SLAS Discovery最新文献

The use of organoid models in biomedical research has grown substantially since their inception. As they gain popularity among scientists seeking more complex and biologically relevant systems, there is a direct need to expand and clarify potential uses of such systems in diverse experimental contexts. Herein we outline a high-content screening (HCS) platform that allows researchers to screen drugs or other compounds against three-dimensional (3D) cell culture systems in a multi-well format (384-well). Furthermore, we compare the quality of robotic liquid handling with manual pipetting and characterize and contrast the phenotypic effects detected by confocal imaging and biochemical assays in response to drug treatment. We show that robotic liquid handling is more consistent and amendable to high throughput experimental designs when compared to manual pipetting due to improved precision and automated randomization capabilities. We also show that image-based techniques are more sensitive to detecting phenotypic changes within organoid cultures than traditional biochemical assays that evaluate cell viability, supporting their integration into organoid screening workflows. Finally, we highlight the enhanced capabilities of confocal imaging in this organoid screening platform as they relate to discerning organoid drug responses in single-well co-cultures of organoids derived from primary human biopsies and patient-derived xenograft (PDX) models. Altogether, this platform enables automated, imaging-based HCS of 3D cellular models in a non-destructive manner, opening the path to complementary analysis through integrated downstream methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z’ = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC₅₀ <10 μM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

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, M^pro (also called 3C-like protease, 3CL^pro), 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, M^pro 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 M^pro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported M^pro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known M^pro 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 spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z’ factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein–protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Emerging highly pathogenic viruses can pose profound impacts on global health, the economy, and society. To meet that challenge, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) centers for early-stage identification and validation of novel antiviral drug candidates against viruses with pandemic potential. As part of this initiative, we established paired entry assays that simultaneously screen for inhibitors specifically targeting SARS-CoV-2 (SARS2), Lassa virus (LASV) and Machupo virus (MACV) entry. To do so we employed a dual pseudotyped virus (PV) infection system allowing us to screen ∼650,000 compounds efficiently and cost-effectively. Adaptation of these paired assays into 1536 well-plate format for ultra-high throughput screening (uHTS) resulted in the largest screening ever conducted in our facility, with over 2.4 million wells completed. The paired infection system allowed us to detect two PV infections simultaneously: LASV + MACV, MACV + SARS2, and SARS2 + LASV. Each PV contains a different luciferase reporter gene which enabled us to measure the infection of each PV exclusively, albeit in the same well. Each PV was screened at least twice utilizing different reporters, which allowed us to select the inhibitors specific to a particular PV and to exclude those that hit off targets, including cellular components or the reporter proteins. All assays were robust with an average Z’ value ranging from 0.5 to 0.8. The primary screening of ∼650,000 compounds resulted in 1812, 1506, and 2586 unique hits for LASV, MACV, and SARS2, respectively. The confirmation screening narrowed this list further to 60, 40, and 90 compounds that are unique to LASV, MACV, and SARS2, respectively. Of these compounds, 8, 35, and 50 compounds showed IC₅₀ value < 10 μM, some of which have much greater potency and excellent antiviral activity profiles specific to LASV, MACV, and SARS2, and none are cytotoxic. These selected compounds are currently being studied for their mechanism of action and to improve their specificity and potency through chemical modification.

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC₅₀ values determined in this assay correlates well with EC₅₀ values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Agonists of the secretin receptor have potential applications for diseases of the cardiovascular, gastrointestinal, and metabolic systems, yet no clinically-active non-peptidyl agonists of this receptor have yet been developed. In the current work, we have identified a new small molecule lead compound with this pharmacological profile. We have prepared and characterized a systematic structure-activity series around this thiadiazole scaffold to better understand the molecular determinants of its activity. We were able to enhance the in vitro activity and to maintain the specificity of the parent compound. We found the most active candidate to be quite stable in plasma, although it was metabolized by hepatic microsomes. This chemical probe should be useful for in vitro studies and needs to be tested for in vivo pharmacological activity. This could be an important lead toward the development of a first-in-class orally active agonist of the secretin receptor, which could be useful for multiple disease states.

Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.