High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2.

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

Protocol for 3D screening of lung cancer spheroids using natural products.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for ∼84% of all lung cancer cases. NSCLC remains one of the leading causes of cancer-associated death, with a 5-year survival rate less than 25%. This type of cancer begins with healthy cells that change and start growing out of control, leading to the formation of lesions or tumors. Understanding the dynamics of how the tumor microenvironment promotes cancer initiation and progression that leads to cancer metastasis is crucial to help identify new molecular therapies. 3D primary cell tumor models have received renewed recognition due to their ability to better mimic the complexity of in vivo tumors and as a potential bridge between traditional 2D culture and in vivo studies. Vast improvements in 3D cell culture technologies make them much more cost effective and efficient largely because of the use of a cell-repellent surfaces and a novel angle plate adaptor technology. To exploit this technology, we accessed the Natural Products Library (NPL) at UF Scripps, which consists of crude extracts, partially purified fractions, and pure natural products (NPs). NPs generally are not very well represented in most drug discovery libraries and thus provide new insights to discover leads that could potentially emerge as novel molecular therapies. Herein we describe how we combined these technologies for 3D screening in 1536 well format using a panel of ten NSCLC cells lines (5 wild type and 5 mutant) against ∼1280 selected members of the NPL. After further evaluation, the selected active hits were prioritized to be screened against all 10 NSCLC cell lines as concentration response curves to determine the efficacy and selectivity of the compounds between wild type and mutant 3D cell models. Here, we demonstrate the methods needed for automated 3D screening using microbial NPs, exemplified by crude extracts, partially purified fractions, and pure NPs, that may lead to future use targeting human cancer.

Crystal Structures of Lignocellulosic Furfuryl Biobased Polydiacetylenes with Hydrogen-Bond Networks: Influencing the Direction of Solid-State Polymerization through Modification of the Spacer Length.

We present the topochemical polymerization of two lignocellulosic biobased diacetylenes (DAs) that only differ by an alkyl spacer length of 1 methylene (n = 1) or 3 methylene units (n = 3) between the diyne and carbamate functionalities. Their crystalline molecular organizations have the distinctive feature of being suitable for polymerization in two potential directions, either parallel or skewed to the hydrogen-bonded (HB) network. However, single-crystal structures of the final polydiacetylenes (PDAs) demonstrate that the resulting orientation of the conjugated backbones is different for these two derivatives, which lead to HB supramolecular polymer networks (2D nanosheets) for n = 1 and to independent linear PDA chains with intramolecular HBs for n = 3. Thus, spacer length modification can be considered a new strategy to influence the molecular orientation of conjugated polymer chains, which is crucial for developing the next generation of materials with optimal mechanical and optoelectronic properties. Calculations were performed on model oligodiacetylenes to evaluate the cooperativity effect of HBs in the different crystalline supramolecular packing motifs and the energy profile related to the torsion of the conjugated backbone of a PDA chain (i.e., its ability to adopt planar or helical conformations).

High throughput screening for compounds to the orphan nuclear receptor NR2F6.

NR2F6 is considered an orphan nuclear receptor since its endogenous ligand has yet to be identified. Recently, NR2F6 has emerged as a novel cancer therapeutic target. NR2F6 has been demonstrated to be upregulated or overexpressed in several cancers. Importantly, Nr2f6-/- mice spontaneously reject tumors and develop host-protective immunological memory, a consequence of NR2F6 acting as an immune checkpoint in effector T cells. Collectively, these data suggest that modulation of NR2F6 activity may have important clinical applications in the fight against cancer. The nuclear receptor superfamily of ligand-regulated transcription factors has proven to be an excellent source of targets for therapeutic intervention of a broad range of diseases. Approximately 15% of FDA approved drugs target NRs, demonstrating their clinical efficacy. To identify small molecule regulators of NR2F6 activity, with the overall goal of immuno-oncology, we developed and initiated a high-throughput cell-based assay that specifically measures the transcriptional activity of NR2F6. We completed automated screening of approximately 666,000 compounds and identified 5,008 initial hits. Further screening efforts, including counterscreening assays, confirmed 128 of these hits, most of which had IC50s of equal to or less than 5µM potencies. Here, we report, for the first time, the identification of several small molecule compounds to the orphan nuclear receptor, NR2F6.

Lead identification using 3D models of pancreatic cancer.

Recent technological advances have enabled 3D tissue culture models for fast and affordable HTS. We are no longer bound to 2D models for anti-cancer agent discovery, and it is clear that 3D tumor models provide more predictive data for translation of preclinical studies. In a previous study, we validated a microplate 3D spheroid-based technology for its compatibility with HTS automation. Small-scale screens using approved drugs have demonstrated that drug responses tend to differ between 2D and 3D cancer cell proliferation models. Here, we applied this 3D technology to the first ever large-scale screening effort completing HTS on over 150K molecules against primary pancreatic cancer cells. It is the first demonstration that a screening campaign of this magnitude using clinically relevant, ex-vivo 3D pancreatic tumor models established directly from biopsy, can be readily achieved in a fashion like traditional drug screen using 2D cell models. We identified four unique series of compounds with sub micromolar and even low nanomolar potency against a panel of patient derived pancreatic organoids. We also applied the 3D technology to test lead efficacy in autologous cancer associated fibroblasts and found a favorable profile for better efficacy in the cancer over wild type primary cells, an important milestone towards better leads. Importantly, the initial leads have been further validated in across multiple institutes with concordant outcomes. The work presented here represents the genesis of new small molecule leads found using 3D models of primary pancreas tumor cells.

