SLAS Discovery最新文献

A rapid drug discovery response to influenza outbreaks with the potential to reach pandemic status could help minimize the virus's impact by reducing the time to identify anti-influenza drugs. Although several anti-influenza strategies have been considered in the search for new drugs, only a few therapeutic agents are approved for clinical use. The cytopathic effect induced by the influenza virus in Madin Darby canine kidney (MDCK) cells has been widely used for high-throughput anti-influenza drug screening, but the fact that the MDCK cells are not human cells constitutes a disadvantage when searching for new therapeutic agents for human use. We have developed a highly sensitive cell-based imaging assay for the identification of inhibitors of influenza A and B virus that is high-throughput compatible using the A549 human cell line. The assay has also been optimized for the assessment of the neutralizing effect of anti-influenza antibodies in the absence of trypsin, which allows testing of purified antibodies and serum samples. This assay platform can be applied to full high-throughput screening campaigns or later stages requiring quantitative potency determinations for structure-activity relationships.

High content screening (HCS) is becoming widely adopted as a high throughput screening modality, using hundred-of-thousands compounds library. The use of machine learning and artificial intelligence in image analysis is amplifying this trend. Another factor is the recognition that diverse cell phenotypes can be associated with changes in biological pathways relevant to disease processes. There are numerous challenges in HCS campaigns. These include limited ability to support replicates, low availability of precious and unique cells or reagents, high number of experimental batches, lengthy preparation of cells for imaging, image acquisition time (45–60 min per plate) and image processing time, deterioration of image quality with time post cell fixation and variability within wells and batches. To take advantage of the data in HCS, cell population based rather than well-based analyses are required. Historically, statistical analysis and hypothesis testing played only a limited role in non-high content high throughput campaigns. Thus, only a limited number of standard statistical criteria for hit selection in HCS have been developed so far. In addition to complex biological content in HCS campaigns, additional variability is impacted by cell and reagent handling and by instruments which may malfunction or perform unevenly. Together these can cause a significant number of wells or plates to fail. Here we describe an automated approach for hit analysis and detection in HCS. Our approach automates HCS hit detection using a methodology that is based on a documented statistical framework. We introduce the Virtual Plate concept in which selected wells from different plates are collated into a new, virtual plate. This allows the rescue and analysis of compound wells which have failed due to technical issues as well as to collect hit wells into one plate, allowing the user easier access to the hit data.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

Type 1 Diabetes mellitus (T1DM) is a chronic metabolic disorder characterized by pancreatic β-cells destruction. Despite substantial advances in T1DM treatment, lifelong exogenous insulin administration is the mainstay of treatments, and constant control of glucose levels is still a challenge. Endogenous insulin production by replacing insulin-producing cells is an alternative, but the lack of suitable donors is accounted as one of the main obstacles to its widespread application.

The research and trials overview demonstrates that endogenous production of insulin has started to go beyond the deceased-derived to stem cells-derived insulin-producing cells. Several protocols have been developed over the past couple of years for generating insulin-producing cells (IPCs) from various stem cell types and reprogramming fully differentiated cells. A straightforward and quick method for achieving this goal is to investigate and apply the β-cell specific transcription factors as a direct strategy for IPCs generation. In this review, we emphasize the significance of transcription factors in IPCs development from different non-beta cell sources, and pertinent research underlies the marked progress in the methods for generating insulin-producing cells and application for Type 1 Diabetes treatment.

The monocarboxylic acid transporter 4 (Mct-4), a downstream biomarker of hypoxia inducing factor (HIF)-1α, is involved in the cellular response to hypoxia, as indicated by the hypoxic response element in its promoter region. Using a tumorsphere assay as an in vitro 3-dimensional (3D) model generated using 384-well ultra-low attachment (ULA) plates for cell proliferation analysis using a plate-based image cytometer, we identify a hypoxic response in the tumorsphere model that is distinct from that of cells grown under 2-dimensional (2D) normoxic conditions and demonstrate a key role for Mct-4 in enabling 3D growth. The tumorsphere model yields evidence of an essential role for Mct-4 in multiple cell lines, which were genetically modified to underexpress and overexpress Mct-4, evidence not apparent in a standard 2D model of growth in the same cell lines. In addition, we identify the effects of overexpressing Mct-4 in cancer cell migration using a transwell chamber assay. We also show that the response to hypoxia may be circumvented by transfection with a CMV promoter driven Mct-4, which confers constitutive 3D growth, wherein tumorsphere growth inhibition by small molecule HIF-1α inhibitors is mitigated. Finally, we demonstrate quantifiable gene/protein expression differences between 2D and 3D cancer models based on the normoxic and hypoxic conditions. Therefore, the tumorsphere 3D model generated using 384-well ULA plates in combination with high-throughput image cytometer is demonstrated to provide a convenient, robust, and reproducible tool and method for the elucidation of mechanisms of action underlying tumor growth and migration in the hypoxic tumor microenvironment.

