Retroviral Integrase: A Novel Target in Antiviral Drug Development and Basic In Vitro Assays with the Purified Enzyme

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21–36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.

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Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development

Virology ◽

10.1016/j.virol.2017.04.001 ◽

2017 ◽

Vol 507 ◽

pp. 32-39 ◽

Cited By ~ 9

Author(s):

Nopporn Chutiwitoonchai ◽

Takafumi Mano ◽

Michinori Kakisaka ◽

Hirotaka Sato ◽

Yasumitsu Kondoh ◽

...

Keyword(s):

Drug Development ◽

Nuclear Export ◽

Influenza A ◽

Antiviral Drug ◽

Nuclear Export Protein ◽

Novel Target ◽

Export Protein

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1341. Development of a Series of High-Throughput Screens to Identify Leads for Nontuberculous Mycobacteria Drug Design

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1205 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S485-S485

Author(s):

Sarah McGuffin ◽

Steven Mullen ◽

Julie Early ◽

Tanya Parish

Keyword(s):

Drug Discovery ◽

Drug Development ◽

High Throughput ◽

Nontuberculous Mycobacteria ◽

Human Infection ◽

Attrition Rate ◽

In Vitro Assays ◽

Vitro Assay

Abstract Background Nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM. Methods We are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria. Results We are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well. Conclusion To enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology. Disclosures All authors: No reported disclosures.

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PTEN expression in endothelial cells is down-regulated by uPAR to promote angiogenesis

Thrombosis and Haemostasis ◽

10.1160/th15-01-0016 ◽

2015 ◽

Vol 114 (08) ◽

pp. 379-389 ◽

Cited By ~ 10

Author(s):

Matthias Unseld ◽

Anastasia Chilla ◽

Clemens Pausz ◽

Rula Mawas ◽

Johannes Breuss ◽

...

Keyword(s):

Endothelial Cells ◽

In Vitro Assays ◽

Soluble Form ◽

Dependent Manner ◽

Drug Interference ◽

Protein Levels ◽

Novel Target ◽

The Impact

SummaryThe tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR- dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.

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A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV-2 main protease

10.1101/2020.07.10.197889 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jeremy D. Baker ◽

Rikki L. Uhrich ◽

Gerald C. Kraemer ◽

Jason E. Love ◽

Brian C. Kraemer

Keyword(s):

Hepatitis C ◽

Drug Development ◽

Large Scale ◽

Previous History ◽

Antiviral Drug ◽

Case Fatality ◽

Drug Repurposing ◽

Main Protease ◽

Approved Drugs

AbstractThe SARS coronavirus type 2 (SARS-CoV-2) emerged in late 2019 as a zoonotic virus highly transmissible between humans that has caused the COVID-19 pandemic 1,2. This pandemic has the potential to disrupt healthcare globally and has already caused high levels of mortality, especially amongst the elderly. The overall case fatality rate for COVID-19 is estimated to be ∼2.3% overall 3 and 32.3% in hospitalized patients age 70-79 years 4. Therapeutic options for treating the underlying viremia in COVID-19 are presently limited by a lack of effective SARS-CoV-2 antiviral drugs, although steroidal anti-inflammatory treatment can be helpful. A variety of potential antiviral targets for SARS-CoV-2 have been considered including the spike protein and replicase. Based upon previous successful antiviral drug development for HIV-1 and hepatitis C, the SARS-CoV-2 main protease (Mpro) appears an attractive target for drug development. Here we show the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ∼6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro. In our primary screen we found ∼50 compounds with activity against Mpro (overall hit rate <0.75%). Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 ≤ 50 μM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including Boceprevir (IC50=0.95 μM), Ciluprevir (20.77μM). Narlaprevir (IC50=1.10μM), and Telaprevir (15.25μM). These results demonstrate that some existing approved drugs can inhibit SARS-CoV-2 Mpro and that screen saturation of all approved drugs is both feasible and warranted. Taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for rapid repurposing.

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Antibacterial Profile of a Microbicidal Agent Targeting Tyrosine Phosphatases and Redox Thiols, Novel Drug Targets

Antibiotics ◽

10.3390/antibiotics10111310 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1310

Author(s):

Kylie White ◽

Gina Nicoletti ◽

Hugh Cornell

Keyword(s):

Drug Development ◽

Plasma Proteins ◽

Tyrosine Phosphatase ◽

Bacterial Species ◽

In Vitro Assays ◽

Antioxidant Systems ◽

Thiol Oxidation ◽

Tyrosine Phosphatases ◽

Activity Profile

The activity profile of a protein tyrosine phosphatase (PTP) inhibitor and redox thiol oxidant, nitropropenyl benzodioxole (NPBD), was investigated across a broad range of bacterial species. In vitro assays assessed inhibitory and lethal activity patterns, the induction of drug variants on long term exposure, the inhibitory interactions of NPBD with antibiotics, and the effect of plasma proteins and redox thiols on activity. A literature review indicates the complexity of PTP and redox signaling and suggests likely metabolic targets. NPBD was broadly bactericidal to pathogens of the skin, respiratory, urogenital and intestinal tracts. It was effective against antibiotic resistant strains and slowly replicating and dormant cells. NPBD did not induce resistant or drug-tolerant phenotypes and showed low cross reactivity with antibiotics in synergy assays. Binding to plasma proteins indicated lowered in-vitro bioavailability and reduction of bactericidal activity in the presence of thiols confirmed the contribution of thiol oxidation and oxidative stress to lethality. This report presents a broad evaluation of the antibacterial effect of PTP inhibition and redox thiol oxidation, illustrates the functional diversity of bacterial PTPs and redox thiols, and supports their consideration as novel targets for antimicrobial drug development. NPBD is a dual mechanism agent with an activity profile which supports consideration of tyrosine phosphatases and bacterial antioxidant systems as promising targets for drug development.

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A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111412183 ◽

2011 ◽

Vol 16 (8) ◽

pp. 852-861 ◽

Cited By ~ 16

Author(s):

Brian J. Geiss ◽

Hillary J. Stahla-Beek ◽

Amanda M. Hannah ◽

Hamid H. Gari ◽

Brittney R. Henderson ◽

...

Keyword(s):

Drug Development ◽

High Throughput ◽

High Throughput Screening ◽

Antiviral Drug ◽

Functional Assay ◽

Screening Assay ◽

Gtp Binding ◽

High Throughput Screening Assay ◽

Capping Enzyme

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5′ end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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