High-throughput Screening Of Small Molecules Research Articles

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

Generation and Analysis of CCM Phenotypes in C. elegans.

This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.

Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening.

High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.

Automated collective motion analysis validates human keratinocyte stem cell cultures

Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential. Image processing and kernel density estimation were used to automatically extract the area of keratinocyte colonies from phase-contrast images of cultures containing feeder cells. The DeepFlow algorithm was then used to calculate locomotion speed of the colony area by analyzing serial images. This image-processing pipeline successfully identified keratinocyte stem cell colonies by measuring cell locomotion speed, and also assessed the effect of oligotrophic culture conditions and chemical inhibitors on keratinocyte behavior. Therefore, this study provides automated procedures for image-based quality control of stem cell cultures and high-throughput screening of small molecules targeting stem cells.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of thesepopulations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations.

Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'>0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

Employing a ZTP Riboswitch to Detect Bacterial Folate Biosynthesis Inhibitors in a Small Molecule High-Throughput Screen.

Various riboswitch classes are being discovered that precisely monitor the status of important biological processes, including metabolic pathway function, signaling for physiological adaptations, and responses to toxic agents. Biochemical components for some of these processes might make excellent targets for the development of novel antibacterial molecules, which can be broadly sought by using phenotypic drug discovery (PDD) methods. However, PDD data do not normally provide clues regarding the target for each hit compound. We have developed and validated a robust fluorescent reporter system based on a ZTP riboswitch that identifies numerous folate biosynthesis inhibitors with high sensitivity and precision. The utility of the riboswitch-based PDD strategy was evaluated using Escherichia coli bacteria by conducting a 128 310-compound high-throughput screen, which identified 78 sulfanilamide derivatives among the many initial hits. Similarly, representatives of other riboswitch classes could be employed to rapidly match antibacterial hits with the biological processes they target.

Diversity of small molecule HIV-1 latency reversing agents identified in low- and high-throughput small molecule screens.

The latency phenomenon produced by human immunodeficiency virus (HIV‐1) prevents viral clearance by current therapies, and consequently development of a cure for HIV‐1 disease represents a formidable challenge. Research over the past decade has resulted in identification of small molecules that are capable of exposing HIV‐1 latent reservoirs, by reactivation of viral transcription, which is intended to render these infected cells sensitive to elimination by immune defense recognition or apoptosis. Molecules with this capability, known as latency‐reversing agents (LRAs) could lead to realization of proposed HIV‐1 cure strategies collectively termed “shock and kill,” which are intended to eliminate the latently infected population by forced reactivation of virus replication in combination with additional interventions that enhance killing by the immune system or virus‐mediated apoptosis. Here, we review efforts to discover novel LRAs via low‐ and high‐throughput small molecule screens, and summarize characteristics and biochemical properties of chemical structures with this activity. We expect this analysis will provide insight toward further research into optimized designs for new classes of more potent LRAs.

Electrochemical biosensors for the detection and study of α-synuclein related to Parkinson's disease – A review

Parkinson's disease (PD) is a long-term degenerative disorder that affects predominately dopaminergic neurons in the substantia nigra, which mainly control movement. Alpha-synuclein (α-syn) is a major constituent of Lewy bodies that are reported to be the most important toxic species in the brain of PD patients. In this critical review, we highlight novel electrochemical biosensors that have been recently developed utilizing aptamers and antibodies in connection with various nanomaterials to study biomarkers related to PD such as α-syn. We also review several research articles that have utilized electrochemical biosensors to study the interaction of α-syn with biometals as well as small molecules such as clioquinol, (−)-epigallocatechin-3-gallate (EGCG) and baicalein. Due to the significant advances in nanomaterials in the past decade, electrochemical biosensors capable of detecting multiple biomarkers in clinically relevant samples in real-time have been achieved. This may facilitate the path towards commercialization of electrochemical biosensors for clinical applications and high-throughput screening of small molecules for structure-activity relationship (SAR) studies.

THE INTEGRATED USE OF HUMAN MODELS OF LEUKEMIA TO IDENTIFY POTENTIAL THERAPEUTIC TARGETS

Acute myeloid leukemia (AML) is a disease that results from the uncontrolled growth of primitive myeloid progenitor cells that are unable to undergo terminal differentiation. To better understand the genetic and epigenetic changes involved in leukemogenesis, we use a human model leukemia system where cord blood donor cells from a single donor are transduced with the human KMT2A-MLLT3 gene fusion to produce leukemias. We have generated multiple model leukemias and compared these to genetically matched pediatric AML patient samples that has allowed us to characterize the step-wise epigenetic and splicing changes that accompany leukemogenesis, allowing us to build an integrated view of this process. Our analysis of these data have revealed a number of candidate biomarker genes that are expressed in KMT2A translocated AMLs but not normal blood cells, some of which are important for leukemic growth. These candidate genes, which have validated diagnostic value, have also been used as targets for monoclonal antibody generation in order to assess their value as potential immunotherapeutics. Having established the value of this approach, we are now expanding our models to include additional oncogenic drivers in order to elucidate the differences in patient outcomes associated with the presence of specific fusions. Moreover, we have recently leveraged these model leukemias and matched patient samples to perform a high-throughput small molecule screen to identify novel therapeutic vulnerabilities and deconvolute the role of secondary mutations in pediatric patients. The establishment of this chemogenomic pipeline to generate, characterize, and then screen model leukemias will provide not only insight into the molecular mechanisms involved in this disease, but also how they can be rationally targeted to improve patient outcomes.

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth invitro and against a xenograft model invivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.

A High-Throughput Screening Method to Identify Compounds Displaying Human Vascular Embryonic Toxicity.

