High-throughput Screening Assay Research Articles

Development of a Fluorescence Probe for High-Throughput Screening of Allosteric Inhibitors Targeting TRAP1.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone implicated in pro-tumorigenic pathways by regulating the folding of substrate proteins (clients) within cancer cells. Recent research has pinpointed a potentially druggable allosteric site within the client binding site (CBS) of TRAP1, suggesting this site might offer a more effective strategy for developing potent and selective TRAP1 inhibitors. However, the absence of reliable assay systems has hindered quantitative evaluation of inhibitors. In this study, we have developed a fluorescent probe, Rho6TPP, designed to target the CBS. Utilizing fluorescence polarization-based high-throughput screening assays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), Z factor (>0.6), and Z' factor (>0.6). Additionally, it facilitates comparative analysis of existing small molecules and discovery of novel binders. MitoTam, a mitochondria-targeted tamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our findings highlight the potential of Rho6TPP as a crucial tool for advancing the development of CBS-targeting TRAP1 inhibitors.

Abstract 4135476: The Cardiomyocyte Hypertrophy Inhibitor RFN-409, Identified by High Throughput Screening Assay, Suppresses Pressure Overload-induced Systolic Dysfunction in Mice by Suppressing p38 Activity

Purpose: When the heart is exposed to stresses such as myocardial infarction or hypertension, it undergoes compensatory hypertrophy in response. However, continuation of the stress causes this compensatory mechanism to fail, and eventually systolic dysfunction or decompensated heart failure occur. As the hypertrophy of individual cardiomyocytes has been observed in this process, controlling cardiomyocyte hypertrophy is a potential target the prevention and treatment of heart failure. In this study, we constructed a high throughput screening (HTS) assay using cardiomyocyte hypertrophy as an index parameter. Compounds that inhibit cardiomyocyte hypertrophy were selected from our low molecular compound library. Methods and Results: In the primary screening, cultured rat primary cardiomyocytes were treated with each compound at a final concentration of 1 µM and then stimulated with 30 µM phenylephrine (PE) for 48 hours. These cells were subjected to fluorescent immunostaining with α-actinin, and cardiomyocyte area was measured using an ArrayScan™ system. The hypertrophy inhibition rate (%) of each compound was calculated as [(PE(+) - compound) / (PE(+) - PE(-))] × 100. The compounds with a hypertrophy inhibition rate greater than 50% and less than 150% were selected as hit compounds. In the secondary screening, these hit compounds were evaluated based on the dose-dependency of cardiomyocyte hypertrophy inhibition and the inhibition of the mRNA levels of the cardiac hypertrophy response genes ANF and BNP using real-time PCR. From the 269 low molecular-weight compounds in the original compound library, eight were selected through the primary and secondary screenings. Among them, we focused on Reference Number 409 (RFN-409). Western blotting indicated that RFN-409 inhibited PE-induced p38 activation. Next, we investigated the effect of RFN-409 on heart failure. Eight-week-old male C57 BL/6J mice were subjected to transverse aortic constriction (TAC) surgery and then randomly assigned to intraperitoneal treatment with RFN-409 (3, 10 mg/kg) or vehicle for eight weeks. RFN-409 at 10 mg/kg significantly prevented TAC-induced increase in left ventricular posterior wall thickness and decrease in left ventricular fractional shortening. Discussion: RFN-409 suppressed TAC-induced development of heart failure, at least partially by inhibiting p38 activity. These findings suggest that RFN-409 may be an effective agent for heart failure therapy.

The Effect of Metformin and Hydrochlorothiazide on Cytochrome P450 3A4 Metabolism of Ivermectin: Insights from In Silico Experimentation.