Screening for modulators of autism spectrum disorder using induced human neurons.

Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder. There are no drugs to treat the core symptoms. De novo mutations often play an important role in ASD and multiple high-risk loci have been identified in the last decade. These mutations range from copy number variants to small insertion/deletion and single nucleotide variants. Large-scale exome sequencing has identified over 100 risk genes that are associated with ASD. Both etiological heterogeneity and unavailability of human neurons remain major hurdles in understanding the pathophysiology of ASD and testing of new drug candidates. Hence, the most achievable and relevant model to screen for potential drugs is human neurons from inducible pluripotent stem cells (iPSCs), including those from individuals with genetic mutations. In this study, we tested stem cells from individuals carrying mutations in ADNP, FOXP1 or SHANK3. They were scaled and reprogrammed to glutamatergic neurons and assessed for the effects of their specific mutations on neurite outgrowth. High Content Analysis allowed us to observe phenotypic differences between ASD neurons compared to controls, in terms of neuron number, neurite number and neurite length per neuron. Further, neurons were derived from both patient derived and genetically modified iPSCs with DDX3X mutation which were tested against 5088 drug like compounds. We assessed individual compound effects on the induced neurons to determine if they elicited changes that would indicate neurite growth (neuroprotection) or, alternatively, reduce outgrowth and hence appear neurotoxic. This report includes all methods, phenotypic outcomes, and results for the largest ASD small molecule screening effort done to date.

Rapid deployment of inexpensive open-source orbital shakers in support of high-throughput screening.

Open-source projects continue to grow in popularity alongside open-source educational resources, software, and hardware tools. The impact of this increased availability of open-source technologies is that end users are empowered to have greater control over the tools that they work with. This trend extends in the life science laboratory space, where new open-source projects are routinely being published that allow users to build and modify scientific equipment specifically tailored to their needs, often at a reduced cost from equivalent commercial offerings. Recently, we identified a need for a compact orbital shaker that would be usable in temperature and humidity-controlled incubators to support the development and execution of a high-throughput suspension cell-based assay. Based on the requirements provided by staff biologists, an open-source project known as the DIYbio orbital shaker was identified on Thingiverse, then quickly prototyped and tested. The initial orbital shaker prototype based on the DIYbio design underwent an iterative prototyping and design process that proved to be straightforward due to the open-source nature of the project. The result of these efforts has been the successful initial deployment of ten shakers as of August 2021. This afforded us the scalability and efficacy needed to complete a large-scale screening campaign in less time and at less cost than if we purchased larger, less adaptable orbital shakers. Lessons learned from prototyping, modifying, validating, deploying and maintaining laboratory devices based on an open-source design in support of a full-scale drug discovery high-throughput screening effort are described within this manuscript.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1-4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

Applications for Open Source Microplate-Compatible Illumination Panels.

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.  Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.).  M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Automating a Magnetic 3D Spheroid Model Technology for High-Throughput Screening.

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

Design of Microplate-Compatible Illumination Panels for a Semiautomated Benchtop Pipetting System.

Microplates are an essential tool used in laboratories for storing research materials and performing assays. Many types of laboratory automation exist that greatly reduce the effort needed to utilize microplates; however, there are cases where the use of such automation is not feasible or practical. In these instances, researchers must work in an environment where liquid handling operations are performed manually with handheld pipetting devices. This type of work is tedious and error-prone as it relies on researchers to manually track a significant amount of metadata, including transfer volumes, plate barcodes, well contents, and well locations. To address this challenge, we have developed an open-source, semiautomated benchtop system that facilitates manual pipetting using visual indicators. This device streamlines the process of identifying the location of wells so that the researcher can perform manual transfers in a more efficient, reliable, and accurate manner. This system utilizes a graphical user interface that allows the user to load worklists and then issues commands to illuminate wells of interest, providing a visual indicator for users to follow in real time. The software and hardware tools utilized for development, along with the implementation techniques used to produce this system, are described within.

The Scripps Molecular Screening Center and Translational Research Institute.