This article describes a screening platform to identify compounds that affect human embryonic vascular development. The procedure comprises the generation of human embryonic-like endothelial cells (ECs) from human pluripotent stem cells (hPSCs) and subsequent maturation under arterial flow conditions; the use of these cells for the high-throughput screening of small molecules that specifically inhibit the survival of embryonic-like ECs; the confirmation of the hits in embryonic-like ECs cultured under flow shear stress; and final validation in mouse embryonic ECs. The embryonic-like ECs express embryonic genes including DLL1, EPHB2, LYN, TEK, ID1, NRP2, CAST, FLT1, IGF1, DKK3, NIN, LEF1, and SORBS3. The entire screening procedure (without the validation step) can be completed within 1 month. This platform is an alternative/complement to standard animal protocols for assessing the effects of chemicals on embryonic vascular development. © 2019 by John Wiley & Sons, Inc.

Small Molecule IL-36\u03b3 Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.

Identification of a small-molecule compound that inhibits homodimerization of oncogenic NAC1 protein and sensitizes cancer cells to anticancer agents

Nucleus accumbens-associated protein-1 (NAC1) is a transcriptional repressor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and plays critical roles in tumor development, progression, and drug resistance. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. However, effective approaches for effective targeting of this nuclear protein remain elusive. In this study, we identified a core unit consisting of Met7 and Leu90 in NAC1's N-terminal domain (amino acids 1-130), which is critical for its homodimerization and stability. Furthermore, using a combination of computational analysis of the NAC1 dimerization interface and high-throughput screening (HTS) for small molecules that inhibit NAC1 homodimerization, we identified a compound (NIC3) that selectively binds to the conserved Leu-90 of NAC1 and prevents its homodimerization, leading to proteasomal NAC1 degradation. Moreover, we demonstrate that NIC3-mediated down-regulation of NAC1 protein sensitizes drug-resistant tumor cells to conventional chemotherapy and enhances the antimetastatic effect of the antiangiogenic agent bevacizumab both in vitro and in vivo These results suggest that small-molecule inhibitors of NAC1 homodimerization may effectively sensitize cancer cells to some anticancer agents and that NAC1 homodimerization could be further explored as a potential therapeutic target in the development of antineoplastic agents.

A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors.

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.

A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

High-throughput screens for agonists of bone morphogenetic protein (BMP) signaling identify potent benzoxazole compounds

Bone morphogenetic protein (BMP) signaling is critical in renal development and disease. In animal models of chronic kidney disease (CKD), re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires harmful doses to achieve efficacy and is costly because of BMPs' complex synthesis. Therefore, alternative strategies are needed to harness the beneficial effects of BMP signaling in CKD. Key aspects of the BMP signaling pathway can be regulated by both extracellular and intracellular molecules. In particular, secreted proteins like noggin and chordin inhibit BMP activity, whereas kielin/chordin-like proteins (KCP) enhance it and attenuate kidney fibrosis or CKD. Clinical development of KCP, however, is precluded by its size and complexity. Therefore, we propose an alternative strategy to enhance BMP signaling by using small molecules, which are simpler to synthesize and more cost-effective. To address our objective, here we developed a small-molecule high-throughput screen (HTS) with human renal cells having an integrated luciferase construct highly responsive to BMPs. We demonstrate the activity of a potent benzoxazole compound, sb4, that rapidly stimulated BMP signaling in these cells. Activation of BMP signaling by sb4 increased the phosphorylation of key second messengers (SMAD-1/5/9) and also increased expression of direct target genes (inhibitors of DNA binding, Id1 and Id3) in canonical BMP signaling. Our results underscore the feasibility of utilizing HTS to identify compounds that mimic key downstream events of BMP signaling in renal cells and have yielded a lead BMP agonist.

Abstract A099: Using high-throughput phenotypic screening to identify therapeutic targets for the inhibition of myeloid-derived suppressor cells

Abstract Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive function, which inhibit the antitumor activity of T-cells and natural killer (NK) cells. MDSC number is greatly increased in tumor-bearing mice and in cancer patients, and in the clinic, MDSC accumulation is associated with cancer progression, recurrence, and poor response to chemo-, radio- and immunotherapies. The increasing evidence for the clinical significance of MDSC has triggered a strong interest in the therapeutic modulation of their function. To date, however, limited progress has been made in this direction, as a major challenge in the field remains the identification of suitable therapeutic targets for the development of novel drugs. Here, we describe a systematic approach in which a small-molecule high-throughput phenotypic screen was used to identify MDSC targets and pathways of therapeutic relevance. This screen was based on a validated in vitro mouse mononuclear MDSC (M-MDSC) model, in which hematopoietic progenitors, immortalized using a NUP98/HOXB4 transgene, were differentiated into immunosuppressive MDSC. Using this model, we developed a 384-well-based phenotypic screening assay, in which the suppressive effect of mouse M-MDSC on CD8+ T-cell proliferation and cytokine secretion was monitored. We screened a small molecule library, comprising 5000+ biologically active compounds with known target(s), and identified 116 compounds that potently disrupted MDSC suppression of T-cell function. With the help of chemoinformatics methods, reported target activities associated with the compounds were annotated, and a set of targets and pathways of potential significance for MDSC-driven immunosuppression was identified. Altogether, this work provides insight into the signaling nodes that could be of relevance for MDSC function, and offers a path forward for the therapeutic targeting of MDSC. Citation Format: Elissaveta Petrova, Sandra Schäffner, Jan-Carsten Pieck, Christian Herhaus, Friedrich Rippmann, Oliver Pöschke, Laura Helming. Using high-throughput phenotypic screening to identify therapeutic targets for the inhibition of myeloid-derived suppressor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A099.

NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules.

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.

Integration of RNAi and Small Molecule Screens to Identify Targets for Drug Development.

Cellular models for siRNA and small molecule high-throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a twofold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screening strategy.