The spread of SARS-CoV-2 has led to an interest in using ivermectin (a potent antiparasitic agent) as an antiviral agent despite the lack of convincing in vivo clinical data for its use against COVID-19. The off-target prophylactic use of ivermectin adds a substantial risk of drug-drug interactions with pharmaceutical medications used to treat chronic conditions like diabetes and hypertension (metformin and hydrochlorothiazide, respectively). Therefore, this study aims to evaluate the potential drug-drug interactions between ivermectin with either metformin or hydrochlorothiazide. In silico experiments and high-throughput screening assays for CYP3A4 were conducted to understand how metformin and hydrochlorothiazide might affect CYP3A4's role in metabolizing ivermectin. The study findings indicated that hydrochlorothiazide is more stable than both ivermectin and metformin. This conclusion was further supported by root mean square fluctuation analysis, which showed that hydrochlorothiazide is more flexible. The variation in the principal component analysis scatter plot across the first three normal modes suggests hydrochlorothiazide has a more mobile conformation than ivermectin and metformin. Additionally, a strong inhibition of CYP3A4 by hydrochlorothiazide was observed, suggesting that hydrochlorothiazide's regulatory effects could significantly impede CYP3A4 activity, potentially leading to a reduced metabolism and clearance of ivermectin in the body. Concurrent administration of these drugs may result in drug-drug interactions and hinder the hepatic metabolism of ivermectin.

Towards maximizing biomass and lipid productivity: high-throughput screening assay for prospecting heterotrophic growth for new microalgal isolates

BackgroundMicroalgae have emerged as sustainable alternatives to fossil fuels and high-value petrochemicals. Despite the commercial potential of microalgae, their low biomass productivity is a significant limiting factor for large-scale production. In the photoautotrophic cultivation of microalgae, achievable cell density levels depend on the light transmittance of the production system, which can significantly decrease the photosynthetic rate and biomass production. In contrast, the mixotrophic cultivation of microalgae using heterotrophic carbon sources enables high-density cultivation, which significantly enhances biomass productivity. The identification of optimal production conditions is crucial for improving biomass productivity; however, it is typically time- and resource-consuming. To overcome this problem, high-throughput screening (HTS) system presents a practical approach to maximize biomass and lipid production and enhance the industrial applicability of microalgae.ResultsIn this study, we proposed a two-step HTS assay that allows effective screening of heterotrophic conditions compatible with new microalgal isolates. To confirm the effectiveness of the HTS assay, three microalgal isolates with distinctive morphological and genetic traits were selected. Suitable cultivation conditions, including various heterotrophic carbon sources, substrate concentrations, and temperatures, were investigated using a two-step HTS assay. The optimized conditions were validated at the flask scale, which confirmed a significant enhancement in the biomass and lipid productivity of each isolate. Moreover, the two-step HTS assay notably enhanced economic and temporal efficiency compared to conventional flask-based optimization.ConclusionsThese results suggest that our two-step HTS assay is an efficient strategy for investigating and optimizing microalgal culture conditions to maximize biomass and lipid productivity. This approach has the potential to enhance the industrial applicability of microalgae and facilitate the seamless transition from laboratory to field applications.

Organoids Research for Colorectal Cancer: Promising Approach for Precision Medicine, their Applications and Future Perspectives

Background: Organoids are three-dimensional (3D) constructs designed to emulate the complexity and functionality of organs in the body. Organoids have recently been used as powerful instruments for modeling and investigating several diseases, including colorectal cancer. Colorectal cancer is caused by altering colonic epithelial cells, which produce adenomas and carcinomas. Objective: The objective of present study was to investigate impact of organoids on colorectal cancer and their therapeutic outcome in cancer research. Organoids can be grown from stem cells in vitro, which closely resemble the structure and function of the organ they are derived from. They have been used in a variety of research applications, including disease modeling, drug screening, and personalized medicine. Organoids have allowed researchers to understand better the mechanisms underlying colorectal cancer initiation, progression, and resistance to therapy. Methods: The literature review was surveyed, and keywords related to cancer management, organoids, modelling, personized medicine, 3D structures were screened for colorectal cancer management were screened in SCI-hub, SCOPUS, WOS, and ABC Journals. Results: The findings of studies suggested that organoids derived from patient tumors can recapitulate the histopathology and genetic alterations of the original tumor, making them a valuable tool for personalized medicine. Conclusion: Organoids have been used to develop high-throughput drug screening assays and investigate the tumor microenvironment's contribution to colorectal cancer progression. In this review, we summarize recent advances in the use of organoids to study colorectal cancer and discuss their potential applications in the clinic.