Micro/nano topological modification is critical for improving the in vivo behaviors of bone implants, regulating multiple cellular functions. Titania (TiO₂) nanotubes show the capacity of promoting osteoblast-related cell differentiation and induce effective osseointegration, serving as a model material for studying the effects of micro/nano-topological modifications on cells. However, the intracellular signaling pathways by which TiO₂ nanotubes regulate the osteogenic differentiation of stem cells are not fully defined. Thy-1 (CD90), a cell surface glycoprotein anchored by glycosylphosphatidylinositol, has been considered a key molecule in osteoblast differentiation in recent years. Nevertheless, whether the micro/nano topology of the implant surface leads to changes in Thy-1 is unknown, as well as whether these changes promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Here, TiO₂ nanotubes of various diameters were prepared by adjusting the anodizing voltage. qPCR and immunoblot were carried out to assess the mechanism by which TiO₂ nanotubes regulate Thy-1. The results revealed Ti plates harboring TiO₂ nanotubes ∼100-nm diameter (TNT-100) markedly upregulated Thy-1. Subsequently, upregulated Thy-1 promoted the activation of Fyn/RhoA/MLC Ⅱ/F-actin axis, which enhanced the nuclear translocation of YAP. After Thy-1 knockdown by siRNA, the Fyn/RhoA/MLC Ⅱ/F-actin axis was significantly inhibited and TiO₂ nanotubes showed decreased effects on osteogenic differentiation. Therefore, Thy-1 upregulation might be a major mechanism by which micro/nano-topological modification of TiO₂ nanotubes promotes osteogenic differentiation in BMSCs. This study provides novel insights into the molecular mechanism of TiO₂ nanotubes, which may help design improved bone implants for clinical application.

The pivotal role of myofibroblast contractility in the pathophysiology of fibrosis is widely recognized, yet HTS approaches are not available to quantify this critically important function in drug discovery. We developed, validated, and scaled-up a HTS platform that quantifies contractile function of primary human lung myofibroblasts upon treatment with pro-fibrotic TGF-β1. With the fully automated assay we screened a library of 40,000 novel small molecules in under 80 h of total assay run-time. We identified 42 hit compounds that inhibited the TGF-β1-induced contractile phenotype of myofibroblasts, and enriched for 19 that specifically target myofibroblasts but not phenotypically related smooth muscle cells. Selected hits were validated in an ex vivo lung tissue models for their inhibitory effects on fibrotic gene upregulation by TGF-β1. Our results demonstrate that integrating a functional contraction test into the drug screening process is key to identify compounds with targeted and diverse activity as potential anti-fibrotic agents.

Cardiovascular toxicity remains a major cause of drug attrition in early drug development, clinical trials, and post-market surveillance. In vitro assessment of cardiovascular liabilities often relies on single cell type-based model systems coupled with functional assays, like calcium flux and multielectrode arrays. Although these models offer high-throughput capabilities and demonstrate good predictivity for functional cardiotoxicities, they fail to consider the vital contribution of non-myocyte cells, thus limiting the potential for integrated risk assessment. Complex 3D hPSC-derived multicellular cardiac model systems have been growing in popularity; however, many of these models are limited to low-throughput with lengthy development timelines and high costs, which hampers their suitability to drug discovery.

To optimize the development of an in vitro multicellular model system containing human-induced pluripotent stem-cell derived cardiomyocytes, endothelial cells and cardiac fibroblasts, we employed the Synthace platform, which enables scientists to express complex experimental intent in a simple format (e.g. Design of Experiments) and to translate this to automation protocols using no-code. Utilizing this approach, we systematically screened the impact of multiple cell culture parameters, including the co-culture of three cell types, on cardiac contractility, with minimal hands-on time. Our platform accelerates the assay development process, providing users with an efficient means to explore and optimize the experimental space for the development of multicellular models. This is particularly valuable in scenarios involving variable biological responses and limited understanding of underling mechanisms. Moreover, users can make better use of resources, streamline their workflows, and drive data-driven decision-making throughout the assay development journey.

Nanobodies are small, single-domain antibodies that have emerged as a promising tool in cancer immunotherapy. These molecules can target specific antigens on cancer cells and trigger an immune response against them. In this mini-review article, we highlight the potential of nanobodies in cell-mediated immunotherapy for cancer treatment. We discuss the advantages of nanobodies over conventional antibodies, their ability to penetrate solid tumors, and their potential to enhance the efficacy of other immunotherapeutic agents. We also provide an overview of recent preclinical and clinical studies that have demonstrated the effectiveness of nanobody-based immunotherapy in various types of cancer.

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3′ to 5′ directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions. Assay development efforts revealed DHX9 can use double stranded RNA with 18-mer poly(U) 3′ overhangs and as well as significantly shorter overhangs at the 5′ or 3′ end as substrates. The enzymatic assays are augmented by a robust SPR assay for compound validation. A mechanism-derived inhibitor, GTPγS, was characterized as part of the validation of these assays and a crystal structure of GDP bound to cat DHX9 has been solved. In addition to enabling drug discovery efforts for DHX9, these assays may be extrapolated to other RNA helicases providing a valuable toolkit for this important target class.