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 Diphenylpyrazole-Derived Compounds Increase Survival Time of Mice after Prion Infection

2011 ◽  
Vol 55 (10) ◽  
pp. 4774-4781 ◽  
Author(s):  
Fabienne Leidel ◽  
Martin Eiden ◽  
Markus Geissen ◽  
Hans A. Kretzschmar ◽  
Armin Giese ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Natural Infection ◽  
New Drugs ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Variant Form ◽  
Screening Assay ◽  
High Throughput Screening Assay

ABSTRACTTransmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative disorders that can be transmitted by natural infection or inoculation. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. The emergence of a variant form of CJD (vCJD), which has been associated with BSE, produced strong pressure to search for effective treatments with new drugs. Up to now, however, TSEs have proved incurable, although many efforts have been made bothin vitroandin vivoto search for potent therapeutic and prophylactic compounds. For this purpose, we analyzed a compound library consisting of 10,000 compounds with a cell-based high-throughput screening assay dealing with scrapie-infected scrapie mouse brain and ScN2A cells and identified a new class of inhibitors consisting of 3,5-diphenylpyrazole (DPP) derivatives. The most effective DPP derivative showed half-maximal inhibition of PrPScformation at concentrations (IC50) of 0.6 and 1.2 μM, respectively. This compound was subsequently subjected to a number of animal experiments using scrapie-infected wild-type C57BL/6 and transgenic Tga20 mice. The DPP derivative induced a significant increase of incubation time both in therapeutic and prophylactic experiments. The onset of the prion disease was delayed by 37 days after intraperitoneal and 42 days after oral application, respectively. In summary, we demonstrate a highin vitroefficiency of DPP derivatives against prion infections that was substantiatedin vivofor one of these compounds. These results indicate that the novel class of DPP compounds should comprise excellent candidates for future therapeutic studies.

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.

Closing the empty anti-Babesia gibsoni drug pipeline in vitro using fluorescence-based high throughput screening assay

2020 ◽  
Vol 75 ◽  
pp. 102054 ◽  
Author(s):  
Mohamed Abdo Rizk ◽  
Shengwei Ji ◽  
Mingming Liu ◽  
Shimaa Abd El-Salam El-Sayed ◽  
Yongchang Li ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Babesia Gibsoni ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Drug Pipeline

scholarly journals Extrapolating In Vitro Screening Assay Data for Thyroperoxidase Inhibition to Predict Serum Thyroid Hormones in the Rat

2019 ◽  
Vol 173 (2) ◽  
pp. 280-292 ◽  
Author(s):  
Iman Hassan ◽  
Hisham El-Masri ◽  
Jermaine Ford ◽  
Amanda Brennan ◽  
Sakshi Handa ◽  
...  
Keyword(s):  
Thyroid Gland ◽  
Time Course ◽  
Ex Vivo ◽  
Response Analysis ◽  
In Vitro Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Serum T4

Abstract Thyroperoxidase (TPO) is an enzyme essential for thyroid hormone (TH) synthesis and a target site for a number of xenobiotics that disrupt TH homeostasis. An in vitro high-throughput screening assay for TPO inhibition, the Amplex UltraRed-TPO (AUR-TPO), has been used to screen the ToxCast chemical libraries for this action. Output from this assay would be most useful if it could be readily translated into an in vivo response, namely a reduction of TH in serum. To this end, the relationship between TPO inhibition in vitro and serum TH decreases was examined in rats exposed to 2 classic TPO inhibitors, propylthiouracil (PTU) and methimazole (MMI). Serum and gland PTU, MMI, and TH levels were quantified using tandem liquid chromatography mass spectrometry. Thyroperoxidase activity was determined in thyroid gland microsomes treated with PTU or MMI in vitro and ex vivo from thyroid gland microsomes prepared from exposed animals. A quantitative model was constructed by contrasting in vitro and ex vivo AUR-TPO results and the in vivo time-course and dose-response analysis. In vitro:ex vivo correlations of AUR-TPO outputs indicated that less than 30% inhibition of TPO in vitro was sufficient to reduce serum T4 by 20%, a degree of regulatory significance. Although further testing of model estimates using other TPO inhibitors is essential for verification of these initial findings, the results of this study provide a means to translate in vitro screening assay results into predictions of in vivo serum T4 changes to inform risk assessment.

Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

2008 ◽  
Vol 13 (6) ◽  
pp. 494-503 ◽  
Author(s):  
Sang-Hyeup Lee ◽  
Jeong Hee Moon ◽  
Eun Ah Cho ◽  
Seong-Eon Ryu ◽  
Myung Kyu Lee
Keyword(s):  
Monoclonal Antibody ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Hypoxia Inducible Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitive Assay ◽  
Screening Assay ◽  
Hypoxic Conditions

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. ( Journal of Biomolecular Screening 2008:494-503)

scholarly journals Identification of Small Molecules That Inhibit the Interaction of TEM8 with Anthrax Protective Antigen Using a FRET Assay

2013 ◽  
Vol 18 (6) ◽  
pp. 714-725 ◽  
Author(s):  
Lorna M. Cryan ◽  
Kaiane A. Habeshian ◽  
Thomas P. Caldwell ◽  
Meredith T. Morris ◽  
P. Christine Ackroyd ◽  
...  
Keyword(s):  
Blood Vessels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protective Antigen ◽  
Resonance Energy ◽  
Cysteine Residue ◽  
Tumor Formation ◽  
Screening Assay ◽  
High Throughput Screening Assay

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z’ value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

scholarly journals Establishment of an In Vitro High-Throughput Screening Assay for Detecting Phospholipidosis-Inducing Potential

2005 ◽  
Vol 90 (1) ◽  
pp. 133-141 ◽  
Author(s):  
Toshihiko Kasahara ◽  
Kazuo Tomita ◽  
Hiroyuki Murano ◽  
Tsuyoshi Harada ◽  
Keisuke Tsubakimoto ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals Practical High-Throughput Method to Screen Compounds for Anthelmintic Activity against Caenorhabditis elegans

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4156
Author(s):  
Aya C. Taki ◽  
Joseph J. Byrne ◽  
Peter R. Boag ◽  
Abdul Jabbar ◽  
Robin B. Gasser
Keyword(s):  
Caenorhabditis Elegans ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Anthelmintic Activity ◽  
Infrared Light ◽  
Parasitic Nematodes ◽  
Screening Assay ◽  
High Throughput Screening Assay

In the present study, we established a practical and cost-effective high throughput screening assay, which relies on the measurement of the motility of Caenorhabditis elegans by infrared light-interference. Using this assay, we screened 14,400 small molecules from the “HitFinder” library (Maybridge), achieving a hit rate of 0.3%. We identified small molecules that reproducibly inhibited the motility of C. elegans (young adults) and assessed dose relationships for a subset of compounds. Future work will critically evaluate the potential of some of these hits as candidates for subsequent optimisation or repurposing as nematocides or nematostats. This high throughput screening assay has the advantage over many previous assays in that it is cost- and time-effective to carry out and achieves a markedly higher throughput (~10,000 compounds per week); therefore, it is suited to the screening of libraries of tens to hundreds of thousands of compounds for subsequent evaluation and development. The present phenotypic whole-worm assay should be readily adaptable to a range of socioeconomically important parasitic nematodes of humans and animals, depending on their dimensions and motility characteristics in vitro, for the discovery of new anthelmintic candidates. This focus is particularly important, given the widespread problems associated with drug resistance in many parasitic worms of livestock animals globally.

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