulotu ◽  
Natalie E. Netzler ◽  
Jennifer H. Lun ◽  
Alice G. Russo ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Throughput Screening ◽  
Polymerase Activity ◽  
In Vitro Assays ◽  
Feline Calicivirus ◽  
Upper Respiratory Tract ◽  
Direct Acting Antivirals ◽  
Ic50 Values ◽  
Direct Acting

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 μM and 2.7 μM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 μM and 2.5 μM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.

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