Enzyme Benchmarking with Polyethylene Furanoate Soluble Scaffolds for Directed Evolution of PEFases.

Plastic waste is a major threat in our industrialized world and is driving research into bioplastics. The success of biobased polyethylene furanoate (PEF) as a viable alternative to polyethylene terephthalate (PET) of fossil origin will depend on designing effective enzymes to break it down, aiding its recycling. Here, a panel of fungal and bacterial cutinases were functionally expressed in a tandem yeast expression system based on Saccharomyces cerevisiae and Pichia pastoris. The activity of the enzyme panel was tested with soluble PEF model scaffolds, observing a correlation with the degradation of real PEF powder. A high-throughput colorimetric screening assay based on the PEF scaffold diethyl furan-2,5-dicarboxylate was developed, establishing the basis for future directed evolution campaigns of PEFases.

Function-driven high-throughput screening and isolation of ester-producing strains for glutinous rice wine fermentation

Ester-producing strains are of great importance for enhancing the quality and flavor profiles of alcoholic beverages. However, traditional methods for screening ester-producing strains are labor-intensive and time-consuming, significantly impeding the development of alcoholic beverages industry. In this study, we selected five brands of Jiuqu to cultivate within different media, results showed that XB (Jiuqu) incubated with malt extract medium possessed the highest ester-producing capability, with the identification of 27 esters at the concentration of 31.44 ± 2.17 mM. Subsequently, fluorescence-activated cell sorting (FACS) was used to screen and isolate the living fungal cells which accounted for 42.80 % of total cells, followed by cultivation utilizing a culturomics approach. High-throughput screening (HTS) assays using 4-Methylumbelliferyl acetate (4-MA) were utilized to evaluate the ester-production potentials of 960 selected strains. The top10 highest ester-producing strains were sequenced, and all were identified as Saccharomyces cerevisiae, Cyberlindnera fabianii, and Wickerhamomyces anomalus. Eventually, three microbial strains were co-incubated with rice wine starter to improve the nutritional and flavor properties of glutinous rice wine. Compared to the control group, Cyberlindnera fabianii could increase reducing sugar content, up to 0.33 ± 0.01 g/mL, and significantly decrease the concentration of bitter amino acids by 55.83 %, resulting in a final concentration of 32.29 ± 1.51 mg/L. Furthermore, the glutinous rice wine with Cyberlindnera fabianii showed 38 distinct ester compounds at the content of 16.22 ± 0.51 mM, which was superior to the control group (30 ester compounds at the concentration of 11.89 ± 1.39 mM). The diversity and concentrations of flavor components, such as alcohols, aldehydes, and ketones, were also enhanced. Our findings would contribute to advancing the rapid screening of ester-producing strains, as well as providing a theoretical basis for improving the quality of glutinous rice wine.

β-sheet Engineering of IsPETase for PET Depolymerization

The enzymatic depolymerization of polyethylene terephthalate (PET) offers a sustainable approach for the recycling of PET waste. Great efforts have been devoted to engineering PET depolymerases on the substrate binding cleft and the surrounding loops/α-helices on the surface. Here, we report the systematic engineering of whole β-sheet regions in the core of IsPETase (a PETase from Ideonella sakaiensis) via a fluorescent high-throughput screening assay. Twenty-one beneficial substitutions were obtained and iteratively recombined. The best variant, DepoPETase β, with an increase in the melting temperatures (Tm) of 22.9 °C, exhibited superior depolymerization performance and enabled complete depolymerization of 100.5 g of untreated post-consumer PET (pc-PET) (0.26% Wenzyme/WPET enzyme loading) in liter-scale bioreactor at 50 °C within 4 d. Crystallization and molecular dynamics simulations revealed that the improved activity and thermostability of DepoPETase β were due to enhanced hydrogen bonds and salt bridges in the β-sheet region, a more tightly packed structure of the core sheets and the surrounding helix, and improved binding of PET to the active sites. This study not only demonstrates the importance of engineering strategy in the β-sheet region of PET hydrolases but also provides a potential PET depolymerase for large-scale PET recycling.