The Scripps Research Molecular Screening Center (SRMSC) was founded in 2004 and comprises more than $22 million of specialized automation. As part of the Translational Research Institute (TRI), it comprises early drug discovery labs and medicinal chemistry. Together with Scripps Research at the La Jolla, California, campus, this represents one of the most competitive academic industrial screening centers worldwide. The SRMSC uses automated platforms, one a screening cell and the other a cherry-picking platform. Matched technologies are available throughout Scripps to allow scientists to develop assays and prepare them for automated screening. The library comprises more than 1 million drug-like compounds, including a proprietary collection of >665,000 molecules. Internal chemistry has included ~40,000 unique compounds that are not found elsewhere. These collections are screened against a myriad of disease targets, including cell-based and biochemical assays that are provided by Scripps faculty or from global investigators. Scripps has proven competence in all detection formats, including high-content analysis, fluorescence, bioluminescence resonance energy transfer (BRET), time-resolved fluorescence resonance energy transfer (TR-FRET), fluorescence polarization (FP), luminescence, absorbance, AlphaScreen, and Ca++ signaling. These technologies are applied to NIH-derived collaborations as well as biotech and pharma initiatives. The SRMSC and TRI are recognized for discovering multiple leads, including Ozanimod.

IoT for Real-Time Measurement of High-Throughput Liquid Dispensing in Laboratory Environments.

Critical to maintaining quality control in high-throughput screening is the need for constant monitoring of liquid-dispensing fidelity. Traditional methods involve operator intervention with gravimetric analysis to monitor the gross accuracy of full plate dispenses, visual verification of contents, or dedicated weigh stations on screening platforms that introduce potential bottlenecks and increase the plate-processing cycle time. We present a unique solution using open-source hardware, software, and 3D printing to automate dispenser accuracy determination by providing real-time dispense weight measurements via a network-connected precision balance. This system uses an Arduino microcontroller to connect a precision balance to a local network. By integrating the precision balance as an Internet of Things (IoT) device, it gains the ability to provide real-time gravimetric summaries of dispensing, generate timely alerts when problems are detected, and capture historical dispensing data for future analysis. All collected data can then be accessed via a web interface for reviewing alerts and dispensing information in real time or remotely for timely intervention of dispense errors. The development of this system also leveraged 3D printing to rapidly prototype sensor brackets, mounting solutions, and component enclosures.

Improved Scalability of Neuron-Based Phenotypic Screening Assays for Therapeutic Discovery in Neuropsychiatric Disorders.

There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Isomorphous crystal structures of chlorodi-acetyl-ene and iododi-acetyl-ene derivatives: simultaneous hydrogen and halogen bonds on carbon-yl.

The crystal structures of tert-butyl (5-chloro-penta-2,4-diyn-1-yl)carbamate, C10H12ClNO2 (II), and tert-butyl (5-iodo-penta-2,4-diyn-1-yl)carbamate, C10H12INO2 (IV), are isomorphous to previously reported structures and accordingly their mol-ecular and supra-molecular structures are similar. In the crystals of (II) and (IV), mol-ecules are linked into very similar two-dimensional wall organizations with anti-parallel carbamate groups involved in a combination of hydrogen and halogen bonds (bifurcated N-H⋯O=C and C≡C-X⋯O=C inter-actions on the same carbonyl group). There is no long-range parallel stacking of diynes, so the topochemical polymerization of di-acetyl-ene is prevented. A Cambridge Structural Database search revealed that C≡C-X⋯O=C contacts shorter than the sum of the van der Waals radii are scarce (only one structure for the C≡C-Cl⋯O=C inter-action and 13 structures for the similar C≡C-I⋯O=C inter-action).

Monitoring of HTS compound library quality via a high-resolution image acquisition and processing instrument.

This report presents the high-resolution image acquisition and processing instrument for compound management applications (HIAPI-CM). The HIAPI-CM combines imaging spectroscopy and machine-vision analysis to perform rapid assessment of high-throughput screening (HTS) compound library quality. It has been customized to detect and classify typical artifacts found in HTS compound library microtiter plates (MTPs). These artifacts include (1) insufficient volume of liquid compound sample, (2) compound precipitation, and (3) colored compounds that interfere with HTS assay detection format readout. The HIAPI-CM is also configured to automatically query and compare its analysis results to data stored in a LIMS or corporate database, aiding in the detection of compound registration errors. To demonstrate its capabilities, several compound plates (n=5760 wells total) containing different artifacts were measured via automated HIAPI-CM analysis, and results compared with those obtained by manual (visual) inspection. In all cases, the instrument demonstrated high fidelity (99.8% empty wells; 100.1% filled wells; 94.4% for partially filled wells; 94.0% for wells containing colored compounds), and in the case of precipitate detection, the HIAPI-CM results significantly exceeded the fidelity of visual observations (220.0%). As described, the HIAPI-CM allows for noninvasive, nondestructive MTP assessment with a diagnostic throughput of about 1min per plate, reducing analytical expenses and improving the quality and stewardship of HTS compound libraries.

High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate.

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.

Rational design and gas-phase characterization of molecular capsules by self-assembly of a symmetric hexasubstituted benzene with seven-membered lactams.

A novel very simple C3-symmetric lactam has been rationally designed to self-assemble as dimers or larger platonic solid capsules. Its core flat benzene ring bears three seven-membered lactams, resulting in tripod molecules that aggregate into robust tetrameric capsules. The self-assembly process was templated by tetraethylammonium cations and proven to be reversible by ESI spectroscopy in various solvents.