Systematic screening of enhancer-blocking insulators in Drosophila identifies their DNA sequence determinants.

Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.

Assay Development for High-Throughput Drug Screening Against Mycobacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

Using Reporter Gene Assays to Screen and Identify Chemical Compounds that Modulate Estrogen Receptor Activity.

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors.

A high throughput screening assay for human Thyroperoxidase inhibitors

Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors.

A robust high-throughput screening system to assess bacterial tyrosine ammonia lyase activity in the context of tyrosine inherited metabolic disorders.

Inborn errors of tyrosine metabolism result in patient's inability to degrade tyrosine. Current treatment consists of a phenylalanine and tyrosine restricted diet and nitisinone, causing a block in the tyrosine degradation pathway. However, tyrosine levels will increase, leading to acquired hypertyrosinemia, implying the need for an add-on treatment. Tyrosine ammonia lyases (TAL) can provide such an add-on treatment as they catalyze the deamination of tyrosine into p-coumaric acid and ammonia. In this study, we developed a robust high-throughput screening (HTS) assay to assess the capacity of bacterial TAL enzymes to decrease excessive tyrosine. The assay is based on the spectrophotometric quantification of p-coumaric acid after conversion of tyrosine by bacterial TAL. As a benchmark, TAL from Flavobacterium johnsoniae (FjTAL) was used to optimize the assay. Optimal growth conditions for high-level protein expression were determined by incubating transformed Escherichia coli BL21 (DE3) cells at different temperatures during various incubation times. Subsequently, assay temperature and pH were optimized followed by testing different ratios of tyrosine assay mixes to bacterial lysate. Finally, assay robustness and functionality were evaluated. Optimal FjTAL expression was obtained after incubation for 24h at 22°C. Ideal assay conditions consist of a 80/20 ratio of 1mM tyrosine assay mix to FjTAL lysate performed at pH 9.2 and 37°C. The robustness test showed Z' values > 0.4 and signal window values > 2 without edge or drift effects. As proof-of-principle, we successfully determined the catalytic activity of two other bacterial TAL enzymes RsTAL (5.718.10-3 ± 0.21.10-3) and SeSAM8 (4.658.10-3 ± 0.37.10-3). A robust, simple and reliable HTS assay was thus developed to evaluate the tyrosine degradation capacity of bacterial TAL enzymes.

Exploring American Elderberry Compounds for Antioxidant, Antiviral, and Antibacterial Properties Through High-Throughput Screening Assays Combined with Untargeted Metabolomics.

American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antibacterial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays was conducted to evaluate 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity against the NL4-3 virus (IC50 = 1.49 μM), followed by isorhamnetin (IC50 = 1.67 μM). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 μM), followed by luteolin (IC50 = 4.38 μM). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 μM), followed by cyanidin 3-O-glucoside (IC50 = 3.7 μM). These findings support and validate the potential health benefits of compounds found in American elderberry juices and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.

164 Combining in silico, in vitro, and in vivo approaches for studying taste perception and preferences of domestic cats and dogs

Abstract Developing food products for pet cats and dogs is an important and challenging area of the food industry. The diet offered must be both balanced and palatable to ensure the amount of food eaten meets their nutritional requirements. The taste perception of pets is different from humans in many cases (Li et al., 2005) and therefore species-specific research is necessary. Hence, understanding the flavor preferences of cats and dogs is an important area of research and requires multiple disciplines and scientific approaches. We have developed a range of complimentary methods for studying taste perception and preferences of domestic cats and dogs In silico: homology models of the pet taste receptors have been developed. These models are used to identify candidate compounds and elucidate the mechanisms of taste perception of pets. In vitro: cell-based high throughput screening assays of pet taste receptors have also been developed. These assays are used to identify taste-active compounds and confirm response to those identified via the in silico method to refine the model. In vivo: finally, a two-bottle choice test for cats has been developed. This test is used to confirm hedonic responses and determine optimum concentrations of single compounds and mixtures identified by the in silico and/or in vitro methods. Using this novel approach, we now have a deeper understanding of the taste perception and preferences of pets, with specific examples including Umami (McGrane et al., 2023) and Kokumi (Laffitte et al., 2021) taste perception of cats and bitter taste perception of dogs (Gibbs et al., 2022). This approach has also enabled us to reduce the number of in vivo tests required, so implementing the 3Rs, by prioritizing the most promising tastants using the in silico and in vitro methods. Gibbs, M., Winnig, M., Riva, I., Dunlop, N., Waller, D., Klebansky, B., Logan, D.W., Briddon, S.J., Holliday, N.D., McGrane, S.J. 2022. Bitter taste sensitivity in domestic dogs (Canis familiaris) and its relevance to bitter deterrents of ingestion. PLoS One. 17:e0277607. Laffitte, A., Gibbs, M., Hernangomez de Alvaro, C., Addison, J., Lonsdale, Z.N., Giribaldi, M.G., Rossignoli, A., Vennegeerts, T., Winnig, M., Klebansky, B., Skiles, J., Logan, D.W., McGrane, S.J. 2021. Kokumi taste perception is functional in a model carnivore, the domestic cat (Felis catus). Sci Reports 2021 111. 11:1–17. Li, X., Li, W., Wang, H., Cao, J., Maehashi, K., Huang, L., Bachmanov, A.A., Reed, D.R., Legrand-Defretin, V., Beauchamp, G.K., Brand, J.G. 2005. Pseudogenization of a sweet-receptor gene accounts for cats’ indifference toward sugar. PLoS Genet. 1:0027–0035.McGrane, S.J., Gibbs, M., Hernangomez De Alvaro, C., Dunlop, N., Winnig, M., Klebansky, B., Waller, D. 2023. Umami taste perception and preferences of the domestic cat (Felis catus), an obligate carnivore. Chem Senses. 48.

Repurposing of a library for high-content screening of inhibitors against Echinococcus granulosus

BackgroundCystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato (E. granulosus), with a worldwide distribution. The current treatment strategy for CE is insufficient. Limited drug screening models severely hamper the discovery of effective anti-echinococcosis drugs.MethodsIn the present study, using high-content screening technology, we developed a novel high-throughput screening (HTS) assay by counting the ratio of propidium iodide-stained dead protoscoleces (PSCs) to the total number of PSCs. In vitro and ex vivo cyst viability assays were utilized to determine the effect of drugs on cyst viability.ResultsUsing the newly established HTS assay, we screened approximately 12,000 clinical-stage or The Food and Drug Administration (FDA)-approved small molecules from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library, as well as the LOPAC1280 and SelleckChem libraries, as a strategic approach to facilitate the drug discovery process. Initial screening yielded 173 compounds with anti-echinococcal properties, 52 of which demonstrated dose–response efficacy against E. granulosus PSCs in vitro. Notably, two agents, omaveloxolone and niclosamide, showed complete inhibition upon further validation in cyst and microcyst viability assays in vitro after incubation for 3 days, and in an ex vivo cyst viability assay using cysts isolated from the livers of mice infected with E. granulosus, as determined by morphological assessment.ConclusionsThrough the development of a novel HTS assay and by repurposing libraries, we identified omaveloxolone and niclosamide as potent inhibitors against E. granulosus. These compounds show promise as potential anti-echinococcal drugs, and our strategic approach has the potential to promote drug discovery for parasitic infections.Graphical

A novel approach to triazole fungicides risk characterization: Bridging human biomonitoring and computational toxicology

Brazil stands as the world's leading coffee producer, where the extensive use of pesticides is economically critical yet poses health and environmental risks due to their non-selective mechanisms of action. Specifically, triazole fungicides are widely used in agriculture to manage fungal diseases and are known to disrupt mammalian CYP450 and liver microsomal enzymes. This research establishes a framework for risk characterization of human exposure to triazole fungicides by internal-dose biomonitoring, biochemical marker measurements, and integration of high-throughput screening (HTS) data via computational toxicology workflows from the Integrated Chemical Environment (ICE). Volunteers from the southern region of Minas Gerais, Brazil, were divided into two groups: farmworkers and spouses occupationally and environmentally exposed to pesticides from rural areas (n = 140) and individuals from the urban area to serve as a comparison group (n = 50). Three triazole fungicides, cyproconazole, epoxiconazole, and triadimenol, were detected in the urine samples of both men and women in the rural group. Androstenedione and testosterone hormones were significantly reduced in the farmworker group (Mann-Whitney test, p < 0.0001). The data show a significant inverse association of testosterone with cholesterol, LDL, VLDL, triglycerides, and glucose and a direct association with HDL (Spearman's correlation, p < 0.05). In the ICE workflow, active in vitro HTS assays were identified for the three measured triazoles and three other active ingredients from the pesticide formulations. The curated HTS data confirm bioactivities predominantly related to steroid hormone metabolism, cellular stress processes, and CYP450 enzymes impacted by fungicide exposure at occupationally and environmentally relevant concentrations based on the in vitro to in vivo extrapolation models. These results characterize the potentially significant human health risk, particularly from the high frequency and intensity of exposure to epoxiconazole. This study showcases the critical role of biomonitoring and utility of computational tools in evaluating pesticide exposure and minimizing the risk.

SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.

The Usnic Acid Analogue 4-FPBUA Enhances the Blood-Brain Barrier Function and Induces Autophagy in Alzheimer's Disease Mouse Models.

Preclinical and clinical studies have indicated that compromised blood-brain barrier (BBB) function contributes to Alzheimer's disease (AD) pathology. BBB breakdown ranged from mild disruption of tight junctions (TJs) with increased BBB permeability to chronic integrity loss, affecting transport across the BBB, reducing brain perfusion, and triggering inflammatory responses. We recently developed a high-throughput screening (HTS) assay to identify hit compounds that enhance the function of a cell-based BBB model. The HTS screen identified (S,E)-2-acetyl-6-[3-(4'-fluorobiphenyl-4-yl)acryloyl]-3,7,9-trihydroxy-8,9b-dimethyldibenzo-[b,d]furan-1(9bH)-one (4-FPBUA), a semisynthetic analogue of naturally occurring usnic acid, which protected the in vitro model against Aβ toxicity. Usnic acid is a lichen-derived secondary metabolite with a unique dibenzofuran skeleton that is commonly found in lichenized fungi of the genera Usnea. In this study, we aimed to evaluate the effect of 4-FPBUA in vitro on the cell-based BBB model function and its in vivo ability to rectify BBB function and reduce brain Aβ in two AD mouse models, namely, 5xFAD and TgSwDI. Our findings demonstrated that 4-FPBUA enhanced cell-based BBB function, increased Aβ transport across the monolayer, and reversed BBB breakdown in vivo by enhancing autophagy as an mTOR inhibitor. Induced autophagy was associated with a significant reduction in Aβ accumulation and related pathologies and improved memory function. These results underscore the potential of 4-FPBUA as a candidate for further preclinical exploration to better understand its mechanisms of action and to optimize dosing strategies. Continued research may also elucidate additional pathways through which 4-FPBUA contributed to the amelioration of BBB dysfunction in AD. Collectively, our findings supported the development of 4-FPBUA as a therapeutic agent against